chondrocyte: a target for the treatment …...osteoarthritis cartilage 2007; in press. 3. lin z,...
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CHONDROCYTE: A TARGET FOR THE TREATMENT
OF OSTEOARTHRITIS
Zhen LIN
This thesis is presented for the degree of
DOCTOR OF PHILOSOPHY Of The University of Western Australia
School of Surgery & Pathology
The University of Western Australia
2007
The work presented in this thesis was performed in The University of
Western Australia, Department of Surgery (Orthopaedics), Queen Elizabeth
II Medical Centre, Nedlands, Western Australia.
DECLARATION
The work detailed in this thesis was performed by the candidate unless otherwise
specified. This thesis is submitted for the degree of Doctorate of Philosophy at The
University of Western Australia and has not been submitted elsewhere for any other
degree.
CANDIDATE
Zhen Lin Signature: ________________________________
SUPERVISORS
Prof. Ming Hao Zheng Signature: ________________________________
A/Prof. Jiake Xu Signature: ________________________________
Prof. David Wood Signature: ________________________________
EXTERNAL SUPERVISOR
Prof. Alan J. Grodzinsky Signature: ________ ______
Date ______24/10/2007______________________
I
ACKNOWLEDGEMENTS
The study described in this thesis was undertaken in the Department of Orthopaedic,
School of Surgery and Pathology, The University of Western Australia. Firstly, I would
like to thank my supervisor Prof. Ming Hao Zheng. Without your guidance, support and
inspiration, I would never have gone this far. Thank you very much for your constant
encouragement and believe in me.
I wish to express my thanks to A/Prof. Jiake Xu for his technical guidance and skillful
assistance throughout my study, to Prof. David Wood for his supply of human tissue
samples. In particularly, I would like to extent my gratitude and appreciation to Dr.
Jonathan Fitzgerald and Prof. Grodzinsky from MIT, U.S. for their invaluable advises
and assistance in statistical analyses.
Also, I would like to thank Rachael Duff and Tina from Australian Neuromuscular
Research Institute for providing the equipment and technical support for Real-time PCR
analysis, and Dr Martin Cake for your useful advises. Many thanks to all the people in
the lab, especially Lyn for initial technical guidance, Nathan for your professional
scientific opinions and assistance, Craig for managing all the paper work, Lesley for
your patience and kindness, Jaime for bringing a lot of sense to the lab, Rina for your
friendship and company, Jim and Sky for sharing the “Perth Survival Guideline”, and
Vincent for providing free meals and Chinese culture reeducation. You guys have made
the lab such a wonderful place to work in and made science so not as boring as it sounds
to be.
Most importantly, thank you and a big huge to my family and friends in China. Thank
you all so very much for the love and support you have always given me throughout my
life. Without you this thesis would never be possible.
II
SPECIAL DEDICATION
This thesis is dedicated to my father for his unconditional and endless love, guidance
and support throughout my life.
III
TABLE OF CONTENTS
page
Declaration I
Acknowledgements II
Special Dedication III
Table of contents IV
Publication, Meetings & Awards IX
Abbreviations XI
List of Figures XVII
List of Tables XX
Thesis Summary XXI
Chapter 1: The chondrocyte: Biology and its Clinical Application (Tissue
Eng. 12, 2006; Published)
1
Chapter 2: Introduction of Osteoarthritis 15
2.1 General Introduction 15
2.2 Clinical Features 15
2.3 Etiology of OA 16
2.4 Pathogenesis 18
2.4.1 Chondrocyte apoptosis and proliferation 19
2.4.2 Alteration of metabolic activity 20
2.4.3 Influences of cytokines and growth factors 21
2.4.4 Phenotypic modulation of chondrocytes 22
2.4.5 Formation of osteophytes 23
IV
2.4.6 Subchondral bone formation 25
2.5 Diagnostic Approaches 26
2.6 Conventional Treatment of OA 26
2.6.1 Early-stage OA treatment 26
2.6.2 Late-stage OA treatment 29
2.7 Novel or Future Therapeutic Targets 29
2.7.1 Cytokine therapies 29
2.7.2 Gene therapies 30
2.7.3 Inhibition of inflammatory signaling pathways 31
2.7.4 Tissue engineering therapies 32
Chapter 3: Research Proposal 34
3.1 Introduction 34
3.2 Hypotheses 36
3.3 Aims 36
Chapter 4: Gene Expression Profiles of Chondrocytes During Passaged
Monolayer Cultivation (JOR, 2007; accepted for publication with revision)
38
Chapter 5: Evidence that Human Cartilage and Chondrocyte do not
Express Calcitonin Receptor (Osteoarthritis and Cartilage, 2007; Peer
review)
59
Chapter 6: Modulation of Human Chondrocyte Functions by Natural
Compounds (Manuscript in preparation)
67
6.1 Introduction 67
6.2 Materials and Methods 72
V
6.2.1 Tissue harvest and cell culture 72
6.2.2 Cell treatments 73
6.2.3 RNA isolation and RT-PCR 73
6.2.4 Real-time PCR and data analyses 74
6.2.5 Determination of proteoglycan content and release 74
6.2.6 Nitric oxide measurement 75
6.2.7 Statistical analyses 76
6.3 Results 76
6.3.1 Parthenolide modulated gene expression profiles of human
chondrocytes
76
6.3.2 Parthenolide prevented proteoglycan degradation induced by
pro-inflammatory cytokines
77
6.3.3 Parthenolide did not reduce nitride oxide production in human
chondrocytes
78
6.3.4 The effect of other natural compounds on chondrocyte gene expression 78
6.4 Discussion 90
6.5 Summary and Conclusion 93
Chapter 7: General Discussion 94
7.1 Discussion 94
7.2 Future directions 97
APPENDICS
Appendix A: Materials and Methods 99
1.1 Materials 99
1.1.1 Chemical reagents 99
1.1.2 Commercially purchased kits 101
VI
1.1.3 PCR Primers 101
1.1.4 Other materials and equipments 101
1.1.5.Nature compounds 103
1.1.6 Antibodies 103
1.1.7 Buffers and solutions 103
1.1.8 Centrifugation 108
1.1.9 Tissue samples and Cells 109
1.1.9.1 Human cartilage and chondrocytes 109
1.1.9.2 RAW264.7 cells 109
1.2 General Methods 109
1.2.1 Primary culture of chondrocyte 109
1.2.1.1 Cartilage samples collection 109
1.2.1.2 Matrix digestion and chondrocyte isolation 110
1.2.1.3 Chondrocyte cultivation and trypsinisation 110
1.2.1.4 Estimation of cell number 111
1.2.2 RNA extraction from cultured chondrocytes 111
1.2.3 Reverse transcription for cDNA preparation 112
1.2.4 Standard PCR 113
1.2.5 Agarose gel electrophoresis 118
1.2.6 Real-time PCR 119
1.2.6.1 Apparatus and reagents 119
1.2.6.2 Standard creation for Real-time PCR 119
1.2.6.3 Melting curve analysis 121
1.2.6.3 Relative quantification 121
1.2.6.4 Data analysis 122
1.2.6.4.1 Data normalization for relative quantitation 122
VII
1.2.6.4.2 Comparative 2^(− ΔΔCT) method 122
1.2.6.4.3 Cluster analysis 122
1.2.7 Immunostaining 123
1.2.7.1 Cartilage tissue preparation 125
1.2.7.2 Gaint cell tumor imprint 125
1.2.7.3 Immunostaining methodology 126
1.2.8 Western-blotting 126
1.2.8.1 Preparation of cell/tissue lysate 126
1.2.8.2 Preparation of SDS-PAGE gel 126
1.2.8.3 Western blot analysis 128
1.2.9 RAW264.7 cell culture and osteoclastgenesis 131
1.2.10 Cyclic AMP ELISA 131
1.2.11 Nitric oxide assay 132
1.2.12 Colorimetric Dimethylmethylene Blue Dye-bingding assay 133
1.2.13 Tartarte resistant acid phosphatase staining 133
Appendix B. Supplementary Figures 135
Appendix C. References 139
Appendix D. Sample of Information Sheet and Consent Form 144
Appendix E. Bone Morphogenesis and Normal Structure (In Chinese) 178
VIII
PUBLICATIONS AND AWARDS
(2004-2007)
Journal Publications:
1. Lin Z, Willers C, Xu J, Zheng MH: The chondrocyte: biology and clinical
application. Tissue Eng 12:1971-84, 2006
2. Lin Z, Pavlos NJ, Cake MA, Wood DJ, Xu J, and Zheng MH. Evidence that
human cartilage and chondrocytes do not express calcitonin receptor.
Osteoarthritis Cartilage 2007; In press.
3. Lin Z, Fitzgerald J, Xu J, Willers C, Wood D, Grodzinsky A, et al. Gene
expression profiles of chondrocyte during passaged monolayer cultivation.
Journal of Orthopaedic Research 2007; In press.
Meeting Abstracts:
1. Lin Z, Fitzgerald J, Xu J, Wood D, Zheng MH: Gene Expression of Passaged
Human Articular Chondrocytes. In: American Society for Bone and Mineral
Research 27th Annual Meeting. Nashville, Tennessee, USA, 2005
2. Lin Z, Fitzgerald J, Xu J, Wood D, Zheng MH: Gene Expression of Passaged
Human Articular Chondrocytes. In: ASMR Medical Research Week Symposium.
Perth, Australia, 2005
3. Lin Z, Fitzgerald J, Xu J, Wood D, Zheng MH: Gene Expression Patterns of
Human Articular Chondrocytes in Monolayer Cultivation. In: Australia and New
Zealand Bone and Mineral Society Annual Meeting. Perth, Australia, 2005
4. Lin Z, Fitzgerald J, Xu J, Wood D, Zheng MH: Gene Expression Patterns of
Human Articular Chondrocytes in Monolayer Cultivation. In: Second Australian
Biotherapeutic and Tissue Regeneration Forum. Margaret River, Western
Australia, 2005
5. Lin Z, Pavlos NJ, Cake MA, Wood DJ, Xu J, and Zheng MH. Evidence that
human cartilage and chondrocytes do not express calcitonin receptor. In: ASMR
Medical Research Week Symposium. Perth, Australia, 2007
IX
X
6. Lin Z, Pavlos NJ, Cake MA, Wood DJ, Xu J, and Zheng MH. Evidence that
human cartilage and chondrocytes do not express calcitonin receptor. In:
Australian & New Zealand Orthopaedic Research Society 13th annual meeting.
Auckland, New Zealand, 2007
Awards:
2005 Student Presentation Award of the Second Australian Biotherapeutic
and Tissue Regeneration Forum
2005 Graduate Research Student Travel Award of UWA
2006 QEII Ad Hoc Scholarship
2007 Australian & New Zealand Orthopaedic Research Society travel grant
LIST OF ABBREVIATIONS
ACI Autologous chondrocyte implantation
ADAMTS A disintegrin and metalloproteinase with thrombospondin
motifs
Ag Antigen
AP-1 Activated protein-1
bFGF Basic fibroblast growth factor
bp Base pair
BMI Body mass index
BMPs Bone morphogenetic proteins
BSA Bovine serum albumin
cAMP Cyclic adenosine monophosphate
CDMP Cartilage-derived morphogenetic protein
cDNA Complementary deoxyribonucleic acid
CAPE Caffeic acid phenethyl ester
CFI Carbon fibre implantation
CO2 Carbon dioxide
COX-2 Cyclooxygenase-2
CRLR Calcitonin-receptor like receptor
Ct Cycle threshold
CTR Calcitonin receptor
°C Degrees Celsius
Da Daltons
DAB Diaminobenzidine
DDW Double deionised water (sterile)
DEPC Diethyl pyrocarbonate
DEPC-treated DDW Diethyl pyrocarbonate treated double deionised water
DMEM Dulbecco’s Modified Eagle’s medium
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
dGTP Deoxyguanosine triphosphate
dNTP Deoxynucleotides triphosphate
dTTP Deoxythymidine triphosphate
ddH20 Double distilled water
ECL Enhanced chemiluminescence
ECM Extracellular matrix
EDTA Ethylenediaminetetracetic acid
ERK Extracellular signal-regulated protein kinase
FBS Fetal bovine serum
FCS Fetal calf serum
FGF Fibroblast growth factor
g Grams
GAG Glycosaminoglycan
GCT Gaint cell tumor
GAPDH Glyceraldehydes-3-phosphate dehydrogenase
HCl Hydrochloride acid
HA Hyaluronan
HMG High-mobility-group
HSP 70 Heat shock protein 70
LPS Lipopolysaccharide
L-NIL n-iminoethyl-L-lysine
IBMX 3-isobutyl-1-methylxanthine
IGF-1 Insulin-like growth factor-1
IκB Inhibitory kappa B
IL-1β Interleukin-1 beta
iNOS Inducible nitric oxide synthase
JNK c-jun N-terminal kinase
μg Microgram
μl Microlitre
k Kilo
M Molar
min Minutes
ml Millimeter
mM Millimolar
MACI Matrix-induced autologous chondrocyte implantation
MAPk Mitogen-active protein kinase
MIT Massachusetts Institute of Technology
MMP Matrix metalloproteinase
MRI Magnetic resonance imaging
MSC Mesenchymal stem cell
n Nano
NaCl Sodium Chloride
NaOH Sodium Hydroxide
NF-κB Nuclear factor kappa B
NO Nitric oxide
NSAIDs Nonsteroidal anti-inflammatory drugs
OA Osteoarthritis
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate Buffered Saline
PDGF Platelet derived growth factor
PGA Polyglycolic acid
PGE2 Prostaglandin E2
PGES Prostaglandin E synthase
PHB Polyhydroxybutyrate
PHBHHx poly(hydroxybutyrate-co-hydroxyhexanoate)
PLA Poly-L-lactic acid
PTH Parathyroid hormone
RA Rheumatoid arthritis
RANKL Receptor activator of NK-κB ligand
RNA Ribonucleic acid
ROS Reactive oxygen species
RT-PCR Reverse Transcription Polymerase chain reaction
sec Seconds
S.D. Standard deviation
SDS Sodium dodecyl sulphate
S.E. Standard error
SSC Standard sodium citrate
TAE Tris-acetate EDTA
TEMED N’N’N’N’-tetramethylene diamine
TBS Tris buffered salime
TBST Tris buffered saline-Tween
TE Tris-EDTA
TGF Transforming growth factor
TIMP Tissue inhibitors of metalloproteinase
TNF-α Tumor necrosis factor-α
TRAP Tartrate resistant acid phosphatase
TRFs Terminal restriction fragment
UWA University of Western Australia
U.S. United State
VEGF Vascular endothelial growth factor
WA Western Australia
LIST OF FIGURES
CHAPTER 2 page
Figure 2.1 Specimen radiagraph of Osteophyte 24 CHAPTER 4 Figure 1 Comparison of gene expression between OA cartilage and
monolayer cultured chondrocytes
53
Figure 2 Comparison of gene expression between cultured OA chondrocytes
and normal chondrocytes
54
Figure 3 Gene expression profiles of matrix proteins for monolayer passaged
human chondrocytes
55
Figure 4 Gene expression profiles of matrix protease. Metalloproteinase
inhibitor and transcription factors for monolayer passaged human
chondrocytes
56
Figure 5 Gene expression profiles of cytokines, growth factors and
intracellular signaling markers for monolayer passaged human
chondrocytes
57
Figure 6 Projection plot of the genes represented by the three main principal
components
58
CHAPTER 5 Figure 1 The mRNA expression of CTR in human cartilage and chondrocytes 61
Figure 2 Western blot analysis of CTR in human giant cell tumor and primary
chondrocytes
62
Figure 3 Immunodetection of CTR in human GCT and articular cartilage 63
Figure 4 The mRNA expression of CRLR in human GCT, human cartilage
and chondrocytes
64
Figure 5 cAMP response of chondrocytes to sCT 64
XVI
Figure 6 The expression profiles of chondrocytic genes (Aggrecan, Type II
collage, MMP-1, 3, 13) in salmon CT treated human chondrocytes
(passage 1) by Real-time PCR
65
CHAPTER 6 Figure 6.1 The effect of parthenolide on the gene expression of COX-2 and
IL-1β in human chondrocytes
79
Figure 6.2 The effect of parthenolide on the expression matrix protein genes
in human chondrocytes detected by Real-time PCR
80
Figure 6.3 The effect of parthenolide on the expression of cytokine genes in
human chondrocytes detected by Real-time PCR
82
Figure 6.4 The GAG content released in culture medium and cell lysate when
chondrocytes were stimulated with LPS after 2hrs parthenolide
treatment
84
Figure 6.5 The GAG content released in culture medium and cell lysate when
chondrocyte were stimulated with IL-1 after 2hrs parthenolide
treatment
85
Figure 6.6 The GAG content released in culture medium and cell lysate when
chondrocyte were stimulated with TNF-α after 2hrs parthenolide
treatment
86
Figure 6.7 Effect of parthenolide on Nitric Oxide released in human
chondrocytes
87
Figure 6.8 The effect of CAPE and Astilbin on the gene expression of IL-1β
and COX in human chondrocytes
88
Figure 6.9 The effect of Naringin and Manigerin on the gene expression of
COX-2 and IL-1β in human chondrocytes
89
APPENDIX B Figure B.1 Standard curves for Real-time PCR (aggrecan, type I collagen, 135
XVII
type II collagen, type X collagen, fibromodulin, fibronectin,
MMP-1, and MMP-3)
Figure B.2 Standard curves for Real-time PCR (MMP-9, MMP-13,
ADAMTS-4, ADAMTS-5, TIMP-1, TIMP-2, TIMP-3, and Sox-9)
136
Figure B.3 Standard curves for Real-time PCR (IL-1β, TGF-β, TNF-α, IGF-1,
HSP-70, Ribosomal, c-fos, and c-jun)
137
Figure B.4 Standard curves for Real-time PCR (COX-2, NOS2, MAPk-1,
Link protein, and GAPDH)
138
Figure B.5 Type I collagen melting curve from 10 samples 139
Figure B.6 Melting curve containing slight primer dimmer interference 140 APPENDIX E Figure 11 Osteoblast differentiation and its intracellular signaling pathway 200
Figure 15 Osteoclast differentiation 201
Figure 16 Intracellular signaling pathway in osteoclast 202
XVIII
LIST OF TABLES
CHAPTER 5 Table 1 The primer sequences used for Real-time PCR 61 APPENDIX A Table A.1 Oligonucleotide primers used in PCR amplification 115
Table A.2 Automated processing run (24hour) for cartilage biopsies 124
XIX
THESIS SUMMARY
Osteoarthritis (OA) is the most common form of arthritis, characterized by
progressively degeneration of articular cartilage. Chondrocyte is the only cell type in
articular cartilage tissue and responsible for cartilage matrix turnover. This thesis
focuses on the biological and genetic behaviors of human chondrocyte and potential
therapeutic strategies that target on chondrocyte.
Chondrocytes have been used for the tissue-engineered cartilage construction, especially
in articular cartilage repair. The technique of chondrocyte-base tissue engineering
utilizes in vitro propagated chondrocytes combined with several manufactured
biomaterials to regenerate cartilage tissue. Although these technologies have been
successfully applied in clinic, the biological characteristics of chondrocyte during in
vitro propagation and after implantation remain unclear. This thesis reviewed the present
studies of chondrocyte biology and its potential uses in tissue engineering. Particularly,
chondrocytes have been shown to de-differentiate into fibroblastic-cells when they are
exposed to inflammatory conditions or cultured on monolayer in vitro. This thesis
investigated the gene expression profile of chondrocytes when they are cultured and
serially passaged on monolayer in vitro. Human chondrocytes obtained from OA
patients were cultured up to passage 6. Twenty-eight chondrocyte associated genes were
measured by Real-time PCR. The results showed that a number of genes were changed
in expression levels at various stages of passage as indications of chondrocyte
de-differentiation. Chondrocytes derived from OA patients or normal donors exhibited a
very similar gene expression pattern. Interestingly, transcription factor Sox-9, which
plays a key role in chondrogenesis remained unchanged with increasing passage number,
indicating that the de-differentiation process of chondrocyte is reversible.
This thesis also focused on the development of novel pharmacological approaches for
OA that target on articular chondrocyte. The clinical feature, etiology, pathogenesis,
diagnostic approaches, conventional and potential future treatments for OA were briefly
XX
reviewed in this thesis. Calcitonin has been recently shown to have a direct protective
effect on articular cartilage against joint degenerative disease. It has been proposed that
calcitonin might act through the calcitonin receptor to activate the cAMP pathway and
protect type II collagen from degradation. This thesis attempted to reveal the cellular
mechanism of calcitonin on human articular chondrocytes. Five human articular
cartilage samples were examined for the expression of calcitonin receptor by multiple
experimental techniques, including polymerase chain reaction, immunostaining and
Western blotting. Cyclic AMP levels in human chondrocytes stimulated with salmon
calcitonin were measured by ELISA. The effect of salmon calcitonin on the gene
expression profiles, including aggrecan, type II collagen, MMP-1, MMP-3 and
MMP-19, of human chondrocytes was also examined by relative quantitative real-time
PCR. The results showed that calcitonin receptor was not detectable in human cartilage
tissue or isolated chondrocytes. The cyclic AMP level in human chondrocytes in vitro
was not induced by salmon calcitonin (10^-7M, 10^-8M, 10^-9M). Real-time PCR
demonstrated that salmon calcitonin tended to reduce the gene expression of MMPs,
though the effect was not statistically significant. The results suggest that there was no
direct effect of salmon calcitonin on human chondrocytes and the chondral protective
effect of calcitonin observed in vivo may be indirect via its effect on subchondral bone
resorptive activity.
Furthermore, this thesis investigated the regulation of chondrocyte functions by natural
compounds. Substances that derived from natural medical plants, including Parthenolide,
Caffeic acid phenethyl ester, Astilbin, Mangiferin or Naringin, were selected to test their
therapeutic value for arthritis. Cells were pretreated with these natural compounds and
stimulated with inflammatory factors. The effects of natural compounds on chondrocyte
gene expression, proteoglycan degradation and nitric oxide production were measured.
The results showed that parthenolide, a NF-κB inhibitor, regulated chondrocyte function
by suppressing the up-regulation of gene expression of inflammatory factors and matrix
proteinases induced by lipopolysaccharide, and down-regulating COX-2 expression.
Parthenolide was able to reduce proteoglycan degradation in human chondrocytes, but
XXI
XXII
had no effect on nitric oxide production. These results suggest that parthenolide
mediates inflammatory-activated NF-κB pathway, and subsequently reduces
inflammatory response, prevents cartilage destruction and relieves pain, and hence may
be useful for OA treatment.
CHAPTER 1
THE CHONDROCYTE:
BIOLOGY AND CLINICAL APPLICATION
Tissue Eng. , Vol.12, 1-14
The Chondrocyte: Biology and Clinical Application
ZHEN LIN, CRAIG WILLERS, JIAKE XU, and MING-HAO ZHENG
ABSTRACT
Chondrocyte is a unique cell type in articular cartilage tissue and is essential for cartilage formation andfunctionality. It arises from mesenchymal stem cells (MSCs) and is regulated by a series of cytokine andtranscription factor interactions, including the transforming growth factor-b super family, fibroblastgrowth factors, and insulin-like growth factor-1. To understand the biomechanisms of the chondrocytedifferentiation process, various cellular model systems have been employed, such as primary chondrocyteculture, clonal normal cell lines (HCS-2/8, Ch-1, ATDC5, CFK-2, and RCJ3.1C5.18), and transformedclonal cell lines (T/C-28a2, T/C-28a4, C-28/I2, tsT/AC62, and HPV-16 E6/E7). Additionally, cell culturemethods, including conventional monolayer culture, three-dimensional scaffold culture, bioreactor cul-ture, pellet culture, and organ culture, have been established to create stable environments for theexpansion, phenotypic maintenance, and subsequent biological study of chondrocytes for clinical appli-cation. Knowledge gained through these study systems has allowed for the use of chondrocytes inorthopedics for the treatment of cartilage injury and epiphyseal growth plate defects using tissue-engineering approaches. Furthermore, the potential of chondrocyte implantation for facial reconstruc-tion, the treatment of long segmental tracheal defects, and urinary incontinence and vesicoureteral refluxare being investigated. This review summarizes the present study of chondrocyte biology and the po-tential uses of this cell in orthopedics and other disciplines.
INTRODUCTION
ALTHOUGH NATIVE CHONDROCYTES OFFER little assistance
to injured articular cartilage, these cells are responsi-
ble for the synthesis and turnover of the cartilage extra-
cellular matrix (ECM), which provides an environment of
nutrition diffusion for chondrocytes and provides the joint
surface with biomechanical competence. Chondrogenic
cells arise from pluripotential mesenchymal stem cells
(MSCs) through a series of differentiation pathways, which
have received extensive study. Subsequently, it has been
shown that a number of cytokines and transcription factors
are involved in chondrocyte maturation and cartilage for-
mation; however, the specific mechanisms regulating these
processes remain unclear.1–6 Hence, the isolation and
growth of chondrocytes outside their matrix housing has
been researched for decades in hope of better understanding
this resilient cell and exploiting its function for therapeutic
gain. Cell culture is a basic experimental approach used in
cellular and molecular biological studies of chondrocytes. Of
these, the monolayer culture of chondrocytes is employed
extensively but fails to maintain the chondrogenic phenotype
during long-term culture.7 Other chondrocyte culture tech-
niques, such as three-dimensional culture, pellet culture, and
bioreactor culture, have been created to obtain large amounts
of cultured chondrocytes with a well-maintained phenotype.
The manipulation of the culture environment for chon-
drocytes presents the most feasible mechanism for opti-
mizing cell behavior and phenotype. Furthermore, various
studies have illustrated the benefits of growth factor inte-
gration toward establishing and maintaining the phenotype
during in vitro cultivation.8,9 In particular, it has been shown
that members of the transforming growth factor (TGF)
family play an important role in chondrocyte development.
Department of Orthopaedic Surgery, Faculty of Medicine and Dentistry, University of Western Australia, Western Australia 6009,
Australia.
TISSUE ENGINEERINGVolume 12, Number 7, 2006# Mary Ann Liebert, Inc.
1
Chondrocyte cultures have been used in numerous disease
and physiology model systems for examining the cell’s
clinical application potential. In recent years, tissue-
engineered cartilage construction has been applied in the
clinical domain, especially in the treatment of chondral and
osteochondral injury (autologous chondrocyte implantation).
Additionally, numerous other attempts have been made to
widen the application of chondrocyte-based reconstruction
to fields outside orthopedics. To this end, this article summa-
rizes the current and potential use of chondrocytes in clinical
practice.
CHONDROCYTE MORPHOLOGYAND FUNCTION
Mature human chondrocytes are predominantly round
cells located in matrix cavities called lacunae. With a mean
diameter of 13 mm, chondrocytes represent only 5% to 10%
of the total cartilage volume but are crucial to the mainte-
nance of a stable ECM.10 In addition, in the epiphy-
seal growth plates, chondrocytes are responsible for the
growth of long bones through endochondral ossification.
Observed under high magnification, the nucleus of the
chondrocyte is usually round or oval and contains one to
several nucleoli, varying between species. Electron micro-
graphs of articular cartilage chondrocytes reveal a juxta-
nuclear cell center with a pair of centrioles and well-
developed Golgi apparatus.11 The surrounding cytoplasm
contains elongated mitochondria, occasional lipid droplets,
and variable amounts of glycogen. When new matrix is
being formed in growing or regenerating cartilage, the cy-
toplasm becomes more basophilic, and the Golgi region
becomes unusually large.11
Chondrocytes anabolize and catabolize ECM macro-
molecules while the matrix in turn maintains a state of
dynamic equilibrium between the cellular environment and
the structure of the cartilage. The 2 main ECM macro-
molecules are type II collagen and large chondroitin sulfate
proteoglycans aggregates, termed aggrecan. Type II colla-
gen primarily endows the cartilage with its tensile strength,
whereas aggrecan provides the osmotic resistance for car-
tilage to resist compressive loads.12 Type II collagen fibrils
associated with other collagens form an endoskeleton.13
Aggrecan monomers consist of a core protein and keratin
sulfate glycosaminoglycan (GAG) chains, filling the inter-
stices of the collagen meshwork by forming large aggre-
gated complexes interacting with hyaluronic acid (HA) and
link proteins.14 Aggrecan, by virtue of its high negative
charge, swells against the collagen network as it draws
water into the tissue to resist compression. Quantitatively,
minor components, such as type IX, XI, and VI collagen;
biglycan; decorin; and cartilage oligomeric matrix protein,
also have important roles in controlling matrix structure
and organization.15 HA is a GAG with repeated disaccha-
ride units of D-glucuronic acid and N-acetyl-D-glucosamine
connected by b-linkages,16 playing essential roles in the
structural organization and function of cartilage, cell adhe-
sion, migration, and differentiation mediated by HA-binding
proteins and cell surface receptors such as CD44.17,18
Overall, the interaction among type II collagen, aggrecan,
link protein, and HA tripartite provides the cartilage with its
ability to continually resist compressive loads.
The composition and structural integrity of ECM in ar-
ticular cartilage is responsible for the ability of joints to
withstand joint biomechanics. The maintenance of ECM
depends on the chondrocyte to mediate synthesis, assembly,
and degradation of matrix proteins. It is also important to
understand the biological and biophysical transduction
mechanisms of the chondrocyte response to mechanical
stimuli. Normal joint loading promotes aggrecan-increased
synthesis and maintains intact functional ECM. Once load-
ing becomes excessive, cartilage ECM degrades, with a loss
of pericellular matrix volume and water content.19 It has
been shown in in vitro cartilage explant models that static
compression significantly inhibits matrix protein bio-
synthesis, whereas dynamic compression promotes matrix
production.20,21 Additionally, immobilization or reduced
loading can cause ECM degradation. Morphologically,
compressive loading flattens chondrocytes in the direction of
loading, and cell volume decreases.22 Alteration also occurs
in cell surface area, nucleus volume and height, and nucleus
surface area.22,23 However, it is still unclear why the trans-
lation of cell deformation leads to ECM degradation.
CHONDROCYTE ONTOGENY
Chondrogenic cells arise from pluripotential MSCs. In
addition to chondrogenic potential, MSCs have the ability
to differentiate into other cell lineages, including osteo-
blasts,fibroblasts, adipocytes, endotheliocytes, andneuronal-
like cells.2,24–29 Chondrogenesis progresses from a morpho-
genetic phase to a cytodifferentiation phase of development
via a number of progenitor and precursor stages to the ma-
ture chondrocyte. Primary chondrocytes arise in the embryo
from mesoderm or from the neural crest, where the facial
skeleton is generated.1 In the head and clavicular skeleton
of higher vertebrates, secondary chondrocytes arise from
the periosteal layer surrounding membranous bone upon
mechanical stimulation.1 Within the morphogenetic phase,
mesenchymal cell populations are recruited to the sites of
chondrogenesis. Mesenchymal–epithelial and cell–cell
matrix interactions lead to cell condensations and differ-
entiation of chondroblasts and osteoblasts (participate in
bone-osteogenesis). In the proceeding cytodifferentiation
phase of development, proliferating chondroblasts differen-
tiate into type II collagen positive chondrocytes synthesiz-
ing highly sulfated cartilage proteoglycans. Chondrocytes
may further experience a dedifferentiation development
with the expression of the hypotrophic chondrocyte unique
marker: type X collagen.1 Cartilage matrix calcification
2 LIN ET AL.
progressively follows the apoptosis of hypotrophic chon-
drocytes, followed by vascular invasion.
Chondrogenesis during embryo development starts with
the condensation of MSCs. The expression of a number of
cartilage-specific molecules signals the process. The tran-
scription factor Sox-9 is one of those signals. Sox-9 belongs
to the Sox family of transcription factors that are char-
acterized by a high-mobility-group (HMG)-box deoxy-
ribonucleic acid (DNA)–binding domain.30 Following
the expression of Sox-9, Col2A, which encodes the
chondrocyte-specific protein type II collagen, becomes in-
volved in the chondrogenesis pathway and regulates the
threshold of the next phase. Essentially, Sox-9 binds and
activates to a consensus sequence in the Col2A enhancer
region, then upregulates Col2A expression. It has been
shown that wild-type mice chimeras carrying Sox-9 null
cells prevent inclusion of the mutant cells into cartilaginous
structures.30 Specifically, haplosufficiency of Sox-9 in hu-
mans leads to the skeletal dysmorphology syndrome cam-
pomelic dysplasia.31 Hence, Sox-9 is considered to be a
crucial factor in chondrocyte differentiation and cartilage
formation and is required to maintain the chondrocyte
phenotype. L-Sox-5 and Sox-6, 2 other members of the Sox
family, may also be involved in chondrocyte differentiation
by complementing the function of Sox-9.1,32 Sox genes are
expressed throughout the chondrogenesis pathway but are
completely turned off in hypertrophic chondrocytes.
It has been shown that there are a number of essential
growth factors involved in chondrocyte maturation and
cartilage formation.32–35 These include the TGF-b family,
bone morphogenetic proteins, cartilage-derived morpho-
genetic proteins, fibroblast growth factors (FGFs), and
insulin-like growth factor (IGF)-1.
TGF-b isoforms
Members of the TGF-b superfamily, TGF-b1, -b2, and -b3are multifunctional peptides that are detected in most cell
types of the body. The expression of TGF-b1 is restricted inthe proliferative and upper hypertrophic zones of cartilage.36
In the cartilage of human long bone, TGF-b2 is the most
extensively detected isoform of TGF-b.36 It has been de-
tected in all zones of cartilage, with greatest expression in
hypertrophic and mineralizing-zone chondrocytes. TGF-b3exhibits the highest level of expression in the hypertrophic
and mineralizing zone chondrocyte. The expression pattern
of the TGF-b isoforms in human cartilage is consistent with
a chick study model that indicated that TGF-b isoforms play
a role in chondrocyte late-stage differentiation and may
participate in bone formation.37 TGF-b1 also induces chon-
drogenesis differentiation in mesenchymal cell lines. The in-
creased expression level of type II collagen after stimulation
byTGF-b1was observed in pluripotentmesenchymal cell line
C3H10T1/2.38 Han et al. reported that TGF-b1 induced the
early stage of chondrogenesis in a pre-chondrogenic cell line,
as indicated by increased production of aggrecan and type II
collagen, although it was unlikely to stimulate further differ-
entiation and possibly inhibit the terminal differentiation
of chondrocytes.39 Additionally, Johnstone et al. reported
100% chondrogenic differentiation in MSCs treated with
TGF-b1, compared with 25% in marrow cell controls.40
TGF-b3 is also thought to play a role in chondrogenic matu-
ration, with human MSCs having been differentiated into
chondrocyte in micromass pellet culture with the supplement
of TGF-b3.41
Bone morphogenetic proteins
Bone morphogenetic proteins (BMPs) are a group of
multi-functional growth factors that belong to the TGF-bsuperfamily.42 They promote several aspects of chondro-
genesis from commitment to terminal differentiation.6 In
the condensation step of the earlier stages of chondrogen-
esis, BMPs promote cell–cell interaction by upregulating
N-cadherin (N-cad) function and expression.43 Also, many
studies have demonstrated the requirement of BMPs for
Sox gene expression in chondrogenesis.44–46 It has been
verified that BMPs upregulate the expression of Sox-9 and
type II collagen in multipotential mesenchymal C3H10T1/2
cells and monopotential chondroprogenitor MC615 cells.47
It has also been shown that BMPs promote Sox gene ex-
pression in vivo with an upregulation of Sox protein in
cartilage after implantation of BMP2.45 Furthermore, it has
been shown that precartilaginous cells in condensation fail
to differentiate into chondrocytes when the BMP signaling
pathway is blocked.48 Additionally, the introduction of BMP
protein increases the expression of the specific hypertrophic
chondrocyte marker type X collagen by inducing type X
collagen promoter activity.49–51
Cartilage-derived morphogenetic proteins
Another subfamily of TGF-b superfamily thought to be
involved in chondrogenesis is the cartilage-derived morpho-
genetic proteins (CDMPs). Three members have been iden-
tified in this subfamily (CDMP-1, -2, and -3), also known as
growth/differentiation factor-5, -6, and -7, respectively.52,53
CDMP-1 gene expresses at the stage of precartilaginous
mesenchymal condensation and throughout the cartilaginous
cores of the developing long bone. It has been shown that
high-dose or low-dose treatment of CDMP-1 combined with
TGF-b1 promotes MSC differentiation into chondrocytes.54
Moreover, CDMP-1 has been found to stimulate aggrecan
and GAG production in vivo and in vitro.55,56 It has been
indicated that CDMP-1 contributes to the early stage of
chondrogenesis and chondrocyte differentiation. Similarly,
CDMP-2 has been detected in the hypertrophic chondrocytes
of ossifying long bone centers, suggesting that it is involved
in the terminal differentiation of chondrocytes and the early
stage of endochondral bone ossification.52 It has also been
shown to stimulate proteoglycan synthesis in a chondrocytic
cell line.57 Additionally, mutations of CDMP genes in mice
lead to a distinct shortening of the limbs due to a lack of
THE CHONDROCYTE 3
chondrogenic signaling by progenitor cells at the stage of
chondrogenesis.58
Fibroblast growth factors
Many studies have shown that FGFs play a key role in
chondrocyte proliferation.59–61 Basic FGF (bFGF) is a known
mitogen that stimulates ribonucleic acid (RNA) and DNA
synthesis and cell division in chondrocytes.60 Also, it has
been shown that bFGF promotes the confluence of rabbit
costal chondrocyte cultures and the synthesis of cartilaginous
ECM in vitro.59 The effects of bFGF on the terminal dif-
ferentiation of chondrocytes and calcification of cartilaginous
matrix have also been investigated. When a low concentra-
tion of bFGF is applied to hypertrophic chondrocytes, which
secrete high amounts of alkaline phosphatase, the activity of
alkaline phosphatase is decreased and calcium deposition is
prevented.62 This indicates that bFGF may be an inhibitor of
terminal chondrocyte differentiation and ossification. It has
been illustrated through different animal cell model systems
that the various FGF subtypes induce differential effects on
chondrocyte proliferation. In an avian chondrocyte culture
system, FGF-2, -4, and -9 strongly stimulated chondrocyte
proliferation, whereas FGF-6 and -8 stimulated proliferation
to a less degree.63 Similarly, in a human growth plate
chondrocyte culture system, FGF-1, -2, and -18 promoted
proliferation, whereas FGF-4 and -9 stimulated proliferation
less, and FGF-10 treatment suppressed chondrocyte prolif-
eration.64 FGF-2 promotes human chondrocyte proliferation
in serum-containing culture medium and serum-free medium
and impedes the dedifferentiation process of chondrocyte in
monolayer culture.8,65
Insulin-like growth factor-1
IGFs are expressed in tissues such as placenta, heart, lung,
testes, brain, and bone. IGF-1 belongs to the IGF family of
peptide hormones including relaxin and insulin and has a
single polypeptide homologous to proinsulin.66 It regulates
many cellular functions by activating cell-surface recep-
tors.67 The expression of IGF-1 is positive in developing
cartilage, mature cartilage, and synovial fluid of the joints.
Studies based on mesenchymal cell lines, embryonic limbs,
and chondrocyte cell lines have shown that IGF-1 induces
chondrocyte differentiation and proliferation.67–69 In mature
cartilage, IGF-1 is believed to participate in ECM anabolism
by enhancing proteoglycan and type II collagen synthesis in
vivo and in vitro.66 It has also been found that IGF-1 inhibits
nitric oxide-induced dedifferentiation and apoptosis of ar-
ticular chondrocytes by modulating multiple pathways.67
CELLULAR MODEL SYSTEMSFOR CHONDROCYTE STUDY
A variety of cultured cell systems have been successfully
used to study the pathways of chondrocyte differentiation,
with each model offering unique advantages toward in-
creasing our understanding of cartilage formation, pro-
liferation, and differentiation; gene expression; pathways
of normal development or pathological degeneration;
ECM turnover; and pharmacotoxicology studies. Com-
monly employed model systems include primary culture of
chondrocyte, established clonal cell lines, non-transformed
immortalized cell lines, experimentally immortalized cell
lines, organ culture of cartilage slices, and growth plate
culture.
Primary cultures of chondrocytes
A primary culture is defined as a culture started directly
from donor cartilage tissue. Primary cultured cells are able
to proliferate in vitro with a limited number of cell divi-
sions. Chondrocytes, usually isolated from the cartilage of
patients undergoing joint replacement surgery for arthritis
or arthroscopic procedure, have been used for the study
of chondrocyte differentiation, articular disease, and gene
expression profiling of human chondrocytes. Human ar-
ticular chondrocyte can be successfully grown from donors
over a wide age range.70 The time to primary confluence is,
however, directly correlated to donor age.70 The primary
chondrocyte is an excellent study model for normal cell
physiology and mechanism of degenerative joint disease,
although it shows low proliferation activity and may dedif-
ferentiate with extended cultivation when it starts to prolif-
erate. Additionally, it is hard to obtain sufficient human
articular chondrocytes because of ethical considerations.
Normal clonal cell lines
Clonal cell lines are non-transformed cells with variable
life spans that descend from a single common ancestor. The
cells from a clonal cell line population remain genetically
identical through serial passage in vitro. They are widely
used to produce a sufficient number of cells for research
purposes. Non-transformed immortalized clonal cell lines
are normally initiated from tumor tissues. HCS-2/8 is a
clonal cell line derived from an aggressive chondrosarcoma
of the proximal humerus of a 72-year-old man.71 The cell
line has been extensively characterized, with synthesis of
proteoglycans and type II, IX, and XI collagen consistent
with chondrocyte phenotype and marked response from a
variety of cytokines, such as tumor necrosis factor (TNF)-a,FGF, IGF, TGF-b, and connective tissue growth fac-
tor.72 The HCS-2/8 cell line was able to maintain the
chondrocyte phenotype for more than 3 years in culture. The
HCS-2/8 cell line provides a study model for understanding
chondrocyte differentiation and proliferation, responses of
cytokine stimulation, and gene expression profile of hu-
man chondrocytes. However, the chondrosarcoma origin of
these cells may affect their biological characteristics.
Chansky et al. have established another human chon-
drosarcoma cell line (Ch-1) for the investigation of the
etiology of human chondrosarcomas and for the synthesis
4 LIN ET AL.
and regulation of cartilage-specific genes.3 The messenger
RNA (mRNA) encoding type XI, XI collagen and proteo-
glycans decorin and aggrecan were presented, whereas
type I, II collagen were not detected. Numerous mRNAs
of TGF-b1 0was detected in the Ch-1 cell line.3 P53, a
tumor-suppressor gene, was also found to be overexpressed
in Ch-1.3 Also, 2 proteins associated specifically with
chondrogenesis were observed: Cart-1, known as a homeo-
box protein involved in cartilage differentiation, and CD-
RAP, a secreted molecule restricted under normal conditions
to differentiated chondrocytes and cartilage.3 The cell line
may be used to investigate the etiology of human chon-
drosarcomas and the gene expression profile of cartilage,
although genomic instability and multiple random muta-
tions of the Ch-1 cell line limit the use of Ch-1 cell line in
chondrocyte study.
ATDC5, isolated from a differentiating culture of AT805
embryonal carcinoma, is an example of a prechondrogenic
stem cell line. The ATDC5 cell line reproduces the dif-
ferentiation stages of chondrocytes observed in vivo during
endochondral bone formation.73 In monolayer culture with
the presence of insulin, ATDC5 cells retain the properties
of chondroprogenitor cells that are at the early phase of
differentiation and forms cartilage nodules when cells reach
condensation. The cartilage nodules enlarge while chon-
drocytes proliferate, lasting about 2 weeks in culture.74
When chondrocytes cease to grow after 3 weeks in culture,
cells at the center of the cartilage nodules become hyper-
trophic in association with type X collagen gene expression
and a dramatic increase in alkaline phosphate activity,
which is characteristic of the late phase differentiation of
endochondral bone formation.74 After a further 2 weeks,
mineralization could be observed without b-glyceropho-sphate supplement.74 Hence, ATDC5 offers a mineralizing
in vitro model to study the molecular mechanism of carti-
lage mineralization during endochondral bone formation.
The ATDC5 cell line is the first example to display the
entire spectrum of chondrocyte differentiation.
CFK2 is a non-transformed clonal chondrocytic cell line
derived from fetal rat calvariae. CFK2 cells deposit a car-
tilaginous matrix and form focal cellular nodes after long-
term culture in monolayer. It has been found that peptide
regulatory factors such as EGF and parathyroid hormone
(PTH) stimulate CFK2 proliferation, whereas steroidal
agents dexamethasone and retinoic acid are inhibitory.75
Meanwhile, PTH and dexamethasone enhance the forma-
tion of focal nodes alone with increased matrix deposition
and link protein expression , whereas EGF and retinoic acid
inhibit them.75 This cell line serves as a reproducible source
of chondrocytic precursor cells and an in vitro model for
studying cartilage matrix deposition and the regulation of
chondrocyte differentiation by various factors.
The clonal cell line RCJ3.1C5.18 (RCJ3) was origi-
nally isolated from rat calvaria undergoing chondrogenic
differentiation after long-term monolayer culture.76 A series
of factors were found to effect its differentiation and
proliferation. Low-contact matrix and dexamethasone
induced RCJ3 cell differentiation, whereas 1,25 (OH)2-
vitamin D3 and retinoic acid inhibited it. Hormone respon-
siveness increases with differentiation for PTH whereas it
decreases with prostaglandins PGE2 and PGE2. Also,
PGF2a enhances proliferation, whereas PGE1 inhibits it.77
Moreover, RCJ3 mimics the growth plate chondrocyte
in vitro, as evidenced by increased type X collagen
expression and mineralization.76 Hence, the cell line can
serve as a study model for growth plate chondrocyte
differentiation.
Immortalized (transformed) clonal cell lines
Transformed cell lines are generally defined as a genet-
ically modified cell line by the introduction of DNA to
cause cell immortalization. T/C-28a2, T/C-28a4, and C-28/
I2 are human chondrocyte cell lines established by retro-
viral-mediated transfection of primary rib chondrocytes
with the large T antigen of Simian virus 49.78 Because
these cell lines represent typical chondrocyte morphol-
ogy for more than 80 passages in monolayer culture with
serum-contained medium, they are commonly applied in
cartilage research.79 Of these 3 cell lines, C-28/I2 is re-
ported to be closest to primary chondrocytes with respect
to anabolic and catabolic gene expression.78 These cell lines
can maintain continuous proliferation in monolayer culture
without morphological loss, although phenotypic chon-
drocyte expression pattern loss has been observed in these
3 cell lines.
A temperature-sensitive immortalized human chon-
drocyte cell line, tsT/AC62, was also developed to examine
the regulation of cartilage-specific matrix gene expression
and study the regulation of chondrocyte functions relevant
to arthritic diseases in adult humans.80,81 Primary adult
articular chondrocytes were transfected by a retrovirus
expressing a temperature-sensitive mutant of SV40-large
T antigen (Tag). The tsT/AC62 cells express Tag at per-
missive temperature (328C) and lose Tag expression at
nonpermissive temperature (37–398C) with decreased cell
proliferation.80
Recently, to overcome potential problems associated
with using osteoarthritic or trauma-damaged cartilage
sources, Grigolo et al. established and characterized a cell
line of HPV-16 E6/E7 immortalized chondrocytes by
transfection of a primary culture of normal human femoral
condyle chondrocytes with a plasmid containing 2 human
papilloma virus type 16 (HPV16) early-function genes (E6
and E7) using the highly efficient cationic liposome-
mediated (lipofection) procedure.82 The immortalized cell
line showed expression of type II, IX, and X collagen in
serum-free medium culture and three-dimensional cell cul-
ture scaffold for up to 6 passages, although it failed to ex-
press cartilage-specific type II collagen in monolayer culture,
a finding consistent with cell lines derived from cartilage
with articular diseases. This cell line displays infinite
THE CHONDROCYTE 5
proliferation capacity and stable phenotype. Additionally, the
use of normal human articular cartilage is preferential to
osteoarthritis or trauma-damaged cartilage, which the cell’s
phenotype alters.82
CHONDROCYTE CELLCULTURE METHODS
In addition to various chondrocytic cell lines providing
model systems for chondrocyte biological studies, a num-
ber of cell culture methods have been created to investi-
gate chondrocytes in various environments. Cell culture of
chondrocytes is a basic experimental approach that has
been developed since 1965 to investigate the cellular and
molecular biology of chondrocytes.83 Many different types
of cell culture models have been developed for the inves-
tigation of chondrocyte biology in vitro and in vivo.
Monolayer cultures, three-dimensional culture systems,
implant models for tissue engineering, and organ culture of
cartilage slices have all been studied. The adoption of a
suitable culture system for chondrocyte study depends on
the scientific question to be answered. More recently, the
cultivation of isolated chondrocytes in suitable tissue scaf-
folds that can be implanted into cartilage defects in patients
has led to the development of tissue engineering.
Monolayer (2D) culture
The monolayer culture of chondrocytes essentially
shares the routine culture environment and maintenance
methodology of most cell culture systems. After harvested
cartilage is enzymatically digested (collagenase II), chon-
drocytes are isolated and filtered to remove any undigested
cartilage particles, then washed with buffer. Cells are then
resuspended in culture medium; supplemented with fetal
calf/bovine or human serum, L-ascorbic acid, gentamicin or
penicillin/streptomycin; and seeded into tissue culture
flasks.84 The choice of antibiotic and serum depends mainly
on the source of chondrocytes. Incubation conditions are
usually maintained at 378C in a humidified atmosphere
with 95% oxygen and 5% carbon dioxide. Culture medium
is changed every 2–3 days. After chondrocyte confluence is
achieved, cells are removed via trypsin treatment to larger
flasks. (An equivalent volume of serum media stops the
trypsin reaction.) The cell suspension is then centrifuged to
isolate the passaged cells before they are resuspended in
culture medium and seeded into larger culture flasks for
further expansion.
Monolayer culture is a basic approach for chondrocyte
propagation. It is economical and technically simple and
allows the culture of large numbers of cells. However,
chondrocytes have been shown to lose their phenotype in
monolayer culture after being passaged.7 The cells become
fibroblast-like, losing their ability to secrete proteoglycans
and changing collagen synthesis from type II to type I. An
elegant experiment by Benya and Shaffer showed that
collagen II expression is lost during monolayer chon-
drocyte culture but that transition to three-dimensional gel
culture can recover the differentiated phenotype.7 Addi-
tionally, serial monolayer culture of chondrocytes is not
precisely representative of native cartilage because it lacks
the cell–matrix interactions that chondrocytes possess
in vivo. As a result of this, other chondrocyte culture sys-
tems have been created to investigate a solution to these
problems.
Serum-free medium culture
Serum-free medium is another culture condition de-
scribed to redifferentiate chondrocytes, because the pres-
ence of serum is thought to impede chondrocyte expansion
and differentiation at higher concentrations. Chondrocytes
cultured in serum-free medium supplemented with an in-
sulin-containing serum substitute show more-active ex-
pression of type II collagen and aggrecan than chondrocytes
in standard monolayer culture.79 In FGF-2-supplemented
serum-free medium, human ear chondrocytes had a higher
expansion rate and greater gene expression ratio of
type II collagen to type I collagen that indicated a well-
maintained differentiated phenotype.8 Serum-free cultured
chondrocytes also exhibit better redifferentiation capacity
when they are subsequently cultivated in three-dimensional
culture systems.8 Sox-9, a specific gene of chondrocyte
lineage, was detected in serum-free cultured chondrocyte
for up to 6 passages, whereas chondrocytes in serum medium
lacked Sox-9 expression.85
Tissue engineering requires large amounts of cultured
chondrocytes; however, routine culture systems contain
animal serum, which appears to be responsible for chon-
drocyte dedifferentiation. Subsequently, dedifferentiated
chondrocytes fail to produce a cartilage matrix when im-
planted in vivo. Calf serum is also considered to be a xe-
nogeneic pathogen to human patients, which may lead to
unpredictable side effects. Currently, human autologous
serum is available to avoid xenogeneic contamination, but
it is available in limited supply. A well-designed serum-free
medium supplemented with combinations of specific growth
factors may be the key to conquering these problems.
Three-dimensional culture
The imperative behind culturing chondrocytes in a three-
dimensional environment is to stabilize their phenotype for
subsequent applications. Chondrocytes are able to prolif-
erate in three-dimensional culture system without pheno-
type loss.86 Varieties of three-dimensional cell culture
techniques using biomaterials such as agarose gel,87–90
alginate,91,92 and fibrin93,94 have been studied. Agarose gel
culture was the first developed three-dimensional culture
technique. Toshihiro and colleagues observed aggrecan
6 LIN ET AL.
formation after 1 day in chondrocyte agarose culture, with
increased production over the culture period. Type II col-
lagen and GAG expression were positive and more intense
than chondrocytes cultured in monolayer culture, indicating
maintenance of the chondrocyte phenotype in agarose
culture.95 Unfortunately, chondrocytes fail to be digested
completely from agarose gel by agarase digestion, leading
to deficient cell numbers at harvested. Alginate is a linear
polysaccharide isolated from marine-grown algae consist-
ing of 2 uronic acids: L-guluronic and D-mannuronic acid
linked by b-1,4 and a-1,4 glycosidic bonds.96 The alginate
polymer undergoes instantaneous ionotrophic gelation in
the presence of divalent cations like calcium. It could be
applied to cultivate chondrocytes while maintaining their
cellular phenotype. It has been observed that dediffer-
entiated human chondrocytes undergo redifferentiation
when cultured in alginate beads.97 Fibrin glue has also been
widely used in the study of cell culture and tissue engi-
neering. Fibrin promotes chondrocyte-specific matrix pro-
duction and chondrocyte proliferation better than other gel
materials do. However, chondrocytes undergo a process of
dedifferentiation while fibrin glue is disintegrated.
Various researchers havealso introduced several alternative
techniques of three-dimensional chondrocyte cultures. A tem-
perature-sensitive copolymer of poly(N-isopropylacrylamide)
and acrylic acid (PNiPAAm-co-Aac), which has the ability to
gelatinize above 378C and become soluble at lower temper-
atures, was developed to promote three-dimensional chon-
drocyte proliferation.98 Chondrocytes isolated from rabbit
scapula were able to duplicate, regain spherical morphology,
and reproduce original matrix secretion patterns in the ther-
mosensitive gel culture. Furthermore, chondrocytes could be
completely recovered from the gel by simply lowering the
temperature below 378C.98
In particular, extensive interest in physically and chemi-
cally modified culture composites for bioengineering carti-
lage has been illustrated in the literature over the last
20 years. Tissue engineering using a construct of a three-
dimensional scaffold and cultured autologous chondrocytes
has been considered to be an effective therapy for cartilage
injury. Numerous attempts have been made to create an ideal
three-dimensional structure suitable for autologous chon-
drocyte therapies. An ideal scaffold for tissue engineering
should be customized, biocompatible, and biodegradable,
with a controllable degradation and resorption rate to match
tissue replacement. It should also allow cell attachment, mi-
gration, and proliferation and should develop into a biome-
chanical tissue-like structure. Chondrocytes should maintain
their differentiated phenotype within the scaffold, and gas
exchange, nutrient diffusion, and waste metabolism should be
facilitated. Also, it should be possible to process the combined
construct into different shapes and sizes using free-form
fabrication.99Varieties of three-dimensional scaffolds that are
currently available may be grouped into natural components
such as collagen, fibrin, gelatin, and alginate and synthetic
components such as polyglycolic acid,100–102 hydroxyapa-
tite,103 poly-L-lactic acid,103,104 polycaprolactone,105 poly-
lactide-co-glycolide,106 poly-L-lactic acid/alginate amal-
gam,107 gelatin-calcium-phosphate,108 polyester-urethane
polymer (DegraPol),109 a blend polymer of poly(hydroxy-
butyrate-co-hydroxyhexanoate)/polyhydroxybutyrate scaf-
fold,110 and photopolymerizing hydrogel.111
Bioreactor culture
A bioreactor is an apparatus in which biological and
biochemical processes develop under finely designed en-
vironmental and operating conditions such as pH, tem-
perature, pressure, nutrient supply, waster removal, and
biomechanical stimuli.112 More recently, bioreactor culture
systems have been used for tissue engineering. Chon-
drocytes have been cultured in three-dimensional vessels
supplemented with biomaterials and culture medium and
maintained under well-designed dynamic conditions. An
ideal bioreactor functions to provide in vivo–like physical
stimulation to the growing tissues using mechanical or
hydrodynamic loading, promoting chondrogenesis, ECM
synthesis and tissue formation, and supplying better oxy-
genation and nutrition than static culture systems.113,114
Bioreactors employed for the production of tissue-engineered
cartilage include conventional spinner flasks,115,116 rotating-
wall vessels,117,118 wavy-walled bioreactors,114 concentric
cylinders,119 perfusion bioreactors,120,121 rotating-shaft bio-
reactors,113 and column reactors.116 In the wavy-walled bio-
reactor, smooth waves that mimic baffles are applied to the
vessel to enhance mixing at controlled shear stress levels. The
culture system promotes chondrocyte aggregation, ECM
formation, and rapid cell–cell attachment and communica-
tion.114 The two-phase rotating-shaft bioreactor was designed
to move the chondrocyte-seeded scaffolds between air and
medium phases for alternating gas and nutrient exchange.113
Different bioreactors provide different dynamic environ-
ments, which leads to varied tissue properties. Bioreactor
cultures of cartilage have been developed to produce cell/
scaffold constructs ready for the dynamic in vivo environ-
ment, before implantation. Moreover, the bioreactor provides
a reliable model system to study tissue biology under con-
trolled healthy or pathological conditions.
Pellet culture of chondrocyte
High cell density is another critical requirement for
chondrocyte phenotype maintenance. Studies have shown
that dedifferentiated chondrocytes regain their phenotype
and re-express chondrocyte-specific type II collagen and
aggrecan when they reach condensation.110,122 Chon-
drocyte pellet culture leads to extremely high cell density
and provides an in vitro model of chondrocyte differenti-
ation. It has been reported that chondrocytes can stabi-
lize their phenotype when cultured at a density of 4� 105
THE CHONDROCYTE 7
cells/cm2.123 Pellet culture was firstly applied to growth
plate chondrocytes.124,125 Rabbit growth plate chon-
drocytes (8� 104 cells/mL) were transferred into a plastic
centrifuge tube, pelleted using centrifugation, and cultured
in eppendorfs.124 After 8 days in culture, the cell mass
formed a cartilage-like tissue used as a growth-plate study
model.124 The use of a pellet culture of hyaline cartilage
chondrocytes has been documented in the literature.126
Several aspects of the pellet culture model of chondrocytes
isolated from hyaline cartilage, such as the functions of
additional cytokines, including BMPs on chondrocyte
phenotype;126 properties of ECM;127 bioenergetics of
chondrocytes;128,129 and histological, immunohistochemi-
cal, and ultrastructural studies of pellet-culture-expanded
chondrocytes have been studied.130 It has been reported
that pellet-cultured chondrocytes share similarities in cel-
lular distribution, matrix composition and density, and tis-
sue ultrastructure with native cartilage.130
Organ culture of cartilage slices
Organ culture has been defined as a technique for the
maintenance or growth of animal organs in vitro. The ad-
vantage of organ culture is that it retains the original his-
tological feature and structural relationship between cells
and their nearby microenvironments. On the other hand, it
is almost impossible to generate an organ in culture larger
than 1 cm3 because of the inherent occlusion of nutrient
supply. Nevertheless, it is feasible to employ organ culture
systems to study the long-term effects of exogenous stimuli
or create models of certain diseases to gain better under-
standing of disease mechanisms. In particular, the organ
culture model has been successfully used in the study of the
biomechanics of cartilage.131 In one study, cartilage slices
3mm in diameter and 1mm thick were expanded in tissue
culture medium, after which compression and tissue shear
deformation were applied to the cultured cartilage slices to
model the physical stimuli on native cartilage in vivo.132
Biomechanical and biochemical properties, gene expres-
sion profile, matrix turnover, and signaling pathway regu-
lation have all been studied using this model.90,131–134
Furthermore, resected mouse coccygeal discs cultured
under static compression were used as an intervertebral disc
degeneration model.135 Chondrocyte apoptosis associated
with mitogen-activated protein kinase and p38 mitogen-
activated protein kinase expression was observed in this
model.135 Hirsch and Svoboda developed a chick sternum
culture model for chondrocyte maturation.136 The whole
chick embryo sternum was cultured in defined serum-free
medium supplemented with dexamethasone, insulin, thy-
roid hormone, and ascorbic acid. Several sites of organ-
cultured chick sterna were compared with sterna grown in
vivo. The study illustrated that the organ-culture tissue
closely resemble in vivo development with respect to mor-
phological characteristics and differentiation patterns.136
CLINICAL APPLICATIONS
Autologous chondrocyte implantation
for treatment of cartilage defect
Autologous chondrocyte implantation (ACI) was first
introduced to treat patients with symptoms of articular
cartilage lesions of the knee in 1987.137 Cartilage biopsy
was obtained from the non-weightbearing surface of the
condyle cartilage in patients under arthroscopy. Primary
chondrocyte culture methodology has been employed to
achieve large numbers of mature cells even though the
technique may produce de-differentiating chondrocytes.
Autologous serum obtained from patients is supplemented
in the culture system. The cultured chondrocytes are later
injected beneath a periosteal or collagen flap, covering the
cartilage defect, and then sealed with fibrin glue. From
1987 to 2001, ACI was used to treat more than 950 patients
with knee injuries in Sweden. Brittberg et al. described the
continuous follow-up of 213 ACI-treated patients.138 The
study reported 84% to 90% good to excellent results with
differential femoral defect types at 2 to 10 years’ follow-up.
An alternative ACI technique, matrix-induced autologous
chondrocyte implantation (MACI), has also been used for
the treatment of cartilage injury since 1999.139 The MACI
technique employs cultured chondrocytes directly inocu-
lated on a resorbable porcine type I/III collagen scaffold. The
cultured autologous chondrocytes are seeded on a highly
purified resorbable porcine collagen type I/III membrane,
which is implanted into the debrided cartilage defect and
fixed with fibrin glue. MACI offers several advantages over
traditional ACT procedure because of the lack of periosteum
use. Cherubino et al. have recently supported the MACI
technique, reporting 6 months of follow-up with no com-
plication, better clinical outcomes, and hyaline-like cartilage
visualized using magnetic resonance scan imaging. It is
thought that the introduction of autologous chondrocytes on
porous collagen membrane increases chondrocyte differen-
tiation within the three-dimensional scaffold.139
Clinical procedures have not been tested adequately. The
future directions of experimental and standard clinical pro-
cedures in terms of functional ACI are required, and the
experimental and clinical outcomes of ACI must be stan-
dardized. Guilak et al. have defined several aspects for the
improvement in assessment of functional outcome after
ACI.140 First, standards of clinical success for cartilage re-
pair must be achieved to compare the safety and efficacy of
various conditions. Second, to improve the engineering of
repair tissue, the biomechanical properties need to be in-
tensively researched. The prioritization of biomechanical
function and physical properties of native cartilage should
be defined to design parameters of engineered healthy hya-
line cartilage. Lastly, investigation of mechanical loading of
implanted engineered tissue in vivo and its consequences is
essential to improve tissue-engineered cartilage outcomes.
8 LIN ET AL.
Another challenge to the development of tissue-engineered
cartilage is to accurately recreate the distinct zonal archi-
tecture of native cartilage. Mature human hyaline articular
cartilage has a unique structural organization, which can be
divided into the superficial, transitional, deep, and calcified
cartilage zones. Histological characteristics, biosynthetic
activity, tissue composition, and mechanical properties vary
to meet the different demands of various functions within the
different zones.141 Chondrocytes in the superficial zone
present a long axis parallel to the tissue surface with less
biosynthetic active, although they are round and more bio-
synthetically active in the middle and deep zones.141 The
distinct zonal arrangement of articular cartilage is essential
to cartilage functionality; however, current articular carti-
lage engineering studies employ homogeneous cell mixtures
containing chondrocytes from a mix of zones, and no ap-
proach is currently available to mimic this zonal structure
with repair by tissue engineering. If the full efficacy of ACI
treatment is to be realized, there must be further study into
possible mechanisms for replicating the zonal architecture
of hyaline cartilage after injury.
Growth plate reconstruction
Lesions of the epiphyseal growth plates caused by frac-
ture, trauma, or inflammation can result in invasion of bone
across the cartilage and localized arrest of long bone
growth. Several attempts at using fat tissue, muscle, poly-
meric silicone, bone wax, bone cement, and cultured au-
tologous chondrocytes as interposition materials after
resecting the bone bridge have been undertaken.142 In a
sheep model of physeal injury, the implantation of cultured
chondrocytes in collagen gel restored the growth arrest in a
small physeal defect of less than 20% of the total physis.143
Chondrocytes have been shown to have a better ability to
synthesize ECM than adipose tissue.142 Lee et al. reported
that cultured allogeneic chondrocytes embedded in agarose
prevented long bone discrepancy and angular deformity in
a rabbit model with up to 50% physeal arrest.144 However,
allogeneic chondrocytes may present antigenic pathogens
to patients. Moreover, the relevant regulatory bodies have
not proved that the use of agarose in humans is safe in
human patients. Tobita et al. modified their methods to
culture autogenous chondrocytes in atelocollagen gel.142
They showed that the chondrocytes in atelocollagen gel
could prevent early ossification and closure and reduce later
angular deformity and length discrepancy in a rabbit model
with 66% physeal injury. Additionally, the atelocollagen
gel was found to be safe and have low immunogenicity.
Cartilage construct for facial reconstruction surgery
Cartilage constructs could be applied for other purposes
than orthopedic use. Attempts have been made to investi-
gate the feasibility of fabricating neo-cartilage in a pre-
determined shape using tissue engineering methods. This
system may be used in the patient-specific shaping of facial
implant tissue engineering. Cui et al. reported the formation
of a construct of human chondrocytes seeded into a non-
woven mesh of polyglycolic acid.145 The construct was
developed into sheets and tubes of neo-cartilage and then
implanted into nude mice after 6 weeks. Similarly, Chang
described a method to create an injection molding of
chondrocyte/alginate complex structures, including the
nose bridge and chin, with good three-dimensional toler-
ance to form cartilage in specific shapes.146 The recon-
struction of the anatomy of the head and neck requires a
high degree of precision with unique geometries. Injectable
chondrocyte-polymer constructs can be remodeled into
specific shapes in the geometry of the original template. A
tissue-engineered auricle has been successfully implanted
in an animal model.147 Autologous chondrocytes combined
with biodegradable polymers were cultured in a hollow
auricle-shaped mold before the molds were implanted
subcutaneously into the animal’s abdominal area. Mor-
phological and histological assessment showed that the
chondrocyte-alginate constructs remodeled the most exact
auricle shape and produced neo-cartilage.147 This potential
achievement of a tissue-engineered auricle may also be
applicable in patients with microtia.
Chondrocyte transplantation for reconstruction
of long segmental tracheal defects
Several animal studies have demonstrated the feasibility
of using cultured chondrocytes combined with bioresorbable
materials for reconstructing long segmental tracheal defects.
Harvested chondrocytes from human trachea cartilage pos-
sess the ability to form new cartilage-like architecture when
cultured onto a three-dimensional DegraPol matrix.109 Hu-
man chondrocytes harvested from tracheal cartilage were
cultured in vitro for 6 to 8 weeks and then seeded onto
DegraPol at a seeding density of 2.4� 107/mL. Human
tracheal chondrocytes proliferated, regained spherical mor-
phology, and formed cartilage-like tissue in the DegraPol.
In addition, survival for up to 20 weeks was achieved using
a polyglycolic acid construct composed of polyglycolic
acid mesh and alginate-encapsulated autologous chon-
drocytes to bridge tracheal defects in New Zealand white
rabbits while patent airways were demonstrated.101 Subse-
quently, tracheal cartilage/polymer constructs have been
considered as tissue substitutes for reconstructing long tra-
cheal defects.
Chondrocyte implantation for treatment of urinary
incontinence and vesicoureteral reflux
Urinary incontinence and vesicoureteral reflux due to
urethral sphincter deficiency often occurs in elderly or
debilitated patients. A surgical approach to immobilize the
bladder neck is considered clinically risky. A substitute
THE CHONDROCYTE 9
therapy of bulking of the suburethral tissue has been de-
veloped since last century.148 The injectable material is in-
jected into the bladder neck as a bulking agent. Several
bulking agents have been investigated. In the early 1970s,
injectable polytetrafluoroethylene (Teflon) paste was intro-
duced for use in the treatment of urinary incontinence,149 but
its use was later stopped because of reports of particle mi-
gration, embolization, and granuloma formation that called
into question the safety of Teflon. In 1993, Contigen (C.R.
Bard, Inc.), a bovine collagen material, was approved for
therapy in the United States and has been used as a standard
material for collagen injection therapy.148 The Contigen
injection therapy has proved to be useful, but only for a short
period. Glutaraldehyde cross-linked bovine collagen is an-
other widely used bulking agent, but it also fails to maintain
its initial volume after injection in vivo and requires revi-
sion.150 Therefore, neither is considered to be a stable ther-
apy. The feasibility of using cultured chondrocytes combined
with calcium alginate gel as a stable bulking substance has
been investigated in animals.151–153 Chondrocytes have an
intrinsic ability to produce ECM and form cartilage structure
in vivo. The injectable alginate gel serves as a chondrocyte
carrier and then degrades after implantation. The neo-car-
tilage formed helps to stabilize the volume of initial injec-
tion. Study has shown that chondrocyte-alginate constructs
are biocompatible, non-antigenic, noncarcinogenic, and non-
migratory and can be delivered endoscopically. Diamond
and Caldamone have reported further clinical trials, with 29
children suffering from varying degrees of vesicoureteral
reflux treated endoscopically with autologous-cultured
chondrocytes combined with injectable alginate gel.149 At 3
months, 57% of the patients were free of reflux after a single
treatment. The overall results of 1 or 2 injections showed an
83% rate of success at 3 months, indicating that the therapy
is safe and effective. A Phase I clinical trial has been per-
formed on 32 women with intrinsic sphincter deficiency.148
The chondrocyte/alginate construct was also used to endo-
scopically correct urinary incontinence, with outcomes fur-
ther confirming previous results. The overall success rate
was 84% at 6 months after a single injection, maintained at
12-month follow-up.154
Only a few groups have attempted tissue-engineered
cartilage-like constructs used for non-orthopedic purposes.
Although the outcomes from in vitro investigation are en-
couraging, their confirmation in vivo is obligatory. Ran-
domized, controlled clinical trials are required for the
application of tissue-engineered cartilage constructs in the
treatment of the various pathologic candidates susceptible
to chondrocyte-based therapy.
CONCLUSIONS
Chondrocytes are the unique cells of cartilage tissue,
responsible for the metabolism and remodeling of ECM. In
the last few decades, attempts have been made to develop
clinical usage of this cell type. Tissue-engineered cartilage
constructs using cultured chondrocytes and biomaterials
have been studied inside and outside the orthopedic arena,
with promising outcomes reported in many disciplines.
From the results reviewed in this article, we perceive that
further chondrocyte application in clinical practice is
encouraging. This review details the myriad studies of
cell-based chondrocyte biology and the challenges to
achieving efficacious and reliable outcomes while high-
lighting the need for further, more-comprehensive research
to validate the widespread clinical acceptance of chon-
drocyte-based approaches. In light of all of the aforemen-
tioned studies, the chondrocyte presents as a possible cell
source for tissue-engineered reconstructive surgery across
many specialties.
ACKNOWLEDGMENT
This work was supported by a National Health and
Medical Research Council development grant toM. H. Zheng
and D. J. Wood.
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Address reprint requests to:
Prof. Ming-Hao Zheng, PhD, DM, FRCPath
Department of Orthopaedic Surgery
School of Surgery and Pathology
University of Western Australia
Nedlands, 6009, WA, Australia
E-mail: [email protected]
14 LIN ET AL.
CHAPTER 2
INTRODUCTION OF OSTEOARTHRITIS
>>Chapter 2 Osteoarthritis<<
15
2.1 GENERAL INTRODUCTION
Osteoarthritis (OA), also known as a complex, chronic degenerative joint disease, is the
most common form of arthritis, which causes severe long-term pain and physical
disability. The disease processes initially degenerate articular cartilage, then affect the
entire joint including subchondral bone (Brandt et al., 1998). OA is extremely prevalent
in all populations, especially in the elderly. Conservative estimate indicated that there
were up to 550,000 patients with severe knee OA in UK, accounting for 200,000 general
practitioner visits (Haq et al., 2003). In 2000, an amount of £405 million had been spent
on hip or knee joint replacement because of arthritis in UK (Haq et al., 2003). In
Australia, about 2.6 millions people suffered from some form of arthritis, counting for
13.6% of the population. Among these Australian sufferers, 1.4 million had
osteoarthritis and 438,200 had rheumatoid arthritis, with prevalence rates of 7.3% and
2.3% respectively (Australia Institution of Health and Welfare, 2001). It causes most of
the disability of the elderly in the western society. While the better understanding and
treatment of other fatal diseases lead to longer average live-span of all population,
nonfatal diseases such as OA become a major hazard of activity limitation for the public.
In Canada, it is projected that the prevalence of arthritis will increase from 2.9 million
in 1991 to 6.5 million in 2031 (Hill et al., 1999). Because of this growing epidemic, OA
has become a major burden on individuals, society and health care systems. Hence, it is
becoming more important to improve identification and understanding of the disease
process and sequentially develop new intervention to delay or even reverse the disease
progress.
2.2 CLINICAL FEATURES
OA patients are usually after middle age, but it is possible that the cartilage change
emerges decades before. The younger patients of OA usually have a history of sport or
accidental joint injury. The clinical features of OA include pain, stiffness, swelling, and
movement limitation. Among them, pain and stiffness are two essential clinical features
>>Chapter 2 Osteoarthritis<<
16
of early OA. Pain starts insidiously and increases slowly over months or years,
exacerbated by exertion and relieved by rest. Stiffness becomes obvious after periods of
rest. It is usually presented in the early morning, lasting for up to 30 minutes. Swelling
and deformity are characteristics of advanced OA, due to osteophyte formation or
effusion caused by synovial fluid accumulation (Brandt et al., 1998). This syndrome is
more obvious in superficial joints. Movement limitation always occurs in the late stage
of OA, and progresses to instability. The fact that OA is unassociated with any systemic
manifestations can be used to distinguish it from other joint inflammatory diseases.
2.3. ETIOLOGY OF OA
Age
Arthritis prevalence is believed to increase with age. The incident of OA increases
significantly after age 40 with every passing decade (Praemer et al., 1999; Buckwalter
et al., 2000, 2001 a,b). According to the Morbidity and Mortality Weekly Report
(MMWR) of 2003 in U.S., it affected approximately 60% of the U.S. population above
the age of 65 years. The number of affected persons with above the age of 65 years
could be doubled by 2030. The aging progress increases the incident of OA by reducing
joint proprioception, cartilage calcification and chondrocyte function. The articular
cartilage age-related change contributes to articular cartilage degeneration that is
characterized by a decrease of mitotic and synthetic activity of chondrocytes, decreased
responsiveness to anabolic growth factors, increased synthesis of mutatedn aggrecan,
and less functional link protein (Martin et al., 2002). Hence, articular cartilage aging is
considered as an important cause for the increasing incident of age related OA.
Sex
It has been reported that OA has a higher prevalence in women than in men. An
Australian survey indicates that the prevalence of OA is higher among women than men
among all age groups (2.95 per 1000 population in women vs. 1.71 per 1000 in men;
Woolf et al., 2003). The prevalence of arthritis among women also rises up with the
increasing of age. For women, the highest incident is among those aged 65-74 years,
>>Chapter 2 Osteoarthritis<<
17
reaching approximately 13.5 per 1000 population per year (Woolf et al., 2003). In
addition, the sex difference of OA incident between women and men increases with age.
The post-menopausal hormone deficiency of women is thought to be responsible for
this gender and age-related prevalence pattern (Brandt et al., 1998).
Obesity
Obesity is a modifiable risk factor for OA. In U.S., a population-based study showed the
rates of prevalence of arthritis were higher for women who were overweight with body
mass index (BMI) greater than 27.3 (MMWR, 1995, May). The excess amount of
loading across joints by overweight could stimulate cartilage breakdown, which
contributes to the progress of OA. Excess mechanical compression can lead to ECM
degradation and chondrocyte morphological alteration (Grodzinsky et al., 2000).
However, people with high BMI are also at high risk of hand OA (Brandt et al., 1998).
The mechanism of excess loading by overweight is not sufficient enough to explain this
phenomenon. The complete understanding of the relationship between overweight and
OA is yet to be sought.
Genetic defects and inheritance
Genetic defects are thought to play a certain role in the initiation and progression of OA.
Genetic epidemiologic studies have suggested that primary OA has a major genetic
component that segregates in families in a complex pattern (Loughlin, 2001). Children
of parents with early onset osteoarthritis are at greater risk. The genetic make up of the
chondrocytes in native cartilage determines the homeostasis and integrity of cartilage
ECM, which is essential for the maintaining of proper cartilage function (Aigner et al.,
2003). In recent year, more and more attempts have been taken to investigate the genetic
aspects of the disease process. Research in genomics of OA chondrocytes could provide
a better understanding of disease mechanisms. The ultimate aim of the genetic study in
OA is to generate gene products or gene-related drugs, which can hinder the disease
process and/or retain joint function.
>>Chapter 2 Osteoarthritis<<
18
Trauma
Cruciate, collateral, meniscal tears and joint facture caused by sudden injury or repeated
stereotyped activity increase risk of OA (Brandt et al., 1998). The Framinghan study
demonstrated that men with a history of major knee injuries have a relative risk of 3.46
for developing knee OA (Brandt et al., 1998). Occupations such as farmers, jackhammer
operators, miners and sport men are at a higher risk of OA, due to excess stress across
the joint or repeat joint use. The joint injury is responsible for most of the OA patients in
young age. People who have a joint injury history are at a high risk of progressive OA
when they become older.
2.4. PATHOGENESIS
The pathogenesis of OA starts initially from abnormality of articular cartilage. Cartilage
is a semi-transparent connective tissue, characterized by an extracellular matrix
enriched with glycosaminoglycans and proteoglycans, macromalacules that interact
with collagen and elastic fibers. The function of cartilage is to provide a smooth,
frictionless articulating surface which can bear compression and even loading. The
ECM secreted by chondrocytes enables cartilage to withstand a considerable degree of
pressure, tension and torsion, while exhibiting considerable degree of elasticity.
Cartilage is supplied by no demonstrable blood vessels, nourished by the diffusion of
nutrients from capillaries in adjacent connective tissue or by synovial fluid from joint
cavities. Hence, the quality of ECM is critical for maintaining the functional properties
of the cartilage. As it may be triggered by an acute or transient injury, gradual
proteolytic degradation of the matrix and alternation of matrix component occur in
articular cartilage. Lost in chondrocytes of the ability of maintaining the balance
between synthesis and degradation of ECM leads to the early morphological changes in
cartilage, such as cartilage surface fibrillation, cleft formation, and late loss of cartilage
volume (Dieppe and Lohmander, 2005). Subsequently, through ossification of cartilage
outgrowths, osteophytes invade the joint margin. Major changes occur in the
subchondral bone as well, which are detailed in Section 2.4.6.
>>Chapter 2 Osteoarthritis<<
19
Some growth factors, cytokines and intercellular signaling molecules released by
cartilage, synovium and bone contribute to the disease process (Fernandes et al., 2002).
It is known that the expression of interleukin-1 (IL-1) and tumor necrosis factor-α
(TNF-α) are increased in OA, inducing nitric oxide and matrix proteases production
(Verschure et al., 1990; Taskiran et al., 1994; Tetlow et al., 2001). Other factors, such as
mechanical loading, also induce catabolic cytokine receptors in chondrocytes (Quinn et
al., 1998). The cellular responses of OA cartilage could be defined in 6 categories: (1)
chondrocyte proliferation and apoptosis, (2) increase of catabolic activity, (3) influences
of cytokines and growth factors, (4) phenotypic modulation of chondrocytes; (5)
formation of osteophyte; (6) subchondral bone remodeling (Sandell et al., 2000).
2.4.1 Chondrocyte apoptosis and proliferation
Cell death is a central event of OA cartilage degeneration featured by lacunar emptying
especially in the calcified cartilage layer (Aigner et al., 2002; Mitrovic et al., 1983).
Chondrocytes were found to undergo apoptosis in human OA and rheumatoid arthritis
(RA) cartilage, with the number of apoptotic chondrocytes being higher in severely
affected OA cartilage than in less affected cartilage (Blanco et al., 1998; Kim et al.,
1999; Hashimoto et al., 1998). Since adult human articular cartilage has no ability to
renew cell population, and chondrocyte is the only cell type in articular cartilage to
synthesis cartilage matrix components, a certain degree of cell death would result in loss
of balance in matrix turnover. The toxicities released from apoptotic cells, such as
pyrophosphate and precipitated calcium, may contribute to the pathological cartilage
calcification in OA (Blanco et al., 1998). However, a recent study revealed that only a
very low fraction of apoptotic cells was found in OA cartilage samples with
approximately 0.1% of the total cell population being apoptotic at certain time point,
which suggests that the death of chondrocytes might only has a limited impact on the
pathogenesis of OA (Sandell et al., 2000).
In addition, many studies have confirmed that the chondrocytes in OA cartilage have a
>>Chapter 2 Osteoarthritis<<
20
very low proliferation rate, when compared to the cells in normal articular cartilage.
This phenomenon leads to chondrocyte clustering which is one of the characteristic
features of OA cartilage (Rothwell et al., 1973; Mankin ey al., 1971).
2.4.2 Alteration of metabolic activity
Many studies have domenstrated that the balance of turnover of the cellular
environment, which is maintained by chondrocytes, is disrupted in OA cartilage. The
destruction of articular cartilage is the hallmark of OA. In the early stage of OA, the
synthesis of matrix component is enhanced. Chondrocytes increase their anabolic
activity, attempting to repair the damaged matrix (Aigner et al., 2001). With the
development of OA, the collagen network and proteoglycan are, however, disrupted and
lost by increased synthesis of catabolic cytokines and matrix degradating enzymes. The
proteases that are involved in OA including matrix metalloproteinase (MMP)-3 (Tetlow
et al., 2001; Monfort et al., 2006), MMP-8 (Shlopov et al., 1997; Chubinskaya ei al.,
1996), MMP-9 (Freemont et al., 1997), MMP-13 (Billinghurst et al., 1997; Aigner et al.,
2001; Mitchell et al., 1996), ADAMTS-4 (Nagase et al., 2003) and ADAMTS-5
(Stanton et al., 2005; Glasson et al., 2005) have been reported to accompany the
increased matrix degradation in OA cartilage. MMP-3, dominantly expressed in normal
articular cartilage, was found to be downregulated in late-stage OA cartilage (Aigner et
al., 2001). It is thought to cleave in the nonhelical telopeptide of type II and type IX
collagens, leading to the disruption of collage crosslink. A significant increase of
MMP-13 expression was observed in late-stage OA, suggesting that MMP-13 might be
the main enzyme responsible for the terminal breakdown of collagen fibers in OA
cartilage destruction (Mitchell et al., 1996). MMP-2, an enzyme known to degrade
denatured collagen, is also significantly elevated in late-stage OA cartilage (Aigner et al.,
2001).
Recent interest has focused on the enzymes responsible for the degradation of the core
protein aggrecan. ADAMTS-4, and -5, defined as aggrecanase 1 and 2, were recently
cloned and characterized. Transgenic mice study by Stanton et al., 2005 suggested that
>>Chapter 2 Osteoarthritis<<
21
ADAMTS-5 is the major aggrecanase in mouse cartilage, which contributes to the
initiation of cartilage destruction in the mouse inflammatory disease model. Apart from
the catabolic activity of metalloproteinases, mutations in type II collagen evidenced by
genetic studies also lead to an unstable collagen network, which eventually lead to
destabilization of the joint (Eyre et al., 1995). On the other hand, the levels of the tissue
inhibitors of metalloproteinases (TIMPs), such as TIMP-1, 2 and 3, are increased in OA
synovial fluid and tissue (Su et al., 1999). The upregulation of TIMPs reflects an
endogenous adaptive response to the increased levels of active proteinase activites,
however the imbalance in the proteinase and inhibitor levels contributes to the
progressive degradation of OA cartilage matrix (Dean et al., 1989)
2.4.3 Influences of cytokines and growth factors
Cellular regulation by cytokines and growth factors is a complex process. Cytokines
that are involved in OA pathogenesis can be classified into 3 categories: anabolic
cytokines, catabolic cytokines, regulatory cytokines. The cartilage pathology is thought
to be the result of overproduction of catabolic cytokines, along with possible
insufficient anabolic stimulation and inadequate control by regulatory factors. The total
net cartilage destruction is more likely to be determined by various factors, rather than
by a certain single mediator (Blom et al., 2007).
In normal adult cartilage, the biosynthesis activity of chondrocytes is stimulated by a
number of anabolic cytokines and growth factors, such as transforming growth factor-β
(TGF-β), bone morphogenetic proteins (BMPs), platelet derived growth factor (PDGF),
basic fibroblast growth factor (bFGF), and insulin-like growth factor1 (IGF-1). In OA
cartilage, these factors contribute to counteract the effect of catabolic cytokines on
articular chondrocytes. For instance, the expression of TGF-β is elevated in OA
chondrocytes to enhance synthesis of proteoglycans and other cartilage matrix
components (van den Berg et al., 1993; van Beuningen et al., 1994).
In OA, catabolic factors, such as tumor necrosis factor-α (TNF-α), and interleukin-1
>>Chapter 2 Osteoarthritis<<
22
(IL-1), are produced by the synovium and chondrocytes. These inflammatory cytokines
act to increase MMPs production, inhibit TIMPs activity, and decrease extracellular
matrix synthesis. Chondrocytes express several inflammatory factors, which could in
turn modulate the cellular functions, when stimulated by IL-1 or in combination with
TNF-α. These factors include inducible nitric oxide synthase (iNOS), cyclooxygenase-2
(COX-2), and phospholipase A2 (Jacques et al., 1999; Clancy et al., 1998; Goldring et
al., 1999). The increased expression of iNOS and COX-2 in chondrocytes leads to the
spontaneous release of nitric oxide (NO) and prostaglandin E2 (PGE2) (Amin et al.,
1997; Jacques et al., 1999).
Regulatory cytokines, such as IL-4, -6, -10, and -13, can inhibit production of catabolic
cytokines or up-regulate inhibitors of those cytokines (Blom et al., 2007). Despite the
inflammatory property in synovium, IL-4 is capable of decreasing iNOS expression
leading to a decrease in NO production, reducing the sensitivity of chondrocytes to IL-1,
and inhibiting IL-1 and TNF-α production by OA synovium in vitro (Lubberts et al.,
2000; Fernandes et al., 2002). Another interesting regulatory cytokine is IL-6. Animal
study revealed that mice with deletion of IL-6 develop more severe OA compared to
controls, suggesting that IL-6 is a negative mediator of cartilage breakdown during OA
(de Hooge et al., 2000; de Hooge et al., 2005). On the other hand, IL-6 can enhance
TIMPs expression in chondrocytes, whilst inhibit the cartilage degeneration caused by
MMPs (Silacci et al., 1998). Further investigation is needed to clarify the regulation and
functions of these regulatory cytokines in human articular cartilage and OA
pathogenesis.
2.4.4 Phenotypic modulation of chondrocytes
Arthritis is characterized by loss of the differentiated phenotype of chondrocyte.
Chondrocytes tend to develop into fibroblastic-like cells when they are cultured on
monolayer or stimulated by retinoic acide, bromodeoxyuridine, or IL-1, which is termed
“de-differentiation”. Phenotypic changes of chondrocyte during differentiation have
been shown in vivo in fetal growth-plate cartilage and in vitro (Sandell et al., 2000;
>>Chapter 2 Osteoarthritis<<
23
Aigner et al., 2002). These cells express type I, III, and V collagen instead of
cartilage-specific anabolic genes, aggrecan and type II collagen. It has been proposed
that the phenotypic alteration contributes to the anabolic failure of articular
chondrocytes in OA cartilage (Aigner et al., 2002). Chondrocyte dedifferentiation can
be induced by IL-1β, which can in turn induce COX-2 expression and suppress
expression of Sox-9, a major transcription factor that regulates type II collagen
expression in cartilage (Hwang et al., 2005).
2.4.5 Formation of osteophytes
One of the most remarkable features of OA articular joint is the development of
prominent osteochondral nodules known as osteophytes. Osteophytes are
cartilage-covered bony projections, which occur at the chondro-synovial junction or
perichondrium where progenitor cells reside (Sandell et al., 2000). Osteophytes show
endochondral ossification surrounding carvities in the OA cartilage and suchondral bone
thickening (Figure 2.1). At the ossification sites, the cartilage is mineralized containing
hypertrophic chondrocytes (Neuman et al., 2003). The formation of osteophytes is
linked to growth factors such as TGF-β1 and basic FGF (bFGF), which are found to be
expressed in osteophytes of the femoral head in OA (Uchino et al., 2000). In a murine
model of OA, osteophytes developed after repeated local injections of TGF-β1 in the
knee and after sustained overexpression of TGF-β in the joint following TGF-β gene
transfer (Menkes and Lane, 2003; van Beuningen et al., 2000).
Interestingly, cartilage lesions are correlated with the degree of osteophyte formation
(Menkes at al., 2004). The formation of osteophyte is thought to be responsive to
mechanical or humoral stimulations caused by joint injury. Although the exact
functional significance of osteophytes remains unclear, osteophytes might contribute to
joint stabilization as compensation after cartilage destruction.
>>Chapter 2 Osteoarthritis<<
Figure 2.1 Specimen radiagraph of Osteophyte. It projects from over the joint surface
in OA as indicated by arrow. (picture origin: http://courses.washington.edu/hubio553/
totrad/SCAR/Images/OAShoulder.jpg)
24
>>Chapter 2 Osteoarthritis<<
25
2.4.6 Subchondral bone remodeling
The progression of joint cartilage degeneration is associated with intensified remodeling
of subchondral bone and increased bone stiffness (Lajeunesse et al., 2003). Yet there
are still diverse opinions about whether subchondral bone remodeling is prior to
cartilage destruction in OA process. Radin et al proposed that changes in subchondral
bone initiate progressive joint degeneration, evidenced by the increasing of subchondral
bone density and stiffness induced by impulsive loading on joints (Radin et al., 1972). It
was suggested that the abnormality of subchondral bone in OA has less capability to
attenuate and distribute forces through the joint, which leads to increased stress in the
articular cartilage and promotes degeneration (Radin et al., 1986). In animal models of
spontaneous OA, increased bone density and osteoid volume often exceed the severity
of cartilage changes (Carlson et al., 1996; Dedrick et al., 1993; Brandt et al., 1991). It
was found that the age-dependent increase in subchondral bone sclerosis is associated
with the development of cartilage degeneration in young guinea pig model; however the
stiffness of subchondral bone did not absolutely lead to future development of OA
(Huebner et al., 2002).
Bone remodeling is tightly controlled by osteoblasts that synthesizes mineralized type I
collagen bone matrix, and osteoclasts that remove the bone mineralized matrix. In OA
partients, both bone formation and resorption are increased, but the rate of
mineralization is low (Lajeunesse et al., 2003). The underlying mechanism of
subchondral bone remodeling in OA is still elusive, yet a number of factors have been
identified to be involved in the biological link between subchondral bone remodeling
and cartilage degeneration. An increased serum level of osteopontin, a bone-specific
matrix protein, was found in patients early after acute articular joint trauma, indicating
that alterations in bone cell activity may occur very early in the disease (Gevers et al.,
1987). Osteocalcin, a marker of bone formation, was found to be elevated in synovial
fluid of patients with OA (Sharif et al., 1995), which may contribute to the increasing of
bone volume without a concomitant increase in bone mineralization (Lajeunesse et al.,
2003).
>>Chapter 2 Osteoarthritis<<
26
There are a number of growth factors and cytokines that are found to affect subchondral
bone tissue in the progression of OA, including IGF-1, TGF-β, IL-1β and IL-6. The
expression levels of IGF-1 and 2, and TGF-β were higher in samples of iliac crest bone
of patients with OA, suggesting a generally increased biosynthetic activity of
osteoblasts in these patients (Dequeker et al., 1993). Among them, IGF-1 is the most
important factor regulating bone formation. In OA disease, the elevated IGF-1 in
subchondral bone by osteoblasts promotes bone remodeling, increases bone stiffness
and exacerbates cartilage matrix degradation (Dequeker et al., 1993; Hilal et al., 1998;
Martel-Pelletier et al., 1998).
2.5 DIAGNOSTIC APPROACHES
Diagnosis of osteoarthritis relies mostly on physical examination and radiographs.
Physical examination of affected joints may demonstrate crepitation, bony enlargement,
limited range of motion, misalignment, joint instability, and gait disturbance. X-ray is
the cheapest radiological diagnostic approach available for OA. The changes of diseased
joint, such as joint space narrowing, osteophytes, bone cysts and subchondral sclerosis,
can be seen on plain radiographs. Magnetic resonance imaging (MRI) is a sensitive
approach of cartilage loss and early OA detection, though it is too expensive to be
included as routine use. Additionally, laboratory tests help to rule out other joint
diseases due to infection, inflammatory disorders, or endocrine and metabolic disorders.
The OA patients demonstrate a normal or only slightly elevated erythrocyte
sedimentation rate, while patients with an underlying malignancy or chronic infection
show a significant elevation. The investigation of serum levels of calcium, phosphorous,
magnesium and thyrotropin may help to determine chondrocalcinosis (Morehead et al.,
2003).
2.6 CONVENTIONAL TREATMENT OF OA
>>Chapter 2 Osteoarthritis<<
27
2.6.1 Early-stage OA treatment
Management
The management of patients is particularly important for early OA treatment. The
principle objectives of it are to help patients understand the disease, relieve pain,
minimize disability and limit the progression of the disease. The strategies of patient
management include patient education, exercise, weight and diet control, applicants, and
local physical treatment (Brandt et al., 1998).
Patient education includes helping patients to better manage their conditions and to
make informed choices between treatment options, and providing information and
therapist contact. Particularly, it is important to educate patients according to the
individual’s situation. As known, inactivity of joints due to pain leads to reduction of
surrounding muscle bulk and causes movement limitation that subsequently contributes
to the degradation of cartilage matrix and the decrease of cartilage volume. Hence,
regular exercise helps to increase the joint’s flexibility and strength and improves its
biomechanics. Because obesity and overweight are both associated with increased
incident of OA, regular exercise can also help to loss weight and alleviate syndrome. A
high saturated fat intake may lead to cartilage destruction. The dietary intervention
could contribute to weight control. People with high BMI who lose a total of 10 lb in 10
years can reduce by half their odds of having knee OA (Felson, 1996). Additionally, it
was suggested that adequate intake of vitamin C, Vitamin E and β-carotene may slightly
reduce the risk of disease progression, while a low intake of vitamin D may increase the
risk of progression (McAlindon et al., 1996). Applicants such as appropriate footwear
could reduce loading on the knee or reduce pain due to OA. Local physical treatment
like heat or cold therapy can reduce the pain threshold and provide local analgesia.
Pharmacologic treatment
Conventional agents of pharmacologic treatments for OA include analgesics,
nonsteroidal anti-inflammatory drugs (NSAIDs), selective cyclooxygenase-2 (COX-2)
inhibitors, glucosamine and chondroitin sulfate, intra-articular glucocorticoids and
>>Chapter 2 Osteoarthritis<<
28
visosupplements (Haq et al., 2003). Paracetamol and paracetamol/opiate combinations
are recommended as initial therapies for OA because of theirs efficacy and safety profile.
Confusion, constipation and increased falls may occasionally occur when patients take
analgesics, so they should be used with caution (Morehead et al., 2003). NSAIDs have
been found to have equal efficacy to paracetamol in most patients. For patients whose
pain has not responded to simple analgesics, NSAIDs are recommended for use. All
NSAIDs are thought to have similar pain relieving effects, with a reduction in pain of
approximately 30% and an improvement in function of approximately 15% (Courtney et
al., 2002; Brandt and Bradley, 2001). However, side effects of NSAIDs are common.
Especially, renal and gastrointestinal side effects of NSAIDs use are often serious.
Elderly or patients at high risk of peptic ulceration should be supplemented with
gastroprotection such as H2 antagonisis, misoprostol or proton pump inhibitors while
NSAIDs are taken. The dose of NSAIDs should be titrated depending on individual
response and side effects.
The new COX-2 selective inhibitors, such as celecoxib and rofecoxib, have equal
efficacy to nonselective NSAIDs in the treatment of OA, but have been claimed to
cause less gastrointestinal side effects. However, there are no studies definitively
demonstrating that COX-2 selective inhibitor is superior to standard NSAIDs in
gastrointestinal side effect prevention. Also, it has been found that COX-2 inhibitors can
precipitate vascular events, causing cardiovascular side effects such as myocardial
infarctions (Haq et al., 2003). It is recommended that aspirin negates should be provided
to all patients who have cardiac risk factors when they take COX-2 inhibitors
(Silverstein et al., 2000).
Glucosamine and chondroitin sulfate are nutrient supplements used to relieve
musculoskeletal symptoms. They are derivatives of glycosaminoglycans in articular
cartilage. A study of 212 patients with primary knee OA demonstrated that there was a
20-25% improvement in symptoms and a reduction in knee medial compartment
changes over three years in those taking glucosamine sulfates (Reginster et al., 2001).
>>Chapter 2 Osteoarthritis<<
29
Intra-articular injection of glucocorticoids and visosupplements provides significant
short term effect of 2-4 weeks over placebo (Lo et al., 2003). The mechanism is unclear,
but some patients achieved a sustained improvement in symptoms. Side effects include
skin atrophy and dermal depigmentation. Excessive use may lead to severe cartilage
destruction.
2.6.2 Late-stage OA treatment
Although the nonpharmacologic and pharmacologic interventions listed above can
provide therapeutic effects, they are not successful for everyone with OA. Some patients
with late-stage OA may need surgical procedure. Arthroscopic debridement and lavage
can improve symptoms in degenerative meniscal tears, but does not halt the progression
of OA.
Total joint replacement surgery can successfully alleviate joint pain in patients whose
medical therapy fail to reduce OA symptoms and altered function. Perioperative
mortality and short-term complications are rare. The failure rate of a joint replacement
associates mostly with the duration of its use (Haq et al., 2003).
2.7 NOVEL OR FUTURE THERAPEUTIC TARGETS
2.7.1 Cytokine therapies
Future directions in the research and treatment of OA will be based on the
understanding of the pathophysiological events that cause the initiation and progression
of OA. Since the progression of OA is initiated from an imbalance between anabolic
and catabolic pathways, to correct the disorder of the imbalance by using cytokines is a
goal for OA treatment in the future.
As mentioned, IL-1 can suppress anabolism by inhibiting synthesis of proteoglycan and
type II collagen, and induce catabolism by enhancing MMPs expression in cartilage. It
was found that inhibition of IL-1 activity or production in OA cartilage results in a
>>Chapter 2 Osteoarthritis<<
30
decrease of cartilage destruction in OA animal model (Melchiorri et al., 1998; Attur et
al., 2000). On the other hand, many studies suggested that TGF-β can also be a
therapeudic target for OA. People with aspirin variant showing inhibition of TGF-β
activity have a markedly greater risk to develop OA than those with the variant without
TGF-β inhibition, indicating the essential role of TGF-β in adult articular cartilage
(Blom et al., 2007). Tanaka et al. reported that injection of TGF-β into the cartilage
defect could accelerate cartilage repair (Tanaka et al., 2005). These studies suggest that
supplementation of TGF-β could be developed as a pharmacological therapy for OA.
The approaches of using cytokines as therapeutic target can also be directed to
interfering the interaction between cytokines and their receptors. This can be achieved
by using receptor antagonists, which compete binding to the receptor cytokine binding
sites without causing subsequent signaling, such as IL-1 receptor antagonist (IL-1ra)
(Blom et al., 2007).
2.7.2 Gene therapies
Gene therapy offers another approach to the treatment of OA. Epidemiological studies
have demonstrated the genetic component is one of the important factors contributing to
both rheumatoid arthritis (RA) and OA. The concept of using gene therapy in the
treatment of orthopaedic diseases was initiated 15 years ago. One goal of gene therapy
is to transfer a wild-type gene that is expected to be free of arthritis related genes or able
to produce functional gene products that could hinder the developmont of articular
diseases. The other goal of gene therapy is to produce a gene product encoding certain
protein that could possibly mediate the cartilage destructive process. It is based on the
complete understanding of the pathway events and in the cartilage regenerative process.
There were several experimental and clinical studies investigating gene products that
are considered as possible therapy approaches for the articular destructive diseases.
These gene products include IL-1 receptor antagonist (IL-1Ra), soluble IL-1 receptor
(sIL-1R), and soluble TNF receptor (sTNFR) (Evans et al., 2004). The introduction of
>>Chapter 2 Osteoarthritis<<
31
IL-1Ra suppressed the MMP gene expression in OA cartilage. In animal studies,
transfer of the IL-1Ra gene to synovium reduced the severity of the cartilagous lesions
(Pelletier et al., 1997; Frisbie et al., 2002). Intraarticular administration of a plasmid
DNA encoding a rat TNF receptor-immunoglobulin Fc (TNFR:Fc) fusion gene
decreased inflammatory cell infiltration, pannus formation, cartilage and bone
destruction, and downregulated the gene expression of joint proinflammatory cytokines
in a streptococcal cell wall (SCW)-induced rat arthritis model (Chan et al., 2002). In
addition, Transfer of IGF-1 cDNA to the synovium of arthritis joint could increase
matrix synthesis by the adjacent articular cartilage (Saxer et al., 2001).
2.7.3 Inhibition of inflammatory signaling pathways
Molecular factors that regulate signal transduction pathways induced by IL-1 and
TNF-α have been proposed as therapeutic targets (Goldring, 2000). These include
protein kinases such as extracellular signal-regulated protein kinase (ERK),
stress-activated protein kinase, c-Jun N-terminal kinase (JNK) and p38
mitogen-activated protein kinase (MAPK), and transcription factors, activated protein-1
(AP-1) and nuclear factor kappa B (NF-κB). These pathways are known to be activated
in chondrocytes (Ding et al., 1998; Shalom-Barak et al., 1998; Martel-Pelletier et al.,
1999). Selective p38 MAPK inhibitor, SB203580, was shown to inhibit IL-1-induced
collagen degradation in a cartilage explant culture model, suppress MMP-1 and MMP-3
expression in chondrocyte culture (Ridley et al., 1997), and prevent cartilage destruction
in an experimental arthritis animal model (Badger et al., 2000). Han et al., demonstrated
the decrease of IL-1-induced AP-1 activity and MMP-3 gene expression by blocking
JNK-MAPK kinase signal transduction pathways in an experimental animal arthritis
model (Han et al., 2001).
NF-κB is of particularly importance in the regulation of expression of various genes in
the initiation of inflammatory response. The NF-κB signaling pathway has been shown
to play a role in the inflammation, cartilage degradation, cell proliferation, angiogenesis
and pannus formation of OA (Roman-Blas et al., 2006). In articular chondrocytes,
>>Chapter 2 Osteoarthritis<<
32
NF-κB activation mediates the response to the major proinflammatory cytokines IL-1
and TNF-α. The blocking of NF-κB in human OA chondrocytes and chondrosarcoma
cells suppress the IL-1 and TNF-α induced MMP-1, MMP-3 and MMP-13 expression,
hence suggesting that inhibitors of NF-κB could be used as pharmacological agents for
OA treatment (Eguchi et al., 2002; Vincenti et al., 2002).
Although development of inhibitors of signal transduction pathways has provided
invaluable insights of the molecular mechanism of the disease, the investigation
however is still at a very early stage. Since OA is a complex disease with many factors
underlying its origin and determining its course, blocking of a single transduction
pathway may not be sufficient to prevent the disease progression. In addition, the
signaling pathways that regulate the inflammatory events in OA are also involved in the
function of many other cells and tissues. The inhibitors of signal transduction in OA
cartilage may also cause other severe side effects. Hence, more specific therapeutic
inhibitions of signaling pathway should be developed to study the local and systemic
responses and underlying mechanism.
2.7.4 Tissue engineering therapy
More recently, tissue engineered neo-cartilage construct successfully employed for the
repair of cartilage defect is being considered as a potential approach of OA treatment.
Cultured autologous chondrocytes seeded on a biomaterial scaffold or bioreactor
produced neo-cartilage construct has been applied for the treatment of cartilage defect
clinically. Currently, several engineered cartilage repair products have been used,
including Carbon Fibre Implantation (CFI; Brittberg et al., 1994; Debnath et al., 2004),
periosteal Autologous Chondrocyte Implantation (ACI; Brittberg et al., 1994; Peterson
et al., 2000), Collagen-covered ACI (Haddo et al., 2004), Matrix-induced ACI (Gigante
et al., 2006; Marlovits et al., 2005), Hyaluronic Acid ACI (Grigolo et al., 2001),
Collagen Gel ACI (Adachi et al., 2004; Katsube et al., 2000), and Allogenic
Chondrocyte Implantation (Kim et al., 2003; Hendrickson et al., 1994). Despite the
diversity of biomaterials used in tissue engineering techniques, the application of
>>Chapter 2 Osteoarthritis<<
33
chondrocytes play an essential role for the clinical effectiveness of cartilage
regeneration in most of the tissue engineering therapies. A large number of
chondrocytes required for cartilage reconstruction are usually achieved by multiple
subculturing of cells in vitro. A detailed description of applying chondrocytes in ACI
technique is included in Chapter 1. Although chondrocyte-based therapies have
demonstrated promising cartilage repair at present, further investigation is needed to
evaluate the long-term effectiveness.
CHAPTER 3
RESEARCH PROPOSAL
>>Chapter 3 Research Proposal<<
34
3.1 INTRODUCTION
Chondrocyte is the unique cell type in articular cartilage tissue and essential for the
cartilage formation and functionality. The cells are responsible for the synthesis and
turnover of cartilage extracellular matrix (ECM), which provides an environment of
nutrition diffusion for chondrocytes and endows the joint surface its biomechanical
competence. More recently, cultured chondrocytes have been extensively utilized in
tissue engineering either for orthopaedic application or non-orthopaedic application (Lin
et al., 2006). Hence, it is important to understand the biology of chondrocytes both in
vivo and in vitro.
The de-differentiational process of articular chondrocytes in vivo plays an essential role
in the degenerative disorders of articular cartilage such as OA. In the disease process,
articular cartilage loses its smooth appearance, develops surface fibrillations and erodes
sub-surface layers progressively (Wheater, 1985). The morbid changes lead to joint pain
and progressive limitation of movement at the joint (Piera-Velazquez et al., 2002). It is
thought that the degradation of articular cartilage in OA is resulted from loss of the
balance between anabolic and catabolic pathway involved in the turnover of its EMC
(Clements et al., 2003). Matrix turnover is solely dependent on chondrocytes, which
are believed to be the main site of production of inflammatory mediators in human OA
(Brandt et al., 1998).
Conventional agents of pharmacologic treatments for OA include analgesics,
nonsteroidal anti-inflammatory drugs (NSAIDs), selective cyclooxygenase-2 (COX-2)
inhibitors, glucosamine and chondroitin sulfate, intra-articular glucocorticoids and
visosupplements. All these current treatments have their pros and cons. Confusion,
constipation and increased falls may occasionally occur when patients take analgesics
(Shanmoon and Hochberg, 2000). All NSAIDs are thought to have similar pain
relieving effects, however, side effects, such as renal and gastrointestinal events are
common (Haq et al., 2003). New COX-2 selective inhibitors have equal efficacy to
>>Chapter 3 Research Proposal<<
35
nonselective NSAIDs in the treatment of OA with less gastrointestinal side effect, but it
has been found that COX-2 inhibitors can precipitate vascular events, causing
cardiovascular side effects such as myocardial infarctions (Shamoon and Hochberg,
2000). The evidences of the efficacy of glucosamine and chondroitin sulfate for OA
treatment have been only shown in knee joint OA, and its mechanism remained
unknown (Towheed and Anastassiades, 1999). Intra-articular glucocorticoids and
visosupplements provide significant short-term effect of 2-4 weeks over placebo;
however, it may cause skin atrophy and dermal depigmentation, or severe cartilage
destruction when excessively used (Ayral, 2001; Haq et al., 2003). Hence, novel
pharmacological compounds derived from natural substance may have fewer side
effects and useful for the treatment of OA.
Another advanced treatment for OA might be tissue engineering approach which utilizes
cultured chondrocyte to generate neo-cartilage to replace the degenerative tissue.
Autologous chondrocyte implantation (ACI) technique, which has been successfully
employed to treat patients with articular cartilage defects, uses in vitro explanded
chondrocytes seeded on a biodegradable substance to form cartilage-like tissue
(Brittberg et al., 2001). From 1987 to 2001, ACI was used to treat more than 950
patients with knee joint injuries in Sweden. The continuous follow up of 213
ACI-treated patients was described by Brittberg M. et al., (2001). About 84-90% cases
reported good to excellent results with functional improvement and histological repair
verified by MRI (Brittberg et al., 1994; Brittberg et al., 2001; Ronga et al., 2004). More
recently, the potential of ACI for the treatment of OA has been seriously considered.
The potential achievement of chondrocyte related tissue engineering is promising.
However, plenty of questions remain unclear. Although much attention is focusing on
the creation of an ideal chondrocyte carrier, the proper selection of implanted cell
source is more critical for successful tissue engineering. With the biomaterial being
degraded after implanted, the chondrocyte is the one that sustains functioning and
synthesizes and develops neo tissue in vivo. A comprehensive understanding of the
biology of the employed chondrocytes would be helpful for optimizing the design of
>>Chapter 3 Research Proposal<<
36
tissue engineered cartilage reconstruction. This research will extensively demonstrate
the gene expression pattern of human passaged articular chondrocytes. Several clusters
of genes will be selected for investigation. These include extracellular matrix proteins
(aggrecan, type I collagen, type II collagen, type X collagen, fibromodulin, fibronectin,
and link protein), matrix proteinases (MMP-1, MMP-3, MMP-9, MMP-13, ADAMTS-4
and ADAMTS-5), proteinase inhibitors (TIMP-1, TIMP-2 and TIMP-3), cytokines
(IL-1β, TGFβ, TNFα, and IGF-1), transcription factors (Sox-9, c-fos and c-jun), and
intracellular signaling (COX-2, MAPK1, and NOS2).
In addition, this thesis will explore the potential of several natural compounds on
osteoarthritis. Particularly, salmon calcitonin has gathered moderate interests due to its
potential capability to directly attenuate cartilage degradation. However, the interaction
between salmon calcitonin and human chondrocyte remains unknown.
3.2 HYPOTHESES:
1. The process of chondrocyte de-differentiation is mediated by the alteration of gene
expression profile of chondrocytes during cultivation.
2. Calcitonin promotes chondrocyte function by calcitonin receptor in human articular
cartilage and chondrocyte.
3. Natural compounds such as parthenolide may have suppressive effects on
inflammatory factors and GAG degradation on chondrocytes and thus have
therapeutic potential in patients with OA.
3.3 AIMS:
Therefore, the aims of this project are:
1. To investigate the gene expression pattern of chondrocytes during serially
monolayer expansion, and to compare with the gene profile of the cartilage
specimen.
>>Chapter 3 Research Proposal<<
37
2. To investigate whether human cartilage or chondrocyte expresses calcitonin receptor
and to examine the response of human chondrocyte to the addition of salmon
calcitonin.
3. To screen the potential beneficial effects of natural compounds on human
chondrocytes.
4. To determine the effects of natural compounds on gene expression, GAG
degradation and nitric oxide production by human chondrocytes.
CHAPTER 4
GENE EXPRESSION PROFILES OF CHONDROCYTE DURING
PASSAGED MONOLAYER CULTIVATION
Journal of Orthopaedic Research, 2007
For Peer ReviewGene Expression Profiles of Chondrocytes During Passaged Monolayer
Cultivation
Journal: Journal of Orthopedic Research Manuscript ID: JOR-07-0024.R1
Wiley - Manuscript type: Research Article Date Submitted by the
Author: n/a
Complete List of Authors: Lin, Zhen; The University of Western Australia, Department of Orthopaedic Research Fitzgerald, Jonathan; Massachusetts Institution of Technology, Biological Engineering Division Xu, Jiake; The University of Western Australia, Department of Orthopaedic Research Willers, Craig; The University of Western Australia, Department of Orthopaedic Research Wood, David; The University of Western Australia, Orthopaedic Surgery, Orthopaedic Surgery, School of Surgery and Pathology Grodzinsky, Alan; MIT Zheng, Ming; The University of Western Australia, Orthopaedic Surgery, Orthopaedic Surgery, School of Surgery and Pathology
Keywords: gene expression, chondrocyte, monolayer cultivation, dedifferentiation
John Wiley & Sons, Inc.
Journal of Orthopaedic Research
For Peer Review
1
Gene Expression Profiles of Human Chondrocytes During Passaged Monolayer Cultivation1
2
Zhen Lin1, Jonathan B. Fitzgerald3, Jiake Xu1, Craig Willers1, David Wood1,2, Alan J. Grodzinsky3, Ming H. 3
Zheng1,24
5
Institution: 6
1Orthopaedic Surgery, University of Western Australia, 2Perth Orthopaedic Institution, Perth, Western Australia, 7
Australia8
3Biological Engineering Division, Massachusetts Institution of Technology, Cambridge, Massachusetts, USA9
10
Correspondence:11
Prof. Ming H. Zheng12
Address: Centre of Orthopaedic Surgery, M Block, QEII Medical Centre, Monash Avenue, Nedlands, WA, 6009,13
Australia.14
Telephone: (618) 9346 405015
Fax number: (618) 9346 321016
Email: [email protected]
18
Running title: Gene expression profiles of chondrocytes 19
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Summary20
Chondrocyte phenotype has been shown to dedifferentiate during passaged monolayer cultivation. Hence, we 21
have investigated the expression profile of 27 chondrocyte-associated genes from both osteoarthritic cartilage 22
tissue and healthy passaged human articular chondrocytes by quantitative Real-time PCR. Our results indicate23
that the gene expression levels of matrix proteins and proteases in chondrocytes from monolayer culture 24
decrease compared with those from cartilage tissue, whilst monolayer cultured chondrocytes from normal and 25
osteoarthritic cartilage exhibit similar gene expression patterns. However, chondrocytic gene expression 26
profiles were differentially altered at various stages of passage. The expression of the matrix proteins aggrecan, 27
type II collagen, and fibromodulin inversely correlated with increasing passage number, while fibronectin and 28
link protein exhibited a marked increase with passage. The expression of matrix proteinases MMP-3/9/13 and 29
ADAMTS-4/5 decreased with passage, whereas proteinase inhibitors TIMP-2/3 were elevated. The cytokine 30
IL-1 also showed increased expression with monolayer chondrocyte culture, while IGF-1 expression levels 31
were diminished. No significant changes in TGF-β, or the chondrogenic transcription factors Sox-9, c-fos, or 32
c-jun were observed. Our data indicates that cultured chondrocytes undergo dedifferentiation during monolayer 33
culture, although the gene expression level of transcription factors necessary for chondrogenesis remains34
unchanged. This data may prove important for the future development of more specific and efficacious 35
cultivation techniques for human articular chondrocyte-based therapies.36
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Introduction 37
The cultivation of chondrocytes has been used for many clinical applications, including correction of urinary 38
incontinence, long segmental tracheal reconstruction, facial tissue reconstruction, and the repair of articular 39
cartilage injury [1]. Tissue engineering for cartilage reconstruction requires large numbers of cultured 40
chondrocytes. In the case of autologous chondrocyte implantation (ACI), chondrocytes removed from patient’s 41
are multiplied in vitro by monolayer culture to reach a minimum density of 10 million cells before they are 42
implanted into the cartilage defect [2]. Although accumulating evidence suggest that the clinical outcome of 43
ACI for cartilage defects is promising and has achieved up to 90% good to excellent functional improvement44
[3-5], the biology of the chondrocytes, in particular the stability of the chondrocyte phenotype during in vitro 45
propagation, has not been well elucidated. 46
47
Monolayer culture of chondrocytes is the most common method for cell propagation in vitro as it is economical 48
and technically simple for tissue engineering applications. Although early passage culture of chondrocytes 49
maintains the phenotype of important chondrocyte genes such as type II collagen and aggrecan [6], it has been 50
well documented that chondrocytes in monolayer culture undergo a change in their expression profile with51
increasing passage [7-10]. Indeed, a pioneering experiment by Benya and Shaffer showed that collagen II 52
expression is lost after three passages of monolayer culture [8], but that this differentiated phenotype of cells 53
can be recovered when they are transferred to a three-dimensional culture environment [8, 11-13]. This change 54
of phenotype, termed “dedifferentiation”, describes the conversion of chondrocytes into fibroblastic-like cells. 55
Typically, after repeated monolayer passage the cell structure changes from a spherical to spindle-shaped 56
morphology, and the production of extracellular matrix shifts from type II collagen to type I collagen [6, 8]. 57
Whilst the exact mechanism of this process is poorly understood, a recent study by Darling et al using goat 58
chondrocytes has suggested that this shift in gene expression is initiated in the early stages of in vitro cultivation59
[14]. Furthermore, another variable in this process is the status of differentiation in osteoarthritic (OA) 60
chondrocytes. As chondrocyte expression in known to undergo change with the degeneration of articular 61
cartilage, the impact of osteoarthritis in donor cartilage on ACI is also poorly researched in the literature [15, 62
16].63
64
Hence, the objective of this study was to compare the gene expression profiles of OA human cartilage tissue and 65
cultured chondrocytes, and to investigate the effect of increasing passage on the expression of chondrogenic66
genes in cultured human chondrocytes. The expression patterns of matrix proteins, matrix proteases, protease 67
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inhibitors, transcription factors, cytokine/growth factors, and intercellular signaling factors in human articular 68
cartilage and isolated chondrocytes were determined by Real-time polymerase chain reaction (PCR). 69
70
Materials and Methods71
Tissue Harvest and Cell Culture72
OA human articular cartilage was obtained from patients (n=20) undergoing knee joint replacement surgery at73
Hollywood Private Hospital (Western Australia) and was used for direct tissue RNA extraction or RNA 74
extraction following passage-varied chondrocyte culture. Healthy chondrocyte cultures (n=10) were also 75
isolated from patients having sustained a traumatic cartilage defect to the knee without any signs of 76
osteoarthritis (clinically and radiographically diagnosed), and subsequently underwent Matrix-induced ACI at 77
the same hospital. The acquisition of tissue and cultured chondrocytes was approved by the Human Research 78
Ethics Committee of The University of Western Australia. 79
80
Chondrocyte culture followed protocols used for ACI treatment. Specimens were mechanically dissected in to 81
200-500µm pieces with a scalpel before being enzymatically digested for 6 hours in type II collagenase 82
(400units/ml) in an orbital mixer incubator at 130rpm in 37°C atmosphere. Digestion was stopped by the 83
addition of fetal bovine serum (FBS; TRACE, Sydney, Australia). Tubes were centrifuged twice at 1,500rpm for 84
10 minutes, then washed in serum-free media. Cells were then resuspended in 25cm2 flask at a density of 5×10485
cells/ml in Dulbecco’s modified Eagle’s medium (DMEM/F-12) medium with GlutaMAX (Gibco, New York, 86
USA) supplemented with 20% of FBS, 0.1% Gentamycin and 50µg/ml L-ascorbic acid (Gibco, New York, 87
USA) at 37°C in a humid environment with 5% CO2. Culture medium was changed every two days. Once 88
chondrocyte confluence was achieved, cells were trypsinised into 75 cm2 flasks to obtain greater cell yield. The 89
term passage number is defined as the number of times that a cell population has been trypsinised from the 90
culture flask and undergone subculture. The first culture following the isolation of chondrocytes from cartilage 91
is defined as the primary culture or passage 0 (P0). Chondrocytes were cultured under same conditions for a 92
total of 2, 4 or 6 passages (P2, P4, or P6 respectively). 93
94
Primer Design and Standard Curve Creation95
Primer sequences for all genes were designed using human mRNA information published on the National 96
Center for Biotechnology Information (NCBI) website. Primer3 software and NCBI’s standard and pairwise 97
BLAST programs were used for primer design. A total of 28 primer pairs for the genes were designed with an 98
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amplification product length of 73-226bp, and an annealing temperature of 60°C (Supplementary Table A). 99
Primer-specific standard curves for relative quantification were created by detecting the quantity of serial 100
dilutions of known concentration purified cDNA amplified from each primer set (Supplementary Figures A-C). 101
The experiment was duplicated, and all primers were applied for further investigation only when the 102
concentration-Ct values were linear [17]. Measured threshold cycles (Ct) were converted to relative copy 103
numbers using these standard curves. 104
105
RNA Extraction, Reverse Transcription and Real-time PCR106
Cartilage tissues from the 20 OA patients were divided into two groups. Firstly, cartilage tissue was freeze 107
pulverized in liquid nitrogen followed by direct extraction with the RNAeasy Mini kit (Qiagen Pty Ltd, 108
Australia) according to manufacturer’s instructions. Secondly, following chondrocyte isolation from the 109
remaining tissue, RNA was extracted from cultured chondrocytes at three different passages (P2, P4, and P6) 110
using the Qiagen RNAeasy Mini kit. The same protocol was used for the healthy chondrocyte cultures. The 111
quantity of the RNA was determined by spectrophotometer. RNA was stored at -80°C immediately after 112
extraction until reverse transcription was performed. Complementary DNA was synthesized by using 6µg of 113
RNA from each sample, 600 units of M-MLV reverse transcriptase, 60 units of Rnasin® Ribonuclease Inhibitor, 114
15mM dNTP (Promega Corp, Madison, Wi, USA), and 300µm Oligo dT in a total volume of 60µl. Relative 115
quantitative Real-time PCR was performed on a 384-well/plate ABI Prism 7000 Sequence Detection machine 116
(Applied Biosystems) using Sybr Green PCR Master Mix (Applied Biosystems). The total volume (10µl) of 117
each PCR reaction contained 5µl Sybr Green PCR Master Mix, 2.5µl ddH2O, 1.5µl cDNA, and 5µM of each of 118
the forward and reverse primers. The Real-time PCR reaction was carried out at 95°C for 10 minutes 119
(activation), 40 cycles of 95°C for 15s, 60°C for 20s and 72°C for 20s (amplification), and 72°C for 1 minute 120
(final extension). GAPDH was used as an internal control. 121
122
Data Normalization and Statistical Analysis123
For analysis of the expression data, the primary gene expression data was captured and normalized by the 124
expression level of the housekeeping gene GAPDH [14, 18]. Expression levels greater than two standard 125
deviations from the mean were considered outliers and removed, since the removal of outlier results in less 126
standard deviation with less effect to the means. The means and standard deviations were then calculated by 127
Excel spreadsheet software (Microsoft, U.S.). The mean expression of each gene in the cartilage tissue samples 128
was used to normalize the gene expression profile to emphasize trends rather than overall amplitudes. The gene 129
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expression levels of different passaged groups were then compared by the non-parametric Mann Whitney U test 130
using SPSS (v12.0) statistical software (SPSS Inc., Chicago, U.S.). P values less than 0.05 were considered 131
significant.132
133
Clustering Analysis134
Clustering analysis was performed to further analyze the expression patterns associated with passage number as135
previously described [17, 19]. Each gene expression profile was normalized by the mean of all three passages. 136
The standardized expression profiles were then iteratively clustered using a k means clustering technique with a137
Euclidean distance metric. This facilitated determining the optimal number of groups to capture the distinct 138
trends,139
140
Results141
Gene Expression of OA Cartilage Tissue vs Cultured OA Chondrocytes142
Twenty-nine genes including the house keeping gene GAPDH were investigated by Real-time PCR. Following 143
normalization of the gene expression data to GAPDH, the relative mean expression levels of each gene were 144
compared between cartilage tissues and cultured chondrocytes (2, 4 and 6 passages). As shown in Figure 1, 145
aggrecan, type II collagen, type I collagen, type X collagen, fibromodulin, fibronection and link protein were 146
highly expressed in OA cartilage tissues. Most genes exhibited higher expression levels in the tissue compared 147
to in culture (P<0.001), besides type I collagen, the common index of chondrocyte dedifferentiation, which had 148
elevated gene expression in cultured chondrocytes compared to intact cartilage (P<0.005). 149
150
By comparison, the metalloproteinases MMP-1, MMP-3, MMP-9 and MMP-13, and two members of the 151
ADAMT enzyme family ADAMTS-4 and ADAMTS-5 were ubiquitously expressed in all tissue and culture152
samples investigated (Figure 1). Protease expression levels were much higher in the cartilage tissue than in the 153
passaged culture chondrocytes (P<0.005). However, the expression of their inhibitors showed somewhat varied154
patterns following isolation and passage of cells. To this end, the expression of TIMP-1 was increased less than 155
1 fold in chondrocytes (P<0.05), whilst TIMP-2 and TIMP-3 were transiently up-regulated in culture passage 2, 156
but down-regulated to the same level as the cartilage tissue with further passage. 157
158
Cytokines, IL-1β and IGF-1 were both significantly down-regulated (P<0.001) in cultured chondrocytes (Figure 159
1). The mean expression of TGFβ and COX-2 were not significantly (P>0.05) different in cartilage tissue or 160
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culture. The expression level of NOS2 was also low in cartilage tissue and further decreased in passaged culture 161
chondrocytes (P<0.001). A slight decrease of HSP70 was found in cultured chondrocytes compared with OA 162
cartilage tissue specimens (P<0.001). Furthermore, tissue expression of ribosomal-6P was more than 30-fold163
higher than in cultured chondrocytes (P<0.001). 164
165
In all specimens, the SOX9 transcription factor and components of the AP-1 signaling complex, c-fos and c-jun,166
were down-regulated in the monolayer cultured chondrocytes (P<0.001). However, the MAPk gene expression 167
was found to be up-regulated 20-fold (P<0.001). 168
169
Gene Expression of Cultured OA Chondrocytes vs Normal Chondrocytes170
The differences in the gene expression patterns between normal and OA chondrocytes were examined. The 171
normal chondrocytes were obtained from patients undergoing treatment for traumatic cartilage injury without 172
OA, while OA chondrocytes were obtained from the patients undergoing total knee replacement. Surprisingly, 173
most gene expression profiles between OA and normal monolayer cultured chondrocyte were not significantly 174
different (P>0.05, Figure 2). The expression levels of the matrix proteins and signaling molecules evidenced175
negligible differences between the two groups. However, ADAMTS-4 was significantly down-regulated in the 176
OA chondrocyte (P<0.001), whilst Cox-2 (P<0.05) and NOS2 (P<0.005) were both significantly up-regulated. 177
178
Gene Expression of Passaged OA Chondrocytes on Monolayer179
The expression level of aggrecan and fibromodulin remained constant between P2 and P4, but significantly 180
decreased at P6 (Figure 3). Type II collagen also showed decreased expression during monolayer cultivation;181
whilst Type I collagen showed no significant change from P2 to P6 (Figure 3). Interestingly, Type X collagen 182
increased expression during in vitro expansion, and the level of fibronectin and link protein dramatically 183
increased after P4 (Figure 3).184
185
Additionally, the gene expression levels of the matrix proteinases all had decreased expression in the later 186
passage of cultivation (Figure 4). In particular, ADAMTS-5 showed a monotonic decrease, while MMP-1 and 187
MMP-3 increased expression from P2 to P4, but then decreased expression to P6. MMP-13 and ADAMTS-4 188
remained constant to P4, but was decreased dramatically to P6. No significant change in TIMP-1 gene 189
expression pattern was shown, whilst TIMP-2 and TIMP-3 were significantly up-regulated with increased 190
passage. Interestingly, the gene expression levels of the three transcription factors, Sox-9, c-fos, and c-jun 191
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showed negligible change during passaged culture (Figure 4).192
193
Cytokine IL-1β expression in cultured chondrocytes was elevated with increasing passage, with greatest 194
elevation after P4 (Figure 5). The expression level of IGF-1 was down-regulated with increasing passage. By 195
comparison, TGF-β showed a similar temporary increase in expression at P4, but no significance was found 196
overall from P2 to P6. Intracellular signaling markers COX-2 and MAPk-1 exhibited a down-regulated 197
expression profile. NOS-2 had extremely low expression during in vitro culture, but showed a temporary 198
increase at P4. The two reference genes, HSP70 and ribosomal-6P, had contrasting results, with HSP70 showing 199
increased expression at P6, and ribosomal-6P constantly down-regulated by increasing passage (Figure 5).200
201
Cluster Analysis of Gene Profiles202
Cluster analysis revealed that the genes were best divided using 3 groups to demonstrate the three main 203
expression patterns of investigated genes with respect to increasing passage number (Figure 6). Cluster 1 204
grouped TIMP-2, TIMP-3, TGFβ, COX-2 and MAPk1, which peak at P2 and remain transience after that. 205
Cluster 2 included the majority of the genes (aggrecan, type II collagen, type X collagen, fibromodulin, 206
fibronectin, link protein, MMP-1, MMP-3, MMP-9, MMP-13, ADAMTS-4, ADAMTS-5, SOX9, c-fos, c-jun, 207
IL-1β, IGF1, NOS2, HSP70 and ribosomal). Most were suppressed in culture. Cluster 3 contains type I collagen 208
and TIMP-1, which were stably decreased with the increasing passage number. 209
210
Discussion211
Current treatments for chondral injury primarily target pain relief and the preservation of joint function via both 212
pharmacological and surgical approaches. In particular, recent interest has been targeted at ACI as a therapeutic213
approach for OA patients since it has already been successfully used for repairing full-thickness cartilage 214
defects [1-5, 20]. In this study, the gene expression profiles of normal and OA chondrocytes were compared. 215
Surprisingly, little significant difference was observed between the two groups, suggesting that the in vitro 216
biosynthetic profile of the chondrocyte is influenced more by the culture conditions, than the disease state of the 217
donor cartilage. 218
219
The clinical application of tissue engineered cartilage requires large numbers of chondrocytes derived from a 220
small cartilage biopsy. Digestion of cartilage and isolation of chondrocytes are both essential to promote cell 221
proliferation [21, 22]. In this study, we have shown that the gene expression profile of chondrocytes 222
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dramatically shifts once entering monolayer culture; whilst chondrogenic transcription factors such as Sox-9 223
remain unchanged. The observed up-regulation of type I collagen and the down-regulation of type II collagen in 224
chondrocytes is consistent with the well-known dedifferentiation phenomenon whereby chondrocytes lose their225
differentiated phenotype and become fibroblastic in monolayer culture. The transcription factor Sox9, 226
considered as a crucial factor in chondrogenesis and cartilage formation in vivo, may not correlate with 227
chondrocyte dedifferentiation during in vitro cultivation [23]. This stable expression of Sox9 in serially 228
passaged chondrocytes suggests that other transcription factors may govern the dedifferentiation process, and 229
that it may be reversible [24-27]. 230
231
Chondrocytes maintain a homeostatic anabolic and catabolic balance of extracellular matrix (ECM) via 232
regulation of secreted matrix proteinases and protease inhibitors to maintain the composition and structural 233
integrity of ECM in articular cartilage [28, 29]. In cultured chondrocytes, the expression levels of the matrix 234
proteinases diminished as compared to that of intact cartilage, however, inhibitors of matrix proteinases 235
exhibited fewer changes. This suggests that cultured chondrocytes have less catabolic activity. This is possibly 236
due to the lack of three-dimensional extracellular matrix microenvironment in the monolayer culture system, or237
that cartilage dissected from the OA joint already had high MMP and ADAMT levels due to the arthritic 238
pathogenesis of the tissue. Inflammatory cytokines such IL-1β and NOS2 are both lower in the gene expression 239
levels of chondrocyte compared to cartilage tissue, which may also subsequently down-regulate the matrix 240
proteinases. Contradictory to our study, Bau et al. has reported that many enzymes had significantly higher 241
expression levels in cultured chondrocytes than in tissue-extracted. However, both the alginate bead and high 242
density culture systems used in their study model have been shown to re-differentiate chondrocytes and 243
up-regulate gene expression [8, 18]. Unfortunately, due to the limitation of healthy chondrocyte donors, we 244
could not include a high density culture control. The variations in gene expression trends between cultured 245
chondrocytes with different cultivation methods suggests that gene expression levels in isolated chondrocytes246
are tightly associated with culture conditions, a factor which may affect the biological properties and hence 247
functionality of chondrocytes. 248
249
When cells are maintained in vitro, their biological activity and gene expression pattern are mostly affected by 250
activated components in the culture medium and the cytokines and growth factors released by the cells. The 251
significant alteration in the level of IL-1β and IGF-1 mRNA in passaged chondrocytes was confirmed by both 252
non-parametric testing and clustering analysis. Interleukin-1β is thought to be one of the major catabolic 253
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pro-inflammatory cytokines relevant to articular destructive diseases [29, 30]. It has also been shown to play a 254
major role in the loss of the differentiated chondrocyte phenotype (dedifferentiation), and alter other cartilage 255
destructive processes [31]. Human IL-1β suppresses the synthesis of hyaline-specific type II collagen, and 256
increases the synthesis of fibroblastic type I and III collagens in human chondrocytes [30]. This 257
dedifferentiation process is though to be due, at least in part, to the up-regulation of IL-1β [32], although we 258
could not rule out the possibility that the profile of IL-1β may also relate to the nature of the donor OA cartilage. 259
TGFβ is also an important pro-inflammatory factor in degenerative joint disease. In our study, TGFβwas 260
constantly expressed, however remain quiescent during chondrocyte dedifferentiation. Similarly, Goessler et al., 261
have suggested that TGF-β1/2 might not be involved in the chondrocyte dedifferentiation process [33]. In 262
addition, IGF-1, a member of the IGF family of peptide hormones believed to induce chondrocyte 263
differentiation and proliferation [34], was found to inhibit the dedifferentiation and apoptosis of articular 264
chondrocytes induced by nitric oxide [35]. Consistent with this finding, our data revealed that IGF-1 was 265
down-regulated in chondrocytes with increasing passage (and dedifferentiation), suggesting that IGF-1 gene 266
expression in passaged monolayer chondrocytes may play a role in protecting the differentiated phenotype 267
human chondrocytes. 268
269
Based on these preliminary data, we conclude that the gene expression profile of the chondrocyte is largely 270
dependent on its cellular passages during cultivation. Chondrocytes demonstrate a rapid change in gene 271
expression profile when isolated from cartilage tissue and placed into a monolayer culture system. The 272
chondrocyte has a differential gene expression profile with increasing passage number, suggesting a varied273
degree of differentiated phenotypes of implanted cells may occur in ACI therapies for articular cartilage repair.274
The further understanding of chondrocyte dedifferentiation in passaged monolayer culture may help to generate 275
new approaches for cultivating more phenotypically stable chondrocytes and/or re-differentiating chondrocytes 276
for increased therapeutic gain. 277
278
Acknowledgement:279
This work is supported by a development grant from National Health Medical Research Council Australia and 280
Verigen/Genzyme Australia. Real-time PCR analysis was done in the Australian Neuromuscular Research 281
Institute, University of Western Australia. The authors would like to thank Dr. Nathan J. Pavlos for his 282
assistance with manuscript preparation. 283
284
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Figure Legends:369
Figure 1. Comparison of gene expression between OA cartilage and monolayer cultured OA chondrocyte: 370
The relative gene expression levels of chondrocytes after 2, 4 or 6 passages were combined for comparison. 371
Mean ± S.E.M. Mann-Whitney U test significance values were * p<0.05, ** p<0.01 and *** p<0.005.372
373
Figure 2. Comparison of gene expression between cultured OA chondrocyte and normal chondrocyte.374
Mean ± S.E.M. Mann-Whitney U test significance values were * p<0.05, ** p<0.01 and *** p<0.005.375
376
Figure 3. Gene expression profiles of matrix proteins for monolayer passaged human chondrocytes. The 377
cells were isolated from human OA cartilage and serially subcultured on monolayer. RNA was extracted from 378
chondrocytes of passage 2, 4 and 6. Real-time PCR was used to quantify the expression levels. The x-axis 379
represents increasing passage number where P2=passage 2 (n=20), P4=passage 4 (n=17), and P6=passage 6 380
(n=14). The y-axis represents the relative expression level of each passage related to the mean of all three 381
passages (mean expression level of each gene normalized to 1). Mann-Whitney U test significance values were 382
* p<0.05, ** p<0.01 and *** p<0.005. 383
384
Figure 4. Gene expression profiles of matrix protease, metalloproteinase inhibitor and transcription 385
factors for monolayer passaged human chondrocytes. The expression trends of the assessed matrix 386
proteinases decreased with increasing passage. In particular, the extracellular matrix proteinases, MMP-13, 387
ADAMTS-4, and ADAMTS-5 showed significant reduction in gene expression with increasing passage. 388
TIMP-2 and TIMP-3 expression were significantly upregulated with increasing passage to P6. 389
390
Figure 5. Gene expression profiles of cytokines, growth factors and intracellular signaling markers for 391
monolayer passaged human chondrocytes. IL-1β exhibited increased expression with increasing passage, 392
whilst IGF-1 showed reduced in expression at P6. TGF-β evidenced no change in expression with passage. 393
COX-2 gene expression was significantly reduced with increasing passage. Ribosomal RNA was uniformly and 394
significantly down-regulated with increasing passage.395
396
Figure 6. Projection plot of the genes represented by the three main principal components. The 397
standardized gene expression vectors were projected onto the three main principal components found using 398
principal component analysis, with groupings found using k means clustering. Group 1: TIMP-2, TIMP-3, TGF399
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β, COX-2 and MAPk1; group 2: aggrecan, type II collagen, type X collagen, fibromodulin, fibronectin, link 400
protein, MMP-1, MMP-3, MMP-9, MMP-13, ADAMTS-4, ADAMTS-5, SOX9, c-fos, c-jun, IL-1β, IGF1, 401
NOS2, HSP70 and ribosomal; group 3: type I collagen and TIMP-1. 402
403
Supplementary Table A: Human primer sequences for examined genes using relative quantitative Real-time 404
PCR (annealing temperature ~60°C).405
406
Supplementary Figures A-C: Standard curves for Real-time PCR. A standard curve was generated from a 407
dilution series of known concentration purified DNA. The DNA samples were amplified by Real-time PCR, and 408
a Ct value was collected. Log-linear standard curves were created by plotting relative copy number on the 409
Y-axis and the Ct value on the X-axis.410
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Figure 1. Comparison of gene expression between OA cartilage and monolayer cultured OA chondrocyte: The relative gene expression levels of chondrocytes after 2, 4 or 6
passages were combined for comparison. Mean S.E.M. Mann-Whitney U test significance values were * p<0.05, ** p<0.01 and *** p<0.005.
150x105mm (300 x 300 DPI)
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Figure 2. Comparison of gene expression between cultured OA chondrocyte and normal chondrocyte. Mean S.E.M. Mann-Whitney U test significance values were * p<0.05, **
p<0.01 and *** p<0.005. 150x99mm (300 x 300 DPI)
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Figure 3. Gene expression profiles of matrix proteins for monolayer passaged human chondrocytes. The cells were isolated from human OA cartilage and serially subcultured on monolayer. RNA was extracted from chondrocytes of passage 2, 4 and 6. Real-time
PCR was used to quantify the expression levels. The x-axis represents increasing passage number where P2=passage 2 (n=20), P4=passage 4 (n=17), and P6=passage 6 (n=14).
The y-axis represents the relative expression level of each passage related to the mean of all three passages (mean expression level of each gene normalized to 1). Mann-Whitney
U test significance values were * p<0.05, ** p<0.01 and *** p<0.005. 199x119mm (300 x 300 DPI)
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Figure 4. Gene expression profiles of matrix protease, metalloproteinase inhibitor and transcription factors for monolayer passaged human chondrocytes. The expression trends of the assessed matrix proteinases decreased with increasing passage. In particular, the extracellular matrix proteinases, MMP-13, ADAMTS-4, and ADAMTS-5 showed significant
reduction in gene expression with increasing passage. TIMP-2 and TIMP-3 expression were significantly upregulated with increasing passage to P6.
199x158mm (300 x 300 DPI)
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Figure 5. Gene expression profiles of cytokines, growth factors and intracellular signaling markers for monolayer passaged human chondrocytes. IL-1 exhibited increased
expression with increasing passage, whilst IGF-1 showed reduced in expression at P6. TGF- evidenced no change in expression with passage. COX-2 gene expression was
significantly reduced with increasing passage. Ribosomal RNA was uniformly and significantly down-regulated with increasing passage.
199x117mm (300 x 300 DPI)
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Figure 6. Projection plot of the genes represented by the three main principal components. The standardized gene expression vectors were projected onto the three main principal components found using principal component analysis, with groupings found using k means clustering. Group 1: TIMP-2, TIMP-3, TGF , COX-2 and MAPk1; group 2: aggrecan, type II collagen, type X collagen, fibromodulin, fibronectin, link
protein, MMP-1, MMP-3, MMP-9, MMP-13, ADAMTS-4, ADAMTS-5, SOX9, c-fos, c-jun, IL-1 , IGF1, NOS2, HSP70 and ribosomal; group 3: type I collagen and TIMP-1.
119x89mm (300 x 300 DPI)
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CHAPTER 5
EVIDENCE THAT HUMAN CARTILAGE AND CHONDROCYTE
DO NOT EXPRESS CALCITONIN RECEPTOR
Osteoarthritis and Cartilage, 2007
Evidence that human cartilage and chondrocytes do not expresscalcitonin receptorZ. Lin M.B.y, N. J. Pavlos B.Sc. (Hons.), Ph.D.y, M. A. Cake B.VMS. (Hons.), Ph.D.z,D. J. Wood B.Sc., M.B.B.S., M.S., F.R.C.S., F.R.A.C.S.y, J. Xu M.D., Ph.D.yand M. H. Zheng Ph.D., D.M., A.R.C.P.A., F.R.C.Pathy*yCentre for Orthopaedic Research, School of Surgery and Pathology,University of Western Australia, Western Australia, AustraliazSchool of Veterinary and Biomedical Sciences, Murdoch University, Western Australia, Australia
Summary
Objective: Calcitonin (CT) has been recently shown to exhibit direct protective effects on articular cartilage against joint degenerative disease.It has been proposed that CT might act via the CT receptor (CTR) to activate the cyclic AMP (cAMP) pathway and protect type II collagendegradation. In this study, we investigated the existence of CTR in human articular cartilage and chondrocytes, and examined the potentialpharmacological effects and transduction pathway of salmon CT (sCT) in human chondrocytes.
Methods: Five human articular cartilage samples were examined for the expression of the CTR by polymerase chain reaction (PCR), immuno-staining and Western blot analysis. cAMP levels in human chondrocyte stimulated with sCT were assessed by ELISA. The effect of sCT on thegene expression profiles, including aggrecan, type II collagen, MMP-1, MMP-3 and MMP-13, of human chondrocytes was also examined byrelative quantitative Real-time PCR.
Results: We failed to detect the CTR at both the transcriptional and protein levels in human chondrocytes and cartilage tissue by PCR, im-munostaining and Western blotting. cAMP levels were significantly elevated in human chondrocytes by forskolin (100 mM) to more than 10-fold(P< 0.001), however, were not induced by sCT (10�7 M, 10�8 M, 10�9 M). Real-time PCR analysis demonstrated that sCT slightly reduced thegene expression of MMPs, although this effect was not statistically significant.
Conclusion: In contrary to previous reports, our data indicate that human cartilage and chondrocytes do not express CTR. Furthermore, sCTdoes not appear to have direct effects on human chondrocytes. We propose that the chondroprotective effect of CT observed in vivo may beindirect via its impact on subchondral bone resorptive activity of osteoclasts.ª 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Key words: Calcitonin, Cartilage, Chondrocyte, Subchondral bone, Osteoarthritis.
Introduction
Calcitonin (CT) is a 32-amino acid peptide hormone whichregulates serum calcium levels. The principal function ofCT is to maintain calcium homeostasis by directly inhibitingosteoclast-mediated bone resorption and enhancing renalcalcium excretion1e3. It is well established that CT can beused for the treatment of osteoporosis and Paget’s disease,principally owning to its inhibitory effect on osteoclasticbone resorption, regulation of mineral metabolism and anti-nociceptive properties4. Recent interest has focused on itsapplication in the treatment of osteoarthritis (OA). CT hasbeen shown to be effective in preventing cartilage degrada-tion and relieving pain, suggesting that it might be an idealpharmacological treatment for OA5,6. Clinical trials of patientswith spinal collapse and knee OA suggest that CT has an anti-nociceptive effects, as evidenced by the reduced consump-tion of analgesic drugs7,8. Interestingly, postmenopausal
women showed a reduction in the excretion of biochemicalmarkers of cartilage degradation following 3 months of CTtreatment9. Furthermore, in a double-blind clinical trial of pa-tients with knee OA, a 1-mg dose of salmon CT (sCT) signif-icantly decreased Lequesne’s algofunctional index scores,and the levels of serum and urinary C-terminal peptide oftype II collagen. Taken together, these data suggest that inaddition to symptom-modifying effects, CT may also improvearticular joint function by attenuating cartilage degradation10.Hence, direct chondroprotective and chondrotheraputicpotential of CT have been proposed11.
Inspired by the clinical observation, a number of studieshave recently been conducted to reveal its physiological im-pact on cartilage and chondrocytes. Studies by Sonder-gaard et al. (2006) demonstrated the existence of CTreceptor (CTR) in bovine cartilage by immunohistochemis-try and suggested that CT may directly inhibit collagenaseactivity by binding to CTR and activating the cyclic AMP(cAMP) pathway, causing a reduction in type II collagendegradation in bovine cartilage12. Similarly, it has been re-ported that CT stimulates calcium efflux, increases cAMPlevel by 2- to 3-fold, and enhances collagen synthesis inrabbit chondrocyte cultures13. Studies employing isolatedchondrocytes or cartilage explants from animals have also
*Address correspondence and reprint requests to: M. H. Zheng,Centre for Orthopaedic Research, M Block, QEII Medical Centre,Monash Avenue, Nedlands, Western Australia 6009, Australia.Tel: 61-8-9346-4050; E-mail: [email protected]
Received 17 April 2007; revision accepted 4 August 2007.
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demonstrated that CT promotes proteoglycan synthesis,chondrocyte proliferation, cartilage formation and matura-tion14e19. In humans, addition of CT to chondrocyte clustercultures increased type II collagen and proteoglycan con-tent in culture medium, but did not enhance cell prolifera-tion16. Other reported effects of CT include an increase ofphospholipase A2 (PLA2) activity, an inflammatory enzymein OA, and a decrease of collagenolytic activity in isolatedhuman OA chondrocytes in vitro20.
Although several positive effects of CT treatment onchondrocytes have been demonstrated, the biologicalmechanism underlying its potential direct effects on humancartilage and chondrocytes remains unclear. It is well ac-cepted that CT acts through the binding to the CTR andsubsequently elicits adenylate cyclase activation. Previousstudies indicate that CTR is expressed in a wide numberof tissues including the central nervous system, ovary, tes-tis, renal epithelial cells and osteoclast (Quinn et al., 1999).Recently, Sondergaard et al. (2006) demonstrated the pres-ence of CTR in bovine cartilage tissue12, however, the pres-ence of CTR in human articular cartilage tissue has notbeen confirmed. In this study, we investigated the expres-sion of CTR in human cartilage and isolated chondrocytesand examined whether CT has a direct functional role inhuman articular chondrocytes.
Methods and materials
TISSUE HARVEST
Human articular cartilage from specimens of femoral condyle was ob-tained from patients (N¼ 5, average age¼ 67.4� 3.8) undergoing totalknee replacement surgery at the Hollywood Private Hospital, Western Aus-tralia. This tissue was used for RNA extraction and chondrocyte culture. Nor-mal articular cartilage tissues (N¼ 3) were obtained from the non-OApatients undergoing arthroscopy for autologous chondrocyte implantationfor knee injury. The acquisition of human tissue was approved by the HumanResearch Ethics Committee of the University of Western Australia.
CHONDROCYTE CULTURE
Chondrocytes were isolated from fresh cartilage tissue and cultivatedin vitro. Specimens were manually dissected into 200e500 mm pieces beforeundergoing enzymatic digestion by type II collagenase (400 units/ml) in an or-bital mixer incubator at 130 rpm at 37�C for 6 h. Matrix digestion was termi-nated by the addition of fetal bovine serum (FBS). Tubes were centrifugedat 1500 rpm for 8 min then washed twice in serum-free medium. Cells werethen resuspended in a tissue culture flask at a density of 5� 104 cells/mland maintained in Dulbecco’s modified Eagle’s medium (DMEM/F-12; Gibco,New York, USA) supplemented with 20% fetal calf serum (FCS; TRACE, Syd-ney, Australia), 0.1% gentamycin and 50 mg/ml L-ascorbic acid (Gibco, NewYork, USA) at 37�C in a humid environment with 5% CO2. The culture mediumwas changed every 2 days. Once chondrocyte confluence was achieved, cellswere either lysed for further RNA/protein extraction or harvested for passageusing trypsin. Primary chondrocytes were used for CTR detection by polymer-ase chain reaction (PCR) and Western blotting, whereas sCT treatment waspreformed on first passage subcultured chondrocytes.
RNA EXTRACTION AND REVERSE TRANSCRIPTION RT-PCR
Qiagen RNeasy� Mini kit (Qiagen) was used to extract RNA from humancartilage tissue and isolated human chondrocytes. Cartilage tissues werepulverized in liquid nitrogen followed by RNA extraction with the RNeasy�
Mini kit according to the manufacturer’s instruction. Cells were washed with1� phosphate buffered saline (PBS) and processed according to the kit’s in-struction. RNA from human giant cell tumor (GCT) tissue was also extractedand served as a source of human osteoclasts and thus a positive control.First strand synthesis was carried out using 2 ml of RNA from each sample,600 U of M-MLV reverse transcriptase, 60 U of RNasin� ribonuclease inhib-itor, 15 mM dNTP (Promega), and 300 mm Oligo dT in a total volume of 20 ml.PCR was performed using 2 ml of the first strand product in a 20-ml reactionvolume containing 10 ml of 2� GoTaq Green Mix (Promega Corp., Madison,WI, USA), 1 ml of each primer (CTR: forward: 50-CTGGGAATCCAGTTTGTCGT-30, reverse: 50-CGCTGGTTCCACTGAATTTT-30; CRLR: forward:
50-AACAGAACCTGGGATGGATG-30, reverse: 50-TCTGTTGCTTGCTGGATGTC-30). The reaction mixture was heated at 95�C for 3 min to denaturethe DNA and then subjected to 35 cycles of PCR (94�C for 40 s, 60�C for40 s, 72�C for 40 s) followed by a final extension at 72�C for 7 min. House-keeping gene 18S was used as an internal control. PCR products wereanalysed by electrophoresis in 2% agarose gel.
WESTERN BLOT ANALYSIS OF CTR
Western blot analysis was used to confirm the expression of CTR in hu-man cartilage. Cells were seeded in a T25 flask and grown to confluence.Primary chondrocytes were then harvested and lysed using modified Ripalysis buffer (5% Tris/pH¼ 7.5, 15% NaCl, 1% Nonidet P-40/IGEPAL, 0.1%sodium dodecyl sulphate, and 1% deoxycholate) supplied with Sigma-FAST� protease inhibitor solution (1:100; SigmaeAldrich, UK). GCT tissuewas used as positive control in the assay. Thirty micrograms of each proteinlysate were electrophoresed onto a nitrocellulose gel and probed witha 1:500 dilution of primary rabbit polyclonal anti-CTR antibody (Abcam,ab11042) and secondary rabbit IgG. Alpha-tubulin (1:2000; SigmaeAldrich,UK) was utilized as an internal control. The membrane was then exposedto Western blotting detection reagents (Amersham, UK) and visualized byexposure to an LAS-3000 imaging system (Fuji Photo Film Co., Ltd., Japan).
IMMUNOSTAINING FOR CTR
Immunohistochemistry was performed on human giant cell tumour (hGCT)of bone, which is known to express CTR21 and normal human articular cartilagesections to further assess the presence of CTR. GCT tissue was moistenedwith PBS and evenly imprinted onto glass slides. Imprints were then fixed in4% paraformaldehyde for 20 min at room temperature. The cartilage tissuewas fixed in 4% paraformaldehyde, embedded with paraffin, and subsequentlysectioned. Paraffin embedded sections were treated with 0.25% trypsin to en-hance antigen presentation, followed by deparaffinization and rehydration.Each slide was then blocked for endogenous peroxidase activity by incubationin 3% H2O2 for 10 min, washed in Tris-buffer saline (TBS), and then pre-incu-bated with 20% FBS for 30 min followed by incubation with a 1:100 dilution ofthe primary anti-CTR antibody at 4�C in a humidified chamber overnight. Slideswere subsequently incubated with anti-rabbit Ig antibody (Dako Pty Ltd., Aus-tralia) followed by three 5-min washes in TBS, then color developed for 5 min ina moist chamber at room temperature using a liquid 3,30-diaminobenzidine(DABþ) substrateechromogen solution kit (Dako Pty Ltd., Australia). Thepreparation in which the primary antibody to CTR was omitted served as a neg-ative control. Slides were counterstained in hematoxylin and mounted in De-pex mounting medium. GCT imprints were co-stained with tartrate-resistantacid phosphatase (TRAP) to identify the osteoclast-like giant cells.
cAMP ELISA ASSAY
It is well established that CT elevates intracellular cAMP in various celltypes. In this study, we employed an Enzyme Immunoassay kit (Assay De-signs Inc., Michigan, USA) to detect intracellular cAMP levels in chondro-cytes after sCT stimulation. Human chondrocytes (passage 1) werecultured in 24-well plates and maintained in serum-free culture medium for12 h. Cells were then pretreated with 100 mM of cAMP phosphodiesterase in-hibitor 3-isobutyl-1-methylxanthine (IBMX; SigmaeAldrich, UK) for 20 min,and subsequently stimulated with sCT (10�7 M, 10�8 M, 10�9 M), or100 mM forskolin (SigmaeAldrich, UK) for 30 min. The cAMP contents fromsupernatant and cell lysis were measured using the cAMP Enzyme Immuno-assay kit (Assay Designs Inc., Michigen, USA) according to the manufactur-er’s instructions. The optical density was read at 405 nm in a POLARstar�
OPTIMA multifunction microplate reader (BMG Labtech, Germany). The con-centration of cAMP was calculated from the raw data using a cAMP standardcurve. All experiments were carried out in triplicate.
REAL-TIME PCR AND DATA ANALYSIS
To measure the gene expression profile of chondrocytes following sCTtreatment, relative quantitative Real-time PCR was performed on a 384-well plate ABI Prism 7000 Sequence Detection machine (Applied Biosys-tems, USA) using SYBR Green PCR Master Mix (Applied Biosystems,USA). Human chondrocytes at first passage were seeded on a 6-well plateand pretreated with sCT at 10�8 M for 24 h, then collected for RNA extractionand reverse transcript reaction. The total volume of each PCR reaction was10 ml, which contained 5 ml of SYBR Green PCR Master Mix, 2.5 ml of ddH2O,1.5 ml of cDNA, and 5 mM of each of the forward and reverse primers(Table I). Real-time PCR was carried out at 95�C for 10 min (activation),40 cycles of 95�C for 15 s, 60�C for 20 s and 72�C for 20 s (amplification)and 72�C for 1 min (final extension). Housekeeping gene (18S) was alwaysincluded in the same plate as an internal control. A cycle threshold (Ct) valuewas obtained for each sample and analysed by the Sequence Detection Sys-tem software (Applied Biosystems). The comparative 2�DDCT method was
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used to calculate the relative expression of each target gene as previouslydescribed22. Briefly, mean Ct value of target genes in the treated groupwas normalized to its averaged Ct values of the housekeeping gene 18Sto give a DCt value, which was further normalized to control samples to ob-tain a DDCt value. The untreated group was used as reference sample.
STATISTICAL ANALYSES
Data are presented as means� SEM. Statistical analysis was performedby Student’s t test using the SPSS12.0 software (SPSS Inc., USA). P valuesless than 0.05 were considered significant.
Table IPrimer sequences used for Real-time PCR (annealing temperature: 60(C)
Gene Accession no. Primer sequences Size
Aggrecan M55172 Fw*: TCCCCTGCTATTTCATCGAC 117rvy: CCAGCAGCACTACCTCCTTC
Type II Collagen NM_001844 fw: CTGGAAAAGCTGGTGAAAGG 105rv: GGCCTGGATAACCTCTGTGA
MMP-1 NM_002421 fw: AGGTCTCTGAGGGTCAAGCA 82rv: CCAGGTCCATCAAAAGGAGA
MMP-3 NM_002422 fw: GAAGCTGGACTCCGACACTC 88rv: GATCCAGGAAAGGTTCTGA
MMP-9 NM_004994 fw: GATGCGTGGAGAGTCGAAAT 87rv: CTATCCAGCTCACCGGTCTC
MMP-13 NM_002427 fw: TTGAGCTGGACTCATTGTCG 84rv: TGCAAACTGGAGGTCTTCCT
18S X03205 fw: CCTGCGGCTTAATTTGACTC 119rv: AACTAAGAACGGCCATGCAC
*Forward.
yReverse.
Fig. 1. The mRNA expression of CTR in human cartilage and chondrocytes. (A) Primary human chondrocytes (passage 0) cultured on mono-layer with spherical phenotype. (B) The gene expression of CTR in five individual human cartilage tissue samples and cultured chondrocytes.Articular cartilage samples were taken from OA patients undergoing total joint replacement. Chondrocytes were isolated from human cartilageand cultured in vitro. RNA extracted from human GCT served as positive control. CTR (200 bp) is expressed in human GCT, but not in any of
the human cartilage tissue or isolated chondrocytes. Housekeeping gene 18S served as internal control.
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Results
IDENTIFICATION OF THE CTR IN HUMAN CARTILAGE
AND CHONDROCYTES
As an initial effort to investigate whether human chondro-cytes express CTR in vitro or in vivo, RT-PCR was used todetect the expression of CTR mRNA in human articular car-tilage and isolated chondrocytes [Fig. 1(A)]. Primer se-quences were specifically designed and blasted to matchthe human CTR sequence. Given that two human CTR iso-forms (C1a and C1b) have been identified23,24, the PCRprimers for human CTR were selected to flank the consen-sus region of all given splice variants. As shown in Fig. 1(B),the expression of CTR was clearly detected in human oste-oclast-like cells derived from GCT, however, no messagewas observed in human cartilage and chondrocyte sampleseven under saturating conditions of PCR cycling (i.e., 35cycles). 18S was served as a housekeeping gene to ensureequivalent loading of samples.
Next, we examined for the expression of CTR at the pro-tein level in human chondrocytes and cartilage tissues. Forthis purpose, a rabbit polyclonal antibody to human CTRwhich, recoginises an epitope within the cytoplasmic do-main that is identical to both C1a and C1b isoforms, wasused. Consistent with our PCR results, Western blot analy-ses revealed abundant expression of human CTR in hGCT(56.9 kDa), but not in the primary chondrocytes at equiva-lent protein levels (Fig. 2). Western blot analysis conductedon the receptor activator of nuclear factor-kappa B ligand(RANKL) stimulated RAW264.7 cell cultures, which areknown to abundantly express CTR25, showed very weakexpression levels 5 days post-culture (data not shown),
suggesting that this antibody is preferentially specific to hu-man but not to mouse CTR.
Immunohistochemical analysis further confirmed the ab-sence of CTR expression in both chondrocytes and cartilagetissues. As shown in Fig. 3, osteoclast-like giant cells fromhGCT imprints stained positively for CTR [Fig. 3(A)]. Osteo-clast-like cells were confirmed by positive reaction to TRAP[Fig. 3(B)]. CTR immunostaining was predominantly local-ized to the osteoclast cell surface and appeared highly spe-cific. When the primary antibody was omitted, no specificstaining was detected in osteoclasts [Fig. 3(C)]. Analysis ofhuman joint tissue by immunohistochemical staining withanti-CTR antibody revealed no specific CTR staining in eithercartilage or chondrocytes [Fig. 3(D, E)], whilst multinucleateosteoclasts in the subchondral bone region (indicated byarrow) were clearly stained with CTR antibody [Fig. 3(F)].
EXPRESSION OF CTR-LIKE RECEPTOR IN HUMAN
CHONDROCYTES
Given that CT has been previously shown to effect chon-drocytes and cartilage, we next explored the possibility thatit might act through an alternative receptor on chondro-cytes. The CTR-like receptor (CRLR) exhibits more than50% identity with CTR, therefore we studied the expressionof CRLR in human cartilage and chondrocytes by RT-PCR.The same tissue and cultured chondrocyte samples weresubjected to PCR using primers specific to CRLR. GCTRNA was used as a positive control. As shown in Fig. 4,CRLR was absent in all human cartilage samples, however,CRLR was weakly expressed in expanded chondrocyte cul-tures. This expression became more evident in late pas-saged chondrocytes suggesting its expression correlatedwith chondrocyte de-differentiation in vitro.
INTRACELLULAR cAMP CONCENTRATION
Having clearly demonstrated that both human cartilageand chondrocytes lack CTR, we next asked whether CT di-rectly effected chondrocytes in vitro. To this end we employeda cAMP assay to measure intracellular responses to CT. Asshown in Fig. 5, sCT failed to elicit a elevation in cAMP in hu-man chondrocytes over a range of concentration (10�7 M,10�8 M, or 10�9 M). On the other hand, forskolin (100 mM),a non-receptor specific cAMP activator, clearly elevatedcAMP levels in chondrocytes by more than 10-fold.
EXAMINATION OF THE EFFECTS OF sCT ON THE GENE
EXPRESSION OF HUMAN CHONDROCYTES
To further investigate whether CT has any direct effect onhuman cartilage or chondrocytes, human chondrocyteswere treated with sCT at a dose of 10�8 M for 24 h, andReal-time PCR was used to study the expression of variouscartilage extracellular matrix proteins (aggrecan and type IIcollagen) and several Matrix Metalloproteases (MMP-1,MMP-3, MMP-13). As shown in Fig. 6, the matrix moleculesaggrecan and type II collagen showed similar levels of geneexpression with or without sCT treatment. sCT slightly de-creased the expression of MMPs, however, this effect wasminimal and did not reach statistical significance (P> 0.05).
Discussion
The present study was prompted by results of re-cent clinical studies, suggesting that CT is a potential
Fig. 2. Western blot analysis of CTR in human giant cell tumor andprimary chondrocytes. A band representing CTR appears atw56.9 kDa only in human GCT tissue. Thirty micrograms of GCTand chondrocyte samples were resolved by SDS-PAGE and trans-ferred overnight onto nitrocellulose before being probed for CTRwith specific antibodies. Alpha-tubulin served as an internal loading
control.
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pharmacological treatment for patients with OA. It has beenpreviously demonstrated that CT has direct effects on artic-ular cartilage and attenuation of cartilage degradation viainteraction with the CT receptor10. Surprisingly, in this studywe failed to detect obvious gene expression of CTR mRNAin either human cartilage or chondrocytes. This result was
strengthened by the lack of detectable CTR protein in nor-mal human articular cartilage as evidenced by Western blot-ting and immunohistochemistry. Furthermore, cAMP ELISAassays demonstrated that CT did not elict a cAMP responsein human chondrocytes over a range of physiologicalconcentrations. Real-time PCR also revealed that sCT
Fig. 3. Immunodetection of CTR in human GCT and articular cartilage. (A) Immunostained osteoclast in human GCT imprint, hematoxylincounterstain, immunostaining is predominantly on the osteoclast plasma membrane (original magnification, 400�); (B) immuno- andTRAP-staining of osteoclasts in human GCT imprint (original magnification, 400�); (C) negative control without primary antibodies in a humanGCT imprint, hematoxylin counterstain (original magnification, 400�); (D) full thickness of human articular cartilage section with subchondralbone, immunostained with anti-CTR antibody and counterstained with hematoxylin (original magnification, 40�); (E) chondrocytes in the samesection show no specific staining, hematoxylin counterstain (original magnification, 400�); (F) osteoclasts (as indicated by arrow) in the sub-
chondral bone region stained positive for CTR (original magnification, 400�).
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treatment had little to no effect on the expression levels ofcartilage matrix proteins, aggrecan and type II collagen,and their associated proteases (MMP-1, MMP-3, MMP-13)in human chondrocytes. Together, these results suggestthat the previous reported effects of CT on chondrocytesmay occur through a mechanism independent of directinteraction with the CTR. Our study challenges thecontempory hypothesis that CT directly mediates attenua-tion of cartilage degradation.
The results of this study are in direct contrast to the re-cent study by Sondergaard et al. who demonstrated thegene expression of CTR and positive immunostaining ofCTR in bovine articular cartilage12. The reason for this dis-crepancy is presently unclear, although it may be attributedto species differences and the primers used. It is possiblethat the use of human primer sequences (sense: 50-GACAACTGCTGGCTGAGTG-30, anti-sense: 50-GAAGCAGTAGATGGTCGCAA-30) to amplify the bovine CTR mayresult in false positives and thus account for the detectedtranscript by Sondergaard et al.12. Alternatively, the tissuedistribution of CTR might simply vary between species. Another possibility may be due to the age difference in the tis-sue origin. Histochemical analysis of pCTR/lacZ expressionin transgenic embryos and foetuses of mice revealed thatCTR expression was positive in cartilage primordium ofthe humerus and ears during fetal development, which is,however, not observed in adult tissue4. It is plausible that
the expression of CTR varies in human with age. However,due to ethical restrictions, human cartilage samples canonly be obtained from adults. Thus, we are unable to di-rectly compare the expression level of CT in cartilagefrom infants.
Previously, CT has been shown to promote cell prolifera-tion of expanded chondrocyte cultures26. Addition of CThas also been shown to enhance chondrocyte adhesion toextracellular matrix in the high-density cultures and alterthe regulation of protease synthesis20. PLA2 activity of hu-man chondrocytes has also been shown to be elevatedupon sCT stimulation in vitro20. The presence of CRLR insubcultured chondrocyte reported in this study might in partaccount for the positive CT response observed in chondro-cyte cultured in vitro. CRLR is a member of the G-protein-coupled receptor family which exhibits up to 55% aminoacid identity with the CTR. Our data indicate that CRLR isupregulated in monolayer cultured chondrocytes. Giventhat the CRLR has been reported to interact with othermembers of the G-coupled protein family, including CT-gene-related peptides and adrenomedullin27, it is temptingto speculate that CRLR might serve as a non-specific recep-tor for CT in isolated human chondrocytes where CTR isabsent. Interestingly, CRLR was not expressed in either car-tilage tissue or primary chondrocytes suggesting that it mightbe involved in the de-differentiation process of in vitro cul-tured chondrocytes. Further study is required to comparethe physiological responses of late passage chondrocyte cul-tures with CT addition.
Despite encouraging evidence of CT treatment for OA,there are large differences between the pharmacologicalaction and the potential presence of receptors. Animal stud-ies suggest that CT treatment reduced the progression ofcartilage damage by preventing the loss of collagen, hyalur-onan and proteoglycan aggregates28e31. A recent pub-lished study on a rabbit model of surgical-induced OAdemonstrated that daily intramuscular injection of sCT helpsto minimize cartilage degeneration, osteophyte formationand subchondral cyst formation11. Furthermore, patientswith regular oral CT treatment have less C-terminal peptideof type II collagen, one of the predominant components ofarticular cartilage matrix, in serum30, However, in the sys-temic models, it is difficult to distinguish whether this chon-droprotective effect is due to direct or indirect action. SinceCTR expression is not found in human cartilage and chon-drocytes, the effect of CT on cartilage matrix might be inde-pendent from chondrocytes. We believe that the beneficialimpact of CT on articular cartilage and chondrocyte ismore likely a secondary effect.
The concept of using CT as a pharmacological treatmentfor OA was initially proposed based on its effects on boneremodeling and mineral metabolism5. Conventionally, OAhas been considered to be primarily a cartilage disorderwith hyaline articular cartilage loss and matrix destruction.A more comprehensive definition of the disease addressesOA as a whole organ disease involving multiple changes incartilage, subchondral bone, periarticular muscle, liga-ments, synovium and the neurosensory system32. In partic-ular, increased subchondral bone turnover compromised byspecific architectural changes is an essential part of prog-ress of the disease. Although there is no conclusive reportabout whether subchondral bone loss occurs prior to thecartilage degeneration in OA, it is well acknowledged thatthe functional integrity of articular cartilage largely dependson the mechanical properties of the underlying subchondralbone. Manicourt et al. reported that the urinary level of de-oxypyridinoline, a specific marker of bone resorption,
Fig. 4. The mRNA expression of CRLR in human GCT, human car-tilage and chondrocytes. CRLR is abundantly expressed in humanGCT and cultured chondrocytes (passage 1), but absent in human
cartilage tissue.
Fig. 5. cAMP response of chondrocytes to sCT. Human chondro-cytes (passage 1) isolated from OA cartilage were cultured on mono-layer and stimulated with sCT (10�7 M, 10�8 M, and 10�9 M) orforskolin (100 mM) for 30 min. The cAMP content from culture super-natant and cell lysis were both measured. sCT does not change thecAMP content in chondrocyte compared to vehicle treatment (IBMX/100 mM), however, forskolin significantly elevates the cAMP level
(P< 0.001).
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64
significantly diminished in surgical-induced OA models by3 weeks subcutaneous injection with CT31. Similarly, histo-morphometric studies have shown that CT treatment sup-pressed the loss of subchondral trabecular bone inducedby anterior cruciate ligament transaction in dog models29.The study suggested that the protective effect of CT on sub-chondral trabecular bone subsequently safeguarded thecartilage from degeneration caused by excessive mechani-cal forces. Additionally, the severity of synovitis in an OAanimal model treated with CT has been shown to be re-duced as evidenced by decreased serum content of anti-genic keratan sulfate and hyaluronan31. These studiessuggest that CT might confer therapeutic benefit to the pa-tients with OA by ameliorating subchondral bone disruptionand/or suppressing joint inflammation. These effects mightbe independent from articular chondrocytes.
In conclusion, this study demonstrates that CTR is not de-tectable in human cartilage or chondrocytes. It suggests thatthe action of CT on cartilage may be indirect via inhibition ofsubchondral osteoclastic bone resorption. We believe thatthe information obtained in this study is important for the fur-ther understanding of CT and its biological behavior, espe-cially on human cartilage and chondrocytes, and for thedevelopment of potential pharmacological treatment for artic-ular degenerative diseases.
Acknowledgments
This work was supported by a National Health and MedicalResearch Council development grant to M. H. Zheng andD. J. Wood. Real-time PCR was performed in the AustralianNeuromuscular Research Institute, University of WesternAustralia. The authors would like to thank Prof. T. J. Martinfor his critical comments on the work.
References
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2. Raisz LG, Niemann I. Early effects of parathyroid hormone and thyrocal-citonin on bone in organ culture. Nature 1967;214:486e7.
3. Warshawsky H, Goltzman D, Rouleau MF, Bergeron JJ. Direct in vivodemonstration by radioautography of specific binding sites for calcito-nin in skeletal and renal tissues of the rat. J Cell Biol 1980;85:682e94.
4. Pondel M. Calcitonin and calcitonin receptors: bone and beyond. Int JExp Pathol 2000;81:405e22.
5. Manicourt DH, Devogelaer JP, Azria M, Silverman S. Rationale for thepotential use of calcitonin in osteoarthritis. J Musculoskelet NeuronalInteract 2005;5:285e93.
6. Karsdal MA, Tanko LB, Riis BJ, Sondergard BC, Henriksen K,Altman RD, et al. Calcitonin is involved in cartilage homeostasis: iscalcitonin a treatment for OA? Osteoarthritis Cartilage 2006;14:617e24.
7. Badurski J, Jeziernicka E, Naruszewicz K, Racewicz A. Comparativeanalysis of three treatment regimens for treating gonarthritis with cal-citonin, naproxen and flavonoids based on EULAR criteria and visualanalogue scale (VAS). Pol Tyg Lek 1995;50:37e40.
8. Punzi L, Schiavon F, Cavasin F, Ramonda R, Gambari PF, Todesco S.The influence of intra-articular hyaluronic acid on PGE2 and cAMP ofsynovial fluid. Clin Exp Rheumatol 1989;7:247e50.
9. Bagger YZ, Tanko LB, Alexandersen P, Karsdal MA, Olson M,Mindeholm L, et al. Oral salmon calcitonin induced suppression ofurinary collagen type II degradation in postmenopausal women:a new potential treatment of osteoarthritis. Bone 2005;37:425e30.
10. Manicourt DH, Azria M, Mindeholm L, Thonar EJ, Devogelaer JP.Oral salmon calcitonin reduces Lequesne’s algofunctional indexscores and decreases urinary and serum levels of biomarkers ofjoint metabolism in knee osteoarthritis. Arthritis Rheum 2006;54:3205e11.
11. Papaioannou NA, Triantafillopoulos IK, Khaldi L, Krallis N, Galanos A,Lyritis GP. Effect of calcitonin in early and late stages of experimen-tally induced osteoarthritis. A histomorphometric study. OsteoarthritisCartilage 2007;15:386e95.
12. Sondergaard BC, Wulf H, Henriksen K, Schaller S, Oestergaard S,Qvist P, et al. Calcitonin directly attenuates collagen type II degrada-tion by inhibition of matrix metalloproteinase expression and activity inarticular chondrocytes. Osteoarthritis Cartilage 2006;14:759e68.
13. Deshmukh K, Kline WG, Sawyer BD. Effects of calcitonin and parathy-roid hormone on the metabolism of chondrocytes in culture. BiochimBiophys Acta 1977;499:28e35.
14. Lyritis G, Boscainos PJ. Calcitonin effects on cartilage and fracture heal-ing. J Musculoskelet Neuronal Interact 2001;2:137e42.
15. Ishikawa Y, Wu LN, Genge BR, Mwale F, Wuthier RE. Effects of calci-tonin and parathyroid hormone on calcification of primary cultures ofchicken growth plate chondrocytes. J Bone Miner Res 1997;12:356e66.
16. Franchimont P, Bassleer C, Henrotin Y, Gysen P, Bassleer R. Effects ofhuman and salmon calcitonin on human articular chondrocytes culti-vated in clusters. J Clin Endocrinol Metab 1989;69:259e66.
17. Di Nino DL, Linsenmayer TF. Positive regulation of endochondral carti-lage growth by perichondrial and periosteal calcitonin. Endocrinology2003;144:1979e83.
Fig. 6. The expression profiles of chondrocytic genes (aggrecan, Type II collagen, MMP-1, MMP-3, MMP-13) in sCT treated human chondro-cytes (passage 1) by Real-time PCR. Mean Ct value of target genes was normalized to it housekeeping gene 18S. Statistic significances were
calculated by Student’s t test. Results are shown as mean� SEM. P< 0.05 is considered statistically significant.
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18. Burch WM, Corda G. Calcitonin stimulates maturation of mammaliangrowth plate cartilage. Endocrinology 1985;116:1724e8.
19. Burch WM. Calcitonin stimulates growth and maturation of embryonicchick pelvic cartilage in vitro. Endocrinology 1984;114:1196e202.
20. Hellio MP, Peschard MJ, Cohen C, Richard M, Vignon E. Calcitonininhibits phospholipase A2 and collagenase activity of human osteo-arthritic chondrocytes. Osteoarthritis Cartilage 1997;5:121e8.
21. Huang L, Xu J, Wood DJ, Zheng MH. Gene expression of osteoprote-gerin ligand, osteoprotegerin, and receptor activator of NF-kappaBin giant cell tumor of bone: possible involvement in tumor cell-inducedosteoclast-like cell formation. Am J Pathol 2000;156:761e7.
22. Livak KJ, Schmittgen TD. Analysis of relative gene expression datausing real-time quantitative PCR and the 2(-Delta Delta C(T)) method.Methods 2001;25:402e8.
23. Kuestner RE, Elrod RD, Grant FJ, Hagen FS, Kuijper JL, Matthewes SL,et al. Cloning and characterization of an abundant subtype of thehuman calcitonin receptor. Mol Pharmacol 1994;46:246e55.
24. Gorn AH, Lin HY, Yamin M, Auron PE, Flannery MR, Tapp DR, et al. Clon-ing, characterization, and expression of a human calcitonin receptorfrom an ovarian carcinoma cell line. J Clin Invest 1992;90:1726e35.
25. Xu J, Wang C, Han R, Pavlos N, Phan T, Steer JH, et al. Evidence ofreciprocal regulation between the high extracellular calcium andRANKL signal transduction pathways in RAW cell derived osteo-clasts. J Cell Physiol 2005;202:554e62.
26. Jones DG, Smith RL. Stimulation of adult chondrocyte metabolism bya thyroid-derived factor. J Orthop Res 1990;8:227e33.
27. McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N,et al. RAMPs regulate the transport and ligand specificity of thecalcitonin-receptor-like receptor. Nature 1998;393:333e9.
28. Badurski JE, Schwamm W, Popko J, Zimnoch L, Rogowski F, Pawlica J.Chondroprotective action of salmon calcitonin in experimental arthrop-athies. Calcif Tissue Int 1991;49:27e34.
29. Behets C, Williams JM, Chappard D, Devogelaer JP, Manicourt DH.Effects of calcitonin on subchondral trabecular bone changes andon osteoarthritic cartilage lesions after acute anterior cruciate ligamentdeficiency. J Bone Miner Res 2004;19:1821e6.
30. El Hajjaji H, Williams JM, Devogelaer JP, Lenz ME, Thonar EJ,Manicourt DH. Treatment with calcitonin prevents the net loss of col-lagen, hyaluronan and proteoglycan aggregates from cartilage in theearly stages of canine experimental osteoarthritis. Osteoarthritis Car-tilage 2004;12:904e11.
31. Manicourt DH, Altman RD, Williams JM, Devogelaer JP, Druetz-VanEgeren A, Lenz ME, et al. Treatment with calcitonin suppresses theresponses of bone, cartilage, and synovium in the early stages of ca-nine experimental osteoarthritis and significantly reduces the severityof the cartilage lesions. Arthritis Rheum 1999;42:1159e67.
32. Felson DT, Neogi T. Osteoarthritis: is it a disease of cartilage or of bone?Arthritis Rheum 2004;50:341e4.
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8 Z. Lin et al.: Calcitonin receptor in human cartilage and chondrocytes
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oarthritis Cartilage (2007), doi:10.1016/j.joca.2007.08.003
66
CHAPTER 6
MODULATION OF HUMAN CHONDROCYTE FUNCTIONS BY
NATURAL COMPOUNDS
>>Chapter 6 Natural compounds<<
67
6.1 INTRODUCTION
As described in Chapter 2, conventional pharmacological management for OA includes
non-opioid analgesics such as paracetamol, non-steroidal anti-inflammatory drugs
(NSAIDs), topical analgesics, opioid analgesics and intra-articular steroid injection of
glucosmin sulphate that mainly target the alleviation of the symptom of the disease such
as pain and stiffness, rather than chondral protection of cartilage. Some of them may
have serious adverse effects, and may be ineffective in some patients. Hence, there is a
need to develop novel pharmacological treatment with optimal efficacy and minimum
toxicity for OA patient. Natural compounds derived from herbal plants, which have less
side effects comparing to those conventional drugs, may be developed as effective and
safe medical treatment for OA patients. This study attempted to identify the natural
extract with therapeutic value for arthritis for a number of organic derived substances.
It is believed that OA is a complicated disease caused by a combination of factors.
Although the whole picture of OA pathogenesis is yet be completed, several factors
have been identified, including injury due to excessive mechanical stress, biochemical
and genetic disorders, dysfunction of chondrocytes, over expression of proteinases, and
degradation of structural macromolecules such as proteoglycan and collagen. In
addition, inflammatory components are believed to be involved in the progression of
OA. Cytokines such as IL-1β and TNF-α were detected in the synovial fluid from OA
patients. Several intracellular signaling pathways, such as AP-1, MAPk and NF-κ, were
shown to mediate the inflammatory response in OA chondrocyte. The inhibition of
NF-κB has been proposed as a potential therapeutic target for patients with OA, due to
its intimate involvement in the modulation of inflammatory events. This chapter focuses
on the modulation of chondrocyte gene expression and function regulated by
pro-inflammatory factors and natural compounds, which have been shown to have
potential NF-κB inhibitory activity in other cell types.
Parthenolide
>>Chapter 6 Natural compounds<<
Me
Me
O
O
H2 C
OH
H
E
R
R
S
S
Formula: C15 H20 O3 CA Index Name: Oxireno[9,10]cyclodeca[1,2-b]furan-9(1aH)-one,
2,3,6,7,7a,8,10a,10b-octahydro-1a,5-dimethyl-8-methylene-, (1aR,4E,7aS,10aS,10bR)-
Other Names: Germacra-1(10),11(13)-dien-12-oic acid,
4,5α-epoxy-6β-hydroxy-, γ-lactone (8CI); Parthenolide (6CI,7CI); (-)-Parthenolide
Sesquiterpene Lactone Parthenolide is a natural compound extracted from the plants of
the Asteraceae family such as feverfew or the leaves of Magnolia grandiflora L.
(el-Feraly et al., 1978). Sesquiterpene Lactone is one of the traditional remedies used for
pain relieving and anti-infection purpose in folk for many years (Hall et al., 1980;
Heinrich et al., 1998; Schinella et al., 1998). Its purified product, parthenolide, is the
active ingredient contributing to the anti-inflammatory and anti-hyperalgesic
(Feltenstein et al., 2004) effect. It has been reported that Sesquiterpene Lactone
Parthenolide inhibits the expression of transcription factor nuclear factor-kappa B
(NF-κB), which is ubiquitously expressed in vivo, activated by a serial of stimuli such
as cytokines, oxidant-free radicals, and some of the bacterial or viral products, and
responsible for the regulation of subsequence inflammatory events. The NF-κB
inhibition effect of parthenolide has been confirmed in several cell lines and disease
models (Yip et al., 2004).
Caffeic acid phenethyl ester (CAPE)
68
>>Chapter 6 Natural compounds<<
PhCH CH C O
O
CH 2
OH
CH 2
HO
Formula: C17 H16 O4 CA Index Name: 2-Propenoic acid, 3-(3,4-dihydroxyphenyl)-, 2-phenylethyl
ester Other Names: β-Phenylethyl caffeate; 2-Phenylethyl caffeate; Caffeic acid
phenethyl ester
Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee
hives, is known to have anti-carcinogenic, anti-inflammatory and immunomodulatory
properties (Chiao et al., 1995; Michaluart et al., 1999; Orban et al., 2000; Natarajan et
al., 1996). It has been shown that CAPE inhibits the activation of one of the common
transcription factors in inflammatory signaling pathway, nuclear factor-kappa B (NF-κB)
by TNF (Natarajan et al., 1996). It was shown that CAPE inhibited osteoclast formation
in vitro via the suppression of RANKL induced activation of NF-κB (Xu, unpublished
data). The effect of CAPE on human chondrocyte function is unclear.
Astilbin
Me
O
O
O
O
HO OH
OH
OH
OH
HO
OH
HR
S
R
R R
SR
Formula: C21 H22 O11 CA Index Name: 4H-1-Benzopyran-4-one, 69
>>Chapter 6 Natural compounds<<
3-[(6-deoxy-β-L-mannopyranosyl)oxy]-2-(3,4-dihydroxyphenyl)-2,3-dihydro-5,7-dihydroxy-, (2R,3R)- (9CI)
Other Names: 4H-1-Benzopyran-4-one,
3-[(6-deoxy-β-L-mannopyranosyl)oxy]-2-(3,4-dihydroxyphenyl)-2,3-dihydro-5,7-dihydroxy-, (2R-trans)-; Astilbin (6CI,7CI,8CI); Taxifolin 3-O-rhamnoside; Taxifolin 3-rhamnoside
Astilbin is the bioactive flavanone from the leaves of Harungana madagascariensis
Lam. ex Poir (Hypericaceae), which has been extensively used in Africa and Asia as
primary remedies due to its antibacterial activity (Moulari et al., 2006). It has also
shown that Astilbin has anti-nociceptive and anti-oedematogenic properties
(Cechinel-Filho et al., 2000), liver protective effect (Wang et al., 2004),
immunomodulatory activity by inhibiting lymphocyte migration (Cai et al., 2003).
Furthermore, Astilbin might have beneficial effect on arthritis since it has been shown to
suppress MMP and NO production in a collagen-induced arthritis model (Cai et al.,
2003). However, no data is available on its effect on human chondrocyte and
osteoarthritis.
Mangiferin
OH
O
O
OH
O
OH
OHHO
OH
OH
HO
SR
R
R
S
Formula: C19 H18 O11 CA Index Name: 9H-Xanthen-9-one,
2-β-D-glucopyranosyl-1,3,6,7-tetrahydroxy- (9CI) Other Names: Mangiferin (6CI,7CI,8CI);
1,3,6,7-Tetrahydroxyxanthone-C2-β-D-glucoside; 70
>>Chapter 6 Natural compounds<<
2-C-β-D-Glucopyranosyl-1,3,6,7-tetrahydroxyxanthone; Alpizarin; Alpizarine; Aphloiol; Chinomin; Chinonin; Hedysarid; NSC 248870; Shamimin
Mangiferin, an ingredient exacted from selected species of Mangifera indica
Linn.(Anacardiaceae), has been reported to show a potent in vivo and in vitro
anti-inflammatory activities (Garrido G. 2001), immunomodulatory avtivity (Garcia D.
2002) on rat macrophages, cardioprotective effect (Prabhu et al., 2006), and a strong in
vitro and in vivo antioxidant effect (Martinez G. 2000; Sanchez G.M. 2000; Rodriguez
et al., 2006). In Ayurveda, an ancient system of India medicine, Mangiferin rich plants
are recommended for the treatment of a number of diseases, such as arthritis, diabetes,
hepatitis, cardiac and mental disorders. People in our lab have previously found that
Mangiferin inhibits osteoclastogenesis in bone marrow cell culture (data not published).
Naringin
Me
O
O
OH
O
O
OHO
HO
O
HO
HO
OH
HO
HO
H
SSR
S
R
S
S
RR
SR
Formula: C27 H32 O14 CA Index Name: 4H-1-Benzopyran-4-one,
7-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl]oxy]-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-, (2S)- (9CI)
71
Other Names: 4H-1-Benzopyran-4-one, 7-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl]oxy]-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-, (S)-; Naringin (6CI,7CI,8CI);
>>Chapter 6 Natural compounds<<
72
(2S)-Naringin; 7-[2-O-(6-Deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyloxy]-2,3-dihydro-4',5,7-trihydroxyflavone; Naringenin 7-neohesperidoside; Naringenin 7-rhamnoglucoside; Naringenin 7β-neohesperidoside; Naringoside
Naringin is extracted from grape fruit juice, which has been reported to alter oral drug
pharmacokinetics by different mechanisms. Naringin exerts a variety of
pharmacological effects such as antioxidant activity, blood lipid-lowering,
anti-carcinogenic activity, and inhibition of selected cytochrome P450 enzymes
including CYP3A4 and CYP1A2, which may result in several drug interactions in vitro.
This study for the first time screened the effect of these natural compounds on human
chondrocytes. It was found that, among these compounds, parthenolide may be the one
that have potential beneficial impact on human OA. Hence, we proposed that
parthenolide may prevent the progress of the disease by suppressing the upregulation of
IL-1β and Nitric Oxide (NO) production induced by pro-inflammatory cytokines
through the inhibition of NF-κB activation.
6.2 MATERIALS AND METHODS
6.2.1 Tissue harvest and cell culture
Human chondrocytes were harvested as previously described and isolated from fresh
cartilage tissue and cultivated in vitro. Specimens were mechanically dissected into
200–500µm pieces by scalpel before being enzymatically digested in type II collagenase
(400units/ml) in an orbital mixer incubator at 130rpm in 37°C atmosphere for 6 hours.
Matrix digestion was ceased by the addition of fetal bovine serum (FBS). Tubes were
centrifuged at 1,500rpm for 8 minutes, and then washed in serum-free media twice.
Cells were then resuspended in tissue culture flask at a density of 5×104 cells/ml and
maintained in DMEM/F-12 medium (Gibco, New York, USA) supplemented with 20%
of FCS (TRACE, Sydney, Australia), 0.1% Gentamicin and 50µg/ml L-ascorbic acid
>>Chapter 6 Natural compounds<<
73
(Gibco, New York, USA) at 37°C in a humid environment with 5% CO2. Culture
medium was changed every two days.
6.2.2 Cell treatments
Extracted CAPE and parthenolide were purchased from Alexis Biochemicals, USA.
Mangiferin and Naringin are commercial available at Sigma-Aldrich, UK. Astilbin was
kindly provided by Prof. Hua Gang Liu from Department of Pharmacology, Guangxi
University of Medical Sciences, China.
The compounds were solubilized in DMSO, stored in 20μl aliquots at -20℃, and added
to culture medium at varying concentrations as indicated below. In each experiment,
cells treated with DMSO served as a vehicle control. LPS was reconstituted in DMSO
and used at final concentration of 10ng/ml. Recombinant human IL-1β and TNF-α were
solubilized in sterilized ddH2O and used at final concentrations of 10ng/ml. For most
experiments, human chondrocytes were plated at 3×105 cells/well in 6-well plates,
1.5×105cells/well in 12 well plate, or 0.8×105cells/well in 24 well plate supplied with
culture medium. Chondrocytes were pretreated with each different natural compound
for 2 hours, and stimulated with pro-inflammatory cytokine of appropriate concentration
for another 16 hours. Then culture mediums and cells were both harvested for further
experiments. A typical experiment was designed as followed.
control Parthenolide 1μM Parthenolide 10μM
IL-1β 10ng/ml Parthenolide 1μM
IL-1β 10ng/ml
Parthenolide 10μM
IL-1β 10ng/ml
6.2.3 RNA isolation and RT-PCR
After the treatment, chondrocytes were lysed, and total RNA was extracted by using the
>>Chapter 6 Natural compounds<<
74
Qiagen Rneasy Mini kit (Qiagen, UK) according to manufactory’s instructions. RNA
was stored at -80°C immediately after extraction until reverse transcription was
performed. Single-stranded cDNA was prepared form 2μg of total RNA using reverse
transcriptase with an oligo-dT primer. Two microliter of each cDNA was subjected to
PCT amplification using specific primers (See Appendix A Table A.1). PCR
amplification was carried out with 30 cycles (94℃ for 40s, 60℃ for 40s, 72℃ for 40s)
except otherwise specified. Housekeeping gene 18S was used as an internal control.
6.2.4 Real-time PCR and Data analyses
Relative quantitative Real-time PCR was preformed on a 384-well/plate ABI Prism
7000 Sequence Detection machine (Applied Biosystems) using Sybr Green PCR Master
Mix as previous described (Applied Biosystems). Briefly, 5µl Sybr Green PCR Master
Mixture, 2.5µl ddH2O, 1.5µl cDNA and 5µM of each of the forward and reverse
primers were mixed to make up a total of 10µl for each PCR reaction. The running
program was 95°C for 10 minutes (activation), 40 cycles of 95°C for 15s, 60°C for 20s
and 72°C for 20s (amplification) and 72°C for 1 minute (final extension). Housekeeping
gene 18S was used as endogenous control and always ran on the same plate. The
comparative 2− ΔΔCT method was used to calculate the relative expression of each target
gene as previously described (Livak et al., 2001). Briefly, mean Ct value of target genes
in experimental group was normalized to it averaged Ct value of housekeeping gene
18S to give a ΔCt value, which was further normalized to control samples to obtained a
ΔΔCt value. All experiments were performed in triplication. The relative expression
levels of the target genes were then compared by the student T test using the SPSS12.0
software (SPSS Inc., Chicago, U.S.). P values less than 0.05 were considered
significant.
6.2.5 Determination of proteoglycan content and release
Human chondrocytes were seeded on a 12 -well plate at the cell density of 1.5×105/ well
with 1ml of DMEM/F-12 supplied with 10% FCS, 0.1% L-ascorbic and 0.1%
Gentamicin for 24hrs. After that, cells were washed with serum-free medium twice and
>>Chapter 6 Natural compounds<<
75
cultured in serum-free growth medium. Parthenolide (1μM) was added 2hrs previous to
pro-inflammatory cytokine (LPS, 10μg/ml; IL-1α, 5ng/ml; TNF-α, 10ng/ml). Culture
mediums were collected at day 2 and day 4 of culture, cells were lysis by the addition of
500μl of Ripa Ripa Buffer at day 4. The content and release of proteoglycan were
determined in cultured human chondrocytes and their culture medium as sulfated
glycosaminoglycan (GAG), which is primarily a measure of proteoglycan aggrecan
content, using a modified colorimetric dimethylmethylene blue dye-binding (DMMB)
assay (Farndale RW et al., 1986; 883:173). A standard curve was constructed using
shark chondroitin sulphate (Sigma-Aldrich, UK) (0, 3, 5, 10, 20, 30, and 40g/mL in
0.15M NaCl), and samples were also diluted appropriately as required in 0.15M NaCl.
The DMMB solution (400μl) was added to all samples and the absorbance of the
mixture was measured at 525nm immediately on a spectrophotometer.
6.2.6 Nitric oxide measurement
Human chondrocytes were seeded on a 48-well plate at the cell density of 0.8×105/well
with 500μl of DMEM/F-12 supplied with 10% FCS, 0.1% L-ascorbic and 0.1%
Gentamicin. The cells were incubated at 37°C in 5% CO2 in a cell incubator for 24hrs to
allow the cells to settle. Cells were then washed with phenol red-free DMEM/F-12
(Gibco, New York, USA) twice and maintained in phenol red-free DMEM/F-12
supplied with 0.1% L-ascorbic, 0.1% Gentamicin, and 1μM parthenolide. The contents
of NO in cell culture mediums were measured. After pretreated with parthenoldie for 2
hours, chondrocytes were stimulated with the pro-inflammatory factor and maintained
at 37℃ in an atmosphere of 5% CO2 for another 48 hours. Subsequently, supernatant
samples were collected from the cell cultures and incubated with equal volume of 1×
Griess reagent (Sigma-Aldrich, UK) in a 96-well plate for 15 minutes at room
temperature. Nitride concentration was calculated with reference to a standard curve
obtained using a serial dilution of NaNO2 (100µM, 50µM, 25µM, 12.5µM, 6.25µM,
3.12µM, 1.56µM, 0.78µM, 0.39µM, and 0.195µM). The optical density was measured
at 550nm in a POLARstar® OPTIMA multifunction microplate reader (BMG
LABTECH, Germany).
>>Chapter 6 Natural compounds<<
76
6.2.7 Statistical analyses
Results shown in the text and figures are expressed as means±standard error (S.E.M.).
Means were compared by student’s t-test. Significance was set at P<0.05, <0.01 and
<0.001.
6.3 RESULTS
6.3.1 Parthenolide modulated gene expression profiles of human chondrocytes
To identify which compound might have beneficial impact on articular cartilage,
semi-quantitative PCR was first used to identify the anti-inflammatory and COX-2
inhibition effects of these natural compounds.
The pilot study revealed that either pro-inflammatory cytokines IL-1β (10ng/ml) or
endotoxin lipopolysaccharide (LPS, 10μg/ml) was able to induce endogenous IL-1β and
COX-2 expression in cultured human chondrocytes. Human chondrocytes was
pretreated with various concentrations of purified natural compounds, and further
incubated with pro-inflammatory cytokines IL-1β (10ng/ml) or LPS (10μg/ml).
However, parthenolide (1μM, 10μM) was shown to markedly lower the IL-1β and
COX-2 expression induced by LPS (Figure 6.1). Hence, parthenolide was selected for
further study.
A wide range of genes were measured by relative quantitative real-time PCR to reveal
the effect of parthenolide on the gene expression profiles of human chondrocytes. The
gene expression of matrix proteins (aggrecan and type II collagen), matrix proteinases
(MMP-1, MMP-3 and MMP-13), cytokines (IL-1β, TGFβ and IGF1), and cellular
signaling markers (COX-2, iNOS and MAPK1) were taken for study. The results
showed that 18hrs parthenolide treatment did not affect aggrecan, type I collagen and
type II collagen on mRNA levels (Figure 6.2A). Consistent with the semi-quantivative
PCR, LPS up-regulated IL-1β gene expression (P<0.05), and the induction was
>>Chapter 6 Natural compounds<<
77
neutralized by the pretreatment of parthenolide (10μM), which also lowered the IGF-1
gene expression induced by LPS (P<0.05) (Figure 6.2B). In terms of matrix proteinases,
MMP-1 and MMP-3 were both elevated by LPS stimulation (P<0.01), and the induction
of MMP-3 was suppressed by parthenolide (P<0.005). Aggrecanase ADAMTS-5 was
slightly increased by LPS (P<0.05), and lowered by the addition of parthenolide
(P<0.05). Other matrix proteinases, MMP-13 and ADAMTS-4, were not affected
(Figure 6.2A). Cellular signaling marker MAPk-1 was up-regulated. In addition,
parthenolide also neutralized the LPS up-regulated inducible nitric oxide synthase
(iNOS) gene expression (Figure 6.2C).
Next, since IL-1β is one of the major pro-inflammatory in osteoarthritis, we introduced
IL-1β into the human chondrocyte culture system and further investigated the
anti-inflammatory effect of parthenolide. The results showed that addition of IL-1β
elevated the gene expression of matrix proteinases (MMP-1, -3 and -13), endogenouse
IL-1β and iNOS. Endogenous IL-1β expression induced by additional IL-1β was
partially suppressed by parthenolide (Figure 6.3).
6.3.2 Parthenolide prevented proteoglycan degradation induced by
pro-inflammatory cytokines.
Furthermore, to investigate the effect of parthenolide on cartilage protection, we
examined the proteoglycan loss induced by LPS and IL-1α. The sulfated
glycosaminoglycan (sGAG) content was assayed in culture medium and tissue extracts.
The result revealed that LPS significantly decreased the GAG content in chondrocyte
(Figure 6.4C), and increased the GAG release into culture medium in 2 days and 4 days
culture (P<0.01, Figure 6.4A, B) . IL-1 promoted the GAG releasing in 2 days and 4
days culture (P<0.05, Figure 6.5A, B), but did not affect its content in cultured human
chondrocytes (Figure 6.5C). TNF-α also markedly stimulated GAG releasing into
cultured medium and reduced GAG content in cell lysate (P<0.05, Figure 6.6A, B, C).
Parthenolide was able to neutralize the GAG releasing induced by LPS (Figure 6.4D),
and reduced 45% of the GAG releasing induced by IL-1 (Figure 6.5D) and
>>Chapter 6 Natural compounds<<
78
approximately 60% of the GAG degradation induced by TNF-α (Figure 6.6D)
6.3.3 Parthenolide did not suppress nitric oxide production induced by
pro-inflammatory cytokines.
To determine whether parthenolide has any effect on the production of nitric oxide by
human chondrocytes, the Griess reaction was employed in this study. As seen, cells
treated with LPS (10μg/ml) or IL-1β (10ng/ml) alone released more nitric oxide into the
medium compared to the untreated group (P<0.05). After 48hrs treatment, parthenolide
(1μM) increased the basal level of nitric oxide (P<0.05) and did not have significant
effect on the evaluation of nitric oxide by LPS or IL-1β (Figure 6.7).
6.3.4 The effect of other natural compounds on chondrocyte gene expression.
CAPE (100nM, 200nM) and Astilbin (0.1mM, 1.0mM) did not alter the gene expression
levels of IL-1β, whilst COX-2 was up-regulated by both (Figure 6.8). Pretreatment of
Naringin (0.1mM, 1.0mM) and Mangiferin (0.1mM, 1.0mM) upregulated IL-1β
expression, and have no effect on COX-2 expression (Figure 6.9).
>>Chapter 6 Natural compounds<<
Figure 6.1 The effect of parthenolide on the gene expression of COX-2 and
IL-1beta in human chondrocytes. The chondrocytes was pretreated with parthenoldie
(1μM, 10μM) for 2hrs, and incubated with or 10μg/ml of LPS for another 16hrs. The
untreated chondrocytes were served as control. Housekeeping gene 18S was used as an
endogenous control.
79
>>Chapter 6 Natural compounds<<
80
>>Chapter 6 Natural compounds<<
81
Figure 6.2 The effect of parthenolide on the gene expression profiles in human
chondrocytes when stimulated with LPS detected by Real-time PCR. The
chondrocytes was pretreated with or without parthenolide 10μM for 2hrs, and incubated
with 10μg/ml of LPS for another 16hrs. The untreated chondrocytes were served as
negative control. The Mean Ct value of target genes was normalized to housekeeping
gene 18S. The gene expression levels were compared using the 2-ΔΔCt method. Statistic
significances were calculated by student T-test. Results are shown as mean±S.E.. *
representing the P value of the experiment groups compared to the negative control, #
representing the P value of the experimental groups compared to the IL-1β-alone treated
group (*, #, P<0.05; **, ##, P<0.01; ***, ###, P<0.001).
>>Chapter 6 Natural compounds<<
82
>>Chapter 6 Natural compounds<<
83
Figure 6.3 The effect of parthenolide on the gene profiles in human chondrocytes
when stimulated with IL-1β detected by Real-time PCR. The chondrocytes were
pretreated with or without parthenolide 10μM for 2hrs, and incubated with 10ng/ml of
IL-1β for another 16hrs. The untreated chondrocytes were served as negative control.
The Mean Ct value of target genes was normalized to housekeeping gene 18S. The gene
expression levels were compared using the 2-ΔΔCt method. Statistic significances were
calculated by student T-test. Results are shown as mean±S.E.. * representing the P value
of the experiment groups compared to the negative control, # representing the P value of
the experimental groups compared to the IL-1β-alone treated group (*, #, P<0.05; **,
##, P<0.01; ***, ###, P<0.001).
>>Chapter 6 Natural compounds<<
Figure 6.4 The GAG content released in culture medium (A, B) and cell lysate (C)
when chondrocytes were stimulated with LPS (10μg/ml) after 2hrs parthenolide
(1μM) treatment. Culture mediums were collected at day 2 and day 4 respectively.
Cells lysates were harvested at day 4. Statistic significances were calculated by student
T-test. Results are shown as mean±S.D. * representing the P value of the experiment
groups compared to the negative control, # representing the P value of the experimental
groups compared to the LPS-alone treated group (*, #, P<0.05; **, ##, P<0.01; ***, ###,
P<0.001).
84
>>Chapter 6 Natural compounds<<
Figure 6.5 The GAG content released in culture medium (A, B) and cell lysate (C)
when chondrocytes were stimulated with IL-α (5μg/ml) after 2hrs parthenolide
(1μM) treatment. Culture mediums were collected at day 2 and day 4 respectively.
Cells lysates were harvested at day 4. Statistic significances were calculated by student
T-test. Results are shown as mean±S.D. * representing the P value of the experiment
groups compared to the negative control, # representing the P value of the experimental
groups compared to the IL-α-alone treated group (*, #, P<0.05; **, ##, P<0.01; ***,
###, P<0.001).
85
>>Chapter 6 Natural compounds<<
Figure 6.6 The GAG content released in culture medium (A, B) and cell lysate (C)
when chondrocytes were stimulated with TNF-α (10μg/ml) after 2hrs parthenolide
(1μM) treatment. Culture mediums were collected at day 2 and day 4 respectively.
Cells lysates were harvested at day 4. Statistic significances were calculated by student
T-test. Results are shown as mean±S.D.. * representing the P value of the experiment
groups compared to the negative control, # representing the P value of the experimental
groups compared to the TNF-α-alone treated group (*, #, P<0.05; **, ##, P<0.01; ***,
###, P<0.001).
86
>>Chapter 6 Natural compounds<<
Figure 6.7 Effect of parthenolide on Nitric Oxide release in human chondrocytes.
Cells were treated with parthenolide (1μM) for 2hrs, added 10μg/ml of LPS or 10ng/ml
IL-1β, and incubated for another 48hours. Supernatants were incubated with the equal
volume of 1× Griess reagent. The nitrate values were calculated according to the
standard curve generated by a serial dilution of NaNO2. Statistic significances were
calculated by student T-test. Results are shown as mean±S.D. * representing the P value
of the experiment groups compared to the negative control (*, P<0.05; **, P<0.01; ***,
P<0.001).
87
>>Chapter 6 Natural compounds<<
Figure 6.8 The effect of CAPE and Astilbin on the gene expression of IL-1beta and
COX-2 in human chondrocytes. The chondrocytes was pretreated with CAPE (100nM,
200nM) or Astilbin (0.1mM, 1.0mM) for 2hrs, and incubated with or without 10ng/ml
of IL-1β for another 16hrs. The untreated chondrocytes were served as control.
Housekeeping gene 18S was used as an endogenous control.
88
>>Chapter 6 Natural compounds<<
Figure 6.9 The effect of Naringin and Manigerin on the gene expression of COX-2
and IL-1beta in human chondrocytes. The chondrocytes was pretreated with Naringin
(0.1mM, 1.0mM) or Mangiferin (0.1mM, 1.0mM) for 2hrs, and incubated with or
10ng/ml of IL-1β for another 16hrs. The untreated chondrocytes were served as control.
Housekeeping gene 18S was used as an endogenous control.
89
>>Chapter 6 Natural compounds<<
90
6.4 DISCUSSION
In this study, we have screened 5 natural compounds for its potential function on human
chondrocytes. CAPE and Astilbin enhance the elevation of COX-2 expression induced
by pro-inflammatory cytokines IL-1β, suggesting that they may have reverse effects on
articular cartilage. Naringin and Mangiferin did not affect the gene expression levels of
COX-2 and IL-1β in human chondrocytes when they are stimulated by additional IL-1β.
However, we found that Sesquiterpene Lactone Parthenolide is able to modulate
chondrocyte function by down-regulating the inflammatory-induced gene expression
levels of IL-1β, COX-2, and iNOS. MMPs, the enzymes that are responsible for the
cartilage degradation, are suppressed by parthenolide. In addition, this thesis showed
that parthenolide can reduce proteoglycan degradation in human chondrocytes when
they are in inflammatory conditions. Our results suggest that this medicinal plant extract
may be useful for the treatment of degenerative articular joint disease.
Parthenolide is an active component purified from feverfew, a medicinal plant
belonging to the Asteracee family. The anti-inflammatory property of parthenolide has
been reported in a number of studies, which have suggested that parthenolide modulates
inflammatory response by inhibiting the activity of nuclear factor-kappaB (NF-κB)
transcription factor. Since inflammatory process plays an important role in the progress
of articular joint degeneration, and NF-κB is prominent for most of inflammatory
process, modulation of the NF-κB pathway could be aimed at as an effective therapy for
OA (Roman-Blas et al., 2006).
NF-κB is located in the cytoplasm of mammalians cells in an inactive form associated
with the inhibitory κB proteins (IκB), which play an essential role in regulating the
NF-κB pathway. When cells are stimulated with pro-inflammatory cytokines such as
TNF-α or IL-1β, the NF-κB pathway is activated by triggering a signaling pathway that
leads to the phosphorylation of IκB, which is carried out by the specific serine/theronine
kinase IκB kinase (IKK). NF-κB activation increases the gene expression of
>>Chapter 6 Natural compounds<<
91
inflammatory cytokines such TNF-α and IL-1β and inducible enzymes including
cyclooxygenase (COX)-2 and iNOS, which are abundantly expressed in articular
chondrocytes with OA disease. It has been reported that the members of the NF-κB
family, NF-κB1 (p50) and RelA (p65), are abundant in rheumatoid and osteoarthritic
synovities. Synovial tissue from arthritis patients show increased NF-κB1+ and Rel+
cells at the cartilage-pannus junction (Benito et al., 2004). Besides inflammatory
response, NF-κB also contributes to progressive extracellular matrix damage and
cartilage destruction. An animal study reported that overexpression of NF-κB in
articular chondrocytes enhanced cartilage destruction in the type II collagen induced
arthritis mice (Eguchi et al., 2002). Liacini, A et al.,2002 demonstrated that inhibition of
NF-κB in IL-1-induced bovine chondrocytes suppress the up-regulation of MMP-1,
MMP-3 and MMP-13, which are critical for the regulation of catalytic degradation of
cartilage extracellular matrix. In vitro study has shown that the NF-κB as well as the
MAP kinase ERK 1/2 kinase pathways mediate inhibition of type II collagen and link
protein gene expression by TNF-α (Liacini et al., 2003). The present study has
demonstrated the inhibitive activity of parthenolide on IL-1β and MMPs expression and
proteoglycan degradation. These results suggest that inhibition of NF-κB could be a
potential therapeutic strategy aimed to reduce articular cartilage degradation in arthritis
(Roman-Blas et al., 2006).
COX-2 is an inducible enzyme that expresses in response to inflammatory cytokines,
and subsequently stimulates Prostaglandin E Synthase (PGES), which is responsible for
Prostaglandin E2 production (Gomez-Hernandez, 2006 #1). The suppression of COX-2
has been shown to be beneficial for the treatment of articular arthritis by repressing joint
inflammatory and cartilage destruction (Gilroy et al., 1998; Myer et al., 2000). The
regulation of COX-2 expression has been shown to occur at both transcriptional and
post-transcritptional levels (Miller et al., 1998; Newton et al., 1998), and structural
analysis of COX-2 gene promoter revealed binding sites for a number of transcriptional
factors including NF-κB (Sheng et al., 1998; Matsuura et al., 1999). Our study suggests
that parthenolide neutralized the up-regulation of COX-2 induced by LPS via the
>>Chapter 6 Natural compounds<<
92
inhibition of NF-κB activity and activation of MAPk pathway.
Reactive oxygen species (ROS) is one of the factors that are thought to responsible for
the cartilage destruction by acting directly on cartilage matrix proteins, activating
zymogen and dysregulating chondrocyte metabolic activity (Mathy-Hartert et al., 2003).
NO is known as a catabolic agent which is synthesized through L-arginine oxidation by
NO synthases. It rapidly reacts with superoxide anions to form a strong oxidant
peraxynitrite. It has been well documented that chondrocytes possess NADPH oxidase
and NO synthase, and produce ROS and NO when exposed to inflammatory factors
such as IL-1β, TNFα, IFNγ or LPS (Tiku et al., 1990; Henrotin et al., 1993; Hayashi et
al., 1997). In vitro study revealed that inhibition of NO production resulted in the
up-regulation of MMPs and suppression of glycosaminoglycan synthesis induced by
IL-1β (Sasaki et al., 1998; Taskiran et al., 1994). A selective inhibitor of iNOS, L-NIL
(n-iminoethyl-L-lysine), reduced the progression of cartilage erosion in an experimental
osteoarthritis animal model (Pelletier et al., 1999). Again, the binding activity of NF-κB
was shown to be involved in the elevation of ROS by IL-1 (Lavrovsky et al., 2000).
Although it is generally agreed that NO is involved in cartilage destruction, the role of
NO playing in the initiation and progression of articular inflammation becomes
controversial more recently. It has been found that NO may has anti-inflammatory under
certain circumstances (Mathy-Hartert et al., 2002). It has been evidenced that inhibition
of NO led to induction of PGE2, which is well documented as a major inflammatory
element to OA (Habib et al., 1997; Patel et al., 1999; Attur et al., 1999). A dual effect of
NO was explanted by Stadler et al., for this discrephancy in the literature, suggesting
that there is a biphasic effect of NO according to its production level, hence antioxidant
therapy might have adverse effects to degenerative joint disease. In the present study,
the elevation of iNOS expression and NO production induced by IL-1β and LPS was
confirmed. Although addition of parthenolide down-regulated the expression of iNOS, it
did not reduce NO production suggesting that the regulation of NO may be independent
of iNOS. The precise function of NO in human chondrocyte and the joint degenerative
>>Chapter 6 Natural compounds<<
93
disease should be further studied, since it is important for the consideration of applying
anti-oxidant as a therapeutic strategy for arthritis.
6.5 SUMMARY AND CONCLUSION
In summary, we demonstrated that parthenolide is capable of protecting articular
cartilage against pro-inflammatory factors such as LPS and IL-1. Parthenolide regulates
human chondrocyte function by suppressing the up-regulation of gene expression of
inflammatory cytokines and matrix proteinases induced by LPS. Particularly, it
down-regulates the expression of COX-2. In addition, parthenolide can reduce
proteoglycan degradation in human chondrocytes. These findings suggest that
parthenolide inhibits inflammatory-activated NF-κB pathway, and subsequently reduces
inflammatory responce, prevents cartilage destruction and relieves pain, and hence may
be used as a pharmacological approach for the treatment of OA.
CHAPTER 7
GENERAL DISCUSSION
>>Chapter 7 General Discussion<<
7.1 DISCUSSION
Osteoarthritis (OA) is the most common chronic joint disease especially in developed
countries. With the increase of life expectancy and ageing population, the burden of
arthritis on individuals and society increases dramatically. It is expected that OA will
become the fourth leading cause of disability by the year 2020 (Woolf and Pfleger,
2003). Because current pharmacological treatments for OA are far less than satisfying,
there is a need to develop a novel pharmacological strategy to eliminate local symptom
and delay or even reverse disease progress with little side effects. In recent years, OA is
more acknowledged as a disease of an organ system which includes articular cartilage,
subchondral bone, synovium, capsule, ligaments and muscles. Several key events in OA
pathogenesis have been identified, such as chondrocyte apoptosis and proliferation,
alteration of metabolic activity, modulation by inflammatory cytokines and growth
factors, phenotypic alteration of chondrocytes, formation of osteophype and
subchondral bone remodeling. Among these features, a progressive loss of articular
cartilage is believed as the major characteristic of OA. Since these pathogenetic
activities in OA cartilage are mainly manipulated by chondrocytes, a great portion of the
drug targeting research endeavor has been concentrated on this only cell type in
articular cartilage tissue (Kim et al., 2007). Thus, this thesis points to the biological
understanding of chondrocytes as well as drug development targeting on chondrocytes
for OA treatment.
Chondrocytic phenotype is regulated by a balance of anabolic and catabolic molecular
reactions which are at the same time involved in maintaining homeostasis of cartilage
tissue. Phenotypic and functional stability of the chondrocytes are essential for the
maintenance of proper cartilage matrix turnover. As known, chondrocytes lose their
phenotype and transform into fibroblast-like cells upon exposure to inflammatory
factors such as IL-1β, TNF-α, and nitric oxide or during serial monolayer subculture. In
this thesis, the genomic investigation of human chondrocytes serially passaged on
monolayer in vitro revealed a number of changes of gene expression at various stages of
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>>Chapter 7 General Discussion<<
passage. With the increase of passage number, the expression of cartilage
macromolecular aggrecan, type II collagen and fibromodulin decreases, whilst proteins
that contribute to the linkage of protein network elevated at mRNA levels. The function
of chondrocyte on maintaining homeostasis of extracellular matrix turnover is disrupted,
evidenced by the down-regulation of proteinases (MMP-3/9/13, ADAMTS-4/5) and
up-regulation of proteinase inhibitors (TIMP-2/3). Inflammatory cytokine IL-1β seems
to be the predominant factor on chondrocyte in vitro de-differentiation, which
demonstrated increased expression with serial chondrocyte culture, and the
chondrogenic transcription factor Sox-9 remains unchanged during in vitro passaging.
Notably, by comparing the gene expression profiles between normal and OA
chondrocytes, little significant difference was observed, implying that the plasticity of
isolated chondrocytes greatly depends on its culture condition.
Another focus of this thesis is the development of novel pharmacological approach for
OA. Clinical studies suggested that calcitonin, an established anti-resorptive agent that
has been used for the treatment of osteoporosis for many years, may also have
beneficial effect on chondrocyte protection in patients with OA. This founding is
encouraging, because currently available treatments for OA mostly target the alleviation
of symptom, but not the cause of disease. A therapeutic approach that could prevent
cartilage degradation, relieve pain and inhibit bone resorption at subchondral region
would be ideal for OA treatment. However, the information of the direct effect of
calcitonin on articular cartilage degradation and its interaction with chondrocytes is very
limited. As known, the biological activity of calcitonin is through the binding of its
receptor to elevate intracellular levels of cAMP, which subsequently activates
down-stream signaling. This thesis attempted to identify and localize the expression of
calcitonin receptor in human chondrocytes, and to investigate the cellular mechanism of
the protective effect of calcitonin on cartilage. However, the result demonstrated that
calcitonin receptor is absent in human chondrocytes and cartilage, assured by multiple
experimental approaches. The treatment of salmon calcitonin did not increase the cAMP
content in human chondrocytes as it did in osteoclasts, a cell type that is well known in
95
>>Chapter 7 General Discussion<<
possession of calcitonin receptor. Real-time PCR study revealed that addition of salmon
calcitonin has little effect on the gene expression of cartilage matrix homeostasis
markers, matrix proteins (Aggrecan and type II collagen) and matrix proteases (MMP-1,
3, 13). These results taken together suggested that there is no evidence of direct
interaction between human chondrocyte and calcitonin, and the protective effect of
salmon calcitonin on cartilage structure could be as secondary. Although these
evidences are negative, the information is valuable for the understanding of calcitonin
activity in human cartilage and the exploration of therapeutic value of organic-derived
calcitonin.
Additional studies of chondrocyte function regulated by natural compounds were
carried out in this thesis. Substances that derived from natural medical plants have been
selected to test their potential for OA treatment. Parthenolide, a NF-κB inhibitor
extracted from feverfew, was found to protect human chondrocytes against
pro-inflammatory factor IL-1 and LPS. The gene expression level of IL-1β, COX-2 and
MMPs was partially down-regulated by the addition of parthenolide as evidenced by
real-time PCR. Parthenolide also suppressed proteoglycan degradation induced by IL-1
and TNF-α. These results suggest that parthenolide inhibits inflammatory-activated
NF-κB pathway and may protect chondrocytes against pro-inflammatory factors. As
mentioned, the NF-κB signaling pathway has been proposed as an important target for
drug development of arthritis due to its fundamental role in the regulation of genes that
participate in inflammatory events. However, there are concerns about using inhibitors
of intracellular molecules as a pharmacological strategy, because these intracellular
molecules are usually ubiquitously expressed. They do not only modulate disease
process but also participate in maintenance of normal metabolism. Systematic blocking
of these pathways may result in severe side effects. Specific inhibitor with moderate
activity may be more suitable for disease control. This thesis revealed that parthenolide
did not alter the gene expression in human chondrocyte at basal level, suggesting that
parthenolide does not interfere with the function of normal chondrocyte and may be
used as a pharmacological approach for OA treatment. The inhibitors of signaling have
96
>>Chapter 7 General Discussion<<
proved valuable for understanding the mechanism of regulation and development of OA
at intracellular level.
7.2 FUTURE DIRECTIONS
In recent years, the understanding of articular cartilage and chondrocyte biology
advance tremendously. Several novel therapeutic strategies for OA treatment have been
developed, including attenuating the impacts of pro-inflammatory factors, regulating the
functions of proteinases and their inhibitors, intervening chondrocyte functions by
genomic techniques, modulating inflammatory signaling pathways, and regenerating
articular cartilage using tissue engineering approach. Current activities of drug
discovery are mostly focusing on single drug component interacting with single targets,
however an ideal drug for OA should be a multi-component drug that would target on
multiple aspects of OA process.
Apart from invention of articular cartilage function, subchondral bone restoration shall
also be considered as a therapeutic strategy for OA treatment. This thesis has
demonstrated that calcitonin does not directly interact with human chondrocytes
because of a lack of calcitonin receptor on cell surface, but the chondral protective
property of calcitonin, which has been demonstrated in a series of clinical and animal
studies, may be as a secondary effect, which is primary through the restoration of
subchondral bone. The subchondral plate is comprised of the mineralized cartilage and
the lamellar subchondral cortical bone that underlie and support the articular cartilage. It
has been well acknowledged that subchondral bone plate provides a physical support to
cartilage and allocates mechanical force to articular joint. More recently, there is
growing knowledge about the role of subchondral bone in the pathogenesis of OA.
Particularly, the abnormal metabolism of osteoblasts may contribute to the disease
process, indicated by the increased level of osteocalcin, a specific marker of bone
formation, in synovial fluid from OA patients (Lajeunesse and Reboul et al., 2003).
Osteocalcin is synthesized by osteoblast and contribute to the increase of bone volume
97
>>Chapter 7 General Discussion<<
98
without elevating bone mineralization, which explains the abnormal remodeling and
low mineralization in OA patients. To further understand the biological function of
subchondral bone in articular cartilage and OA disease, we need to understand the
interaction between osteoblasts and chondrocytes. Jiang et al. have introduced an
indirect co-culture model to study the effect of cell-cell interaction between osteoblasts
and chondrocytes on their phenotype maintenance (Jiang et al., 2005). This co-culture
model may be used to discover drugs that could target both cartilage and subchondral
bone in degenerative joint disease. Since the chondral protective effect of calcitonin is
most likely a secondary impact on OA cartilage according to the finding of this thesis, it
is very likely to act through bone cells that are well known to possess calcitonin receptor.
The use of this co-culture model will enable investigation of the interaction of
osteoblast and chondrocyte following administration of calcitonin, and hence aid in
delineating the complex biologic link between subchondral bone remodeling and
articular cartilage degeneration in OA process.
APPENDIX A
MATERIALS AND METHODS
>>Appendix A. Materials and Methods<<
99
1.1 MATERIALS
1.1.1 Chemical reagents
All chemical reagents used during the course of this experiment were of analytical grade,
and were purchased from the following manufactures.
Reagent Manufactory
Acrylamide 99.9% Bio-Rad Laboratories, Herules, C.A.
Agarose powder Promega Corp, Madison, Wi, USA
α-MEM TRACE, Sydney, NSW, Australia
Aprotinin Sigma Chemicals Co, St Louis, Mo,
USA
APS (Ammonium Persulfate) Bio-Rad Laboratories, Herules, C.A
Disodium IN, USA
D-MEM/F-12 Medium Gibco, New York, USA
DMSO (Dimethyl sulphoxide) BDH Laboratory Supplies, Poole,
Dorset, England
DNA ladder Promega Corp, Madison, Wi, USA
dNTP (ATP, TTP, CTP, GTP nucleotides) Promega Corp, Madison, Wi, USA
di-sodium hydrogen orthophosphate BDH, Poole, England
EDTA (Ethylene tera-acetic acid) 0.5 M stock, brought to pH 8.0 with
NaOH and stored at room
temperature.
Ethanol (100%) Merck, Victoria, Australia
Ethidium Bromide
(2,7-diamino-10-ethyl-9-phenylphenanthridium
bromide; homodium bromide)
Sigma Chemicals Co, St Louis, Mo,
USA
Fetal calf serum (FCS) TRACE, Sydney, Australia
Glacial Acetic Acid BDH Laboratory Supplies, Poole,
>>Appendix A. Materials and Methods<<
100
Dorset, England
Glycine BDH Laboratory Supplies, Poole,
Dorset, England
Glycerol BDH Laboratory Supplies, Poole,
Dorset, England
Hydrocholoric acid BDH Laboratory Supplies, Poole,
Dorset, England
L- Ascorbic Acid Gibco, New York, USA
L-Glutamine Gibco BRL, Life Technologies,
Melbourne, Australia
Methanol BDH Laboratory Supplies, Poole,
Dorset, England
β-Mercaptoethanol Sigma Chemical Co., St Louis, Mo.,
USA
M-MLV Reverse Transcriptase
(Rnase H Minus)
Promega Corp, Madison, Wi, USA
M-MLV RT 5 × Reaction Buffer Promega Corp, Madison, Wi, USA
Paraformaldehyde Merck, Damstadt, Germany
PCR grade water Baxter Healthcare Pty Ltd, Australia
Penicillin/Streptomycin (2x) Gibco BRL, Life Technologies,
Melbourne, Australia
Phenol red-free D-MEM/F-12 Medium Gibco, New York, USA
PMSF (Phenylmethylsulfony fluoride) Boehringer Mannheim Corp, Indpl,
IN, USA
Rnasin robonuclease inhibitor Promega Corp, Madison, Wi, USA
SDS (Sodium Dodecyl sulphate) BDH Laboratory Supplies, Poole,
Dorset, England
Sterile DD Water Baxter, NSW, Australia
Sodium Chloride BDH Laboratory Supplies, Poole,
>>Appendix A. Materials and Methods<<
101
Dorset, England
Sodium hydroxide (NaOH) BDH Laboratory Supplies, Poole,
Dorset, England
Sodium phosphate, dibasic, anhydrous
(Na2HPO4)
Sigma Chemical Co., St Louis, Mo.,
USA
Sodium phosphate, monobasic, anhydrous
(NaH2PO4)
Sigma Chemical Co., St Louis, Mo.,
USA
TEMED
(N’,N’,N’,N’-tetra-methyl-ethylenediamine)
Bio-Rad Laboratories, Hercules,
CA, USA
Tris (Hydroxymethyl methane) BDH Laboratory Supplies, Poole,
Dorset, England
Trypan Blue Promega Corp, Madison, Wi, USA
Trypsin Invitrogen Corporation
Tween-20 Sigma Chemical Co., St Louis, Mo.,
USA
1.1.2 Commercially purchased kits
ECL plus Western Blotting Detection Kit I Amersham Biosciences, Little Chalfont,
Buckinghamshire, UK
Rneasy Mini® Kit QIAGEN Gmbh, Australia
Taq DNA polymerase kit QIAGEN Gmbh, Australia
SYBR® Green PCR Master Mix Applied Biosystems, USA
1.1.3. PCR primers
All oligo primers were purchased from Proligo, Australia Pte Ltd. They were stored at
-20°C and diluted to 10µmol/ml prior to use.
1.1.4. Other materials and equipment
>>Appendix A. Materials and Methods<<
102
Materials and Equipment Manufacturer
ABI PRISM 7000 Sequence Detection
System
Applied Biosystems, USA
AdventureTM Pro Weighing Balance Ohaus Corp, New Jersey, USA
Baxter ddH2O Baxter, Sydney Australia
Carbon Dioxide (CO2) gas, food grade Aligal® 2, Air Liquid, WA, Australia
Cell Culture Flask (T-25) NuncTM A/S, Denmark
Cell Culture Falsk (T-75) Sarstedt Inc, Newton, USA
Cell Scraper Sarstedt, Germany
Centrifuge tubes: 10 ml, 50 ml Sarstedt, Germany
Cling Film Glad, Australia
CK30 Micrpscope Olympus
DNA ladder 100kb (250µl) Promega Corp, Madison, Wi, USA
Filter paper Whatmans International, England
GeneAmp PCR system 2400 Perkins-Elmer
Grant W-14 water bath Selby Scientific and Medical, USA
Hybond-C® N+ membrane Amersham Biosciences, Little Chalfont,
Bukinghanshire, UK
Liquid Nitrogen (N2) Air Liquid, WA, Australia
Microcentrifuge tubes: 1.5 ml Sarstedt, Germany
Microcentrifuge tubes: 0.5 ml, 2.0 ml TRACE Bioscience, Australia
Micropore filters (0.2, 0.45 & 0.8 μm) Crown Scientific, Australia
MiniSpin® centrifuge Eppendorf AG
P1210 Weighing Balance Metler, Zurich, Switzerland
Parafilm Laboratory film American National CanTM, Menasha,
Wi, USA
PCR 0.2 ml thin-walled tubes Unimed Australia Pty Ltd, Australia
>>Appendix A. Materials and Methods<<
103
PC-960C Colled Thermal cycler Corbett Research, Australia
PH211 microprocessor pH meter Hanna Intruments INC, Rhode Island,
USA
PolarStar Optima BMG Labtechnologies, Germany
Prestained SDS-PAGE standards Bio-Rad Laboratories, Herules, CA,
USA
PowerPac HC high-current power supply Bio-Rad, Hercules, CA, USA
Serological pipettes: 5 ml, 10 ml Life Technologies, Australia
Thermomixer comfort heating block Eppendorf AG
Transfer pipette, 1 ml Sarstedt, Germany
10 × 10 cm2 and 7 × 10 cm2 UV
transparent gent tray; 8 and 15 well comb
Bio-Rad, Hercules, CA, USA
1.1.5 Nature compounds
Astilbin Donated by Prof. Hua Gang Liu,
Department of Pharmacology, Guangxi
University of Medical Sciences, China
Caffeic acid phenethyl ester (CAPE) Alexis Biochemicals, USA
Mangiferin Sigma-Aldrich, UK
Naringin Sigma-Aldrich, UK
Parthenolide Alexis Biochemicals, USA
1.1.6 Antibodies
Anti-rabbit calcitonin receptor polyclonal Abcam, USA
Anti-rabbit α-tubulin polyclonal Sigma Chemical Co, St Louis, Mo, USA
Anti-rabbit IgG Sigma Chemical Co, St Louis, Mo, USA
1.1.7 Buffers and solutions
>>Appendix A. Materials and Methods<<
104
Solutions and Buffers were prepared using highly purified millique (milli-Q) double
distilled water and, where appropriate, were sterilized by autoclaving. All chemicals
were weighed using a P1210 Weighing Balance. Adjustments of pH were made using a
PH211 microprocessor pH meter, calibrated with appropriate pH standards (pH 4.0 or
7.0). All solutions were poured into autoclaved bottles and autoclaved (at 121ºC for
20mins) again where appropriate.
70% Ethanol
100% Ethanol 700ml
The solution was made up to 1L in MilliQ water, and stored at -20°C
100% Ethanol 700ml
The solution was made up to 1L in DEPC treated water, and stored at -20°C
Collagenase type II (4000 Ü/ml stock)
Collagenase type II powder (277Ü/mg) 1g
1 × PBS 69.25 ml
Solution is mixed completely, divided into 1ml aliquots and stored at -20°C
Collagenase type II (400 Ü/ml working conc.)
Stock Collagenase type II (4000 Ü/ml) 1ml
Serum-free DMEM-F/12 medium 9ml
Solution is mixed, divided into 5ml aliquots and stored at -20°C
Growth Medium (20% FCS) for chondrocyte culture
DMEM-F/12 400ml
Fetal Calf Serum (FCS) 100ml
L-Ascorbic Acid 0.5ml
Penicillin-Streptomycin (2X) 2.5ml
Solution mixed and stored at 4°C
>>Appendix A. Materials and Methods<<
105
Growth Medium for RAW246.7 cell culture
α-MEM 450ml
Fetal Calf Serum (FCS) 50ml
L-Glutamine 100mM 5ml
Penicillin-Streptomycin (2X) 2.5ml
Solution mixed and stored at 4°C
dNTP (5mM)
dATP 10 μl
dGTP 10 μl
dTTP 10 μl
dCTP 10 ul
ddH20 60 μl
Mixed, spin, and then stored at -20°C
Phosphate Buffered Saline (1× PBS)
Sodium Chloride 4.25g
Sodium phosphate monobasic 1.925g
di-sodium hydrogen orthophosphate 5.09g
Dissolved completely in ddH2O/Milli Q water to 500ml volume, adjust to PH 7.45 and
autoclave
RIPA RIPA
1M Tris (pH 7.5 or 8.0) 25g
NaCl 75g
Nonidet P-40/IGEPAL 5.0g
SDS 0.5g
Deoxycholate 5.0g
Autoclaved MilliQ ddH2O 500ml
>>Appendix A. Materials and Methods<<
106
Dissolved completely and stored at 4ºC.
Serum-Free chondrocyte culture Medium
DMEM-F/12 500ml
L-Ascorbic Acid 0.5ml
Penicillin-Streptomycin (2X) 2.5ml
Mixed and stored at 4°C
10 × SDS-PAGE Buffer
Tris 30g
Glycine 144g
SDS 10g
MilliQ ddH2O 1L
Placed in a 60ºC water bath for 30 minutes to dissolve.
Separating Gel Buffer
Tris 90.75g
SDS 2g
MilliQ ddH2O 450ml
Dissolved completed, adjust pH to 8.8, topped with MilliQ ddH2O to 500ml, and stored
at room temperature.
Stacking Gel Buffer
Tris 30.25g
SDS 2g
MilliQ ddH2O 500ml
Mixed completely and stored at room temperature.
Stripping Buffer
100mM β-mercaptoethanol 0.5ml
>>Appendix A. Materials and Methods<<
107
2% (w/v) SDS 10.0g
1M Tris-HCl (pH 6.7) 31.25ml
Topped up with MilliQ ddH2O to 500ml, and stored at room temperature.
50 × TAE Buffer
Tris Base 242g
Glacial Acetic Acid 57.1ml
0.5 M EDTA (pH 8.0) 100ml
Mixed completely, top up with MilliQ water to 1L and stored at room temperature.
1 × TAE Buffer
50 × TAE Buffer 40ml
MilliQ ddH2O 1960ml
Mixed completely, and stored at room temperature.
10 × TBS Buffer
Tris 30.275g
NaCl 43.83g
Add 450ml MilliQ ddH2O, adjust pH to 7.4, and top up with MilliQ ddH2O to 500ml.
Stored at room temperature.
Transfer Buffer (Western Blot)
Glycine 7.57g
Tris 36g
MilliQ ddH2O 1.6L
Topped up to 2L with Methenol, mixed completely, and stored at room temperature.
1 × TBS Buffer
10 × TBS Buffer 50ml
MilliQ ddH2O 450ml
>>Appendix A. Materials and Methods<<
108
Mixed completely, and stored at room temperature.
1 × TBS-tween Buffer
1 ×TBS buffer 500ml
Tween-20 5ml
Mixed completely, and stored at room temperature.
1M Tris-HCl
Tris 60.55g
MilliQ ddH2O 500ml
Add HCl to a pH of 6.8, mixed completely, and stored at room temperature.
0.05% Trypsin
2.5% Trypsin 10ml
Sterile PBS 90ml
Solution mixed, divided into 5ml aliquots and stored at -20°C
0.05% Trypsin-EDTA
0.5% Trypsin-EDTA 10ml
Sterile PBS 90ml
Solution mixed, divided into 5ml aliquots and stored at -20°C
1.1.8 Centrifugation
All micro-centrifugation (0.2 ml, 0.5 ml, 1.5 ml and 2.0 ml) procedures at room
temperature were performed using a Biofuge A microcentrifuge. Micro-centrifugation
at 4°C was carried out in a Centra-M2 centrifuge. Centrifugation of solutions in 10 and
50 ml centrifuge tubes were performed using a Hettich Universal 2S centrifuge. All high
speed and large volume (250 ml and 500 ml) centrifugation procedures were performed
in the J-2 Series High Speed Centrifuges (Beckman Instruments) at 4°C unless
otherwise specified.
>>Appendix A. Materials and Methods<<
109
1.1.9 Tissue Samples and Cells
1.1.9.1 Human cartilage and chondrocytes
The human cartilage used in this project was from the discarded tissue of the knee joint
from patients undergoing joint replacement surgery in the Hollywood Private Hospital.
The normal chondrocytes used as a control sample were from patients in the Hollywood
Private Hospital or from Verigen, Australia. The utilization of human tissues in this
project has been approved by Human Research Ethics Committee Research Services of
UWA (reference No. RA/4/1/1046).
1.1.9.2 RAW264.7 cells
The cell line was purchased from American Type Culture Collection.
1.1.10 Computer Softwares
Endnote 12.0
Microsoft Office Excel 2003
Microsoft Office PowerPoint 2003
Microsoft Office Word 2003
Adobe Photoshop CS2
SPSS 12.0
1.2 GENERAL METHODS
1.2.1 Primary Culture of Chondrocyte
1.2.1.1 Cartilage Samples collection
Immediately after knee joints released from patients undergoing joint replacement
surgery of OA, harvest cartilage tissue samples were stored into sterile biopsy jars
containing serum-free medium and transported from operation theatre to fume hood
conditions for further treatment. Perioperatively, cartilage tissue samples were cleaned
of any adherent muscular, connective or subchondral bone tissue, and then placed into
>>Appendix A. Materials and Methods<<
110
50ml tubes containing serum-free DMEM-F/12 medium, since serum inhibits the
efficacy of following collagenase digestion.
1.2.1.2 Matrix digestion and chondrocyte isolation
In order to successfully cultivate chondrocytes, they must first be isolated from their
dense extracellular matrix. Cartilage was first washed in serum-free DMEM-F/12
medium three times. Harvest cartilage was then transferred from the 50ml tubes into
petri dishes with a few drops of warm type II collagenase (400units/ml). Before
digestion, the cartilage must first be mechanically broken down to maximize the
effectiveness of enzymatic digestion. Using two 23-grade surgical scalpels, harvest
cartilage was diced into fine pieces approximately 200μm to 500μm in size. The diced
pieces were then transferred (using sterile pasteur pipettes) into a 25ml flask. Totally
10ml warmed type II collagenase (800units/ml) was added to the flask. Flasks were
then placed upright on shaker at 37°C for 3 hours. After digestion is complete, flasks
were removed from the incubator and collagenase digestion was ceased by addition of
an equal volume of warm growth medium. Cell suspension was transferred to sterile
tubes and twice centrifuged at 2000rpm for 10 minutes then washed in serum-free
medium to remove supernatant matrix debris. Cells were then resuspended in 5ml fresh
growth medium (20% FCS) and seeded into a T25 tissue culture flask. Cells were
incubated at 37°C in 5% CO2 in a cell incubator. Half the volume of growth medium
was replaced after 24 hours, and then every 2-3 days following until cells were totally
confluent over the flask surface. Once chondrocyte confluence was achieved, cells were
trypsinised into T75 tissue culture flasks to obtain a greater cell yield.
1.2.1.3 Chondrocyte cultivation and trypsinisation
After chondrocyte confluence was achieved in T25 tissue culture flask, cells were
removed via trypsin treatment to larger T75 tissue culture flasks. Growth medium was
removed from the T25 tissue culture flask and the well was washed with Ca/Mg free
PBS to ensure total serum evacuation (trypsin is inhibited by serum). Once wells were
serum free, about 3ml of prewarmed trypsin was added via the side of the well until the
>>Appendix A. Materials and Methods<<
111
cell surface was covered. Flasks were then incubated at 37°C in the cell incubator for
2-3 minutes in 5% CO2 until the cell layer could be observed as a detached mass in the
trypsin solution. Total detachment was achieved by tapping the bottom of the flask to
dislodge any resilient cell populations. After total detachment was complete, the trypsin
reaction was ceased by addition of FCS and flasks were stood upright to allow all the
cells to drain to the bottom of the flask. To gain the highest cell yield, the monolayer
was washed by repeat pipetting of the serum/trypsin solution. Then, the cell solution
were transferred to a 50 ml tube and centrifuged at 2000rpm for 10 min to remove the
trypsin solution. Cells were then resuspended in 25ml growth medium (10% FCS) and
seeded into larger T75 tissue culture flasks for further expansion. The growth medium
was changed every two days.
1.2.1.4 Estimation of Cell Number
After cells were centrifuged to remove the trypsin solution, a 500μL PBS aliquot of
cells was removed in three samples and stained with 500μL of Trypan Blue (0.4%).
Cells were left in Trypan Blue for 5 minutes then delivered to both sides of a
haemocytometer by capillary action using pasteur pipettes. Cells were counted using a
Neubauer haemocytometer under a Nikon Diaphot phase-contrast microscope. Cells
were counted in five of the nine large squares in each of the two sides of the
haemocytometer for a total of 10 squares. The number of cells in ten chambers is
calculated to give the number of cells in 1 x 103 ml. To calculate the number of cells per
millilitre, the total number of cells is multiplied by 1000, and then multiplied by 2 to
account for the dilution of 500μL PBS aliquot of cells in 500μL of Trypan Blue. Only
blue (viable) cells were included in cell number estimation. The haemocytometer is
rinsed with DDW and 70% ethanol between uses and dried with lens paper.
1.2.2 RNA extraction from cultured chondrocytes
RNA was isolated from cultured chondrocytes using RNeasy Mini kit (QIAGEN)
according to the manufacture’s protocol. Human chondrocytes, grown as monolayers in
T-75 culture flasks, were lysed directly in the culture dish by the addition of Buffer RLT.
>>Appendix A. Materials and Methods<<
112
All steps of the protocol were performed at room temperature. Firstly, the growth
medium was removed from the culture flask by using transfer pipet. Cells were
washed in 5ml of sterile Ca2+, Mg2+ free PBS 2 times to get rid of any trace of serum.
600µl of Buffer RLT was added to per 75 cm2 flask of chondrocytes (approximately
6×106). The cell lysate were then transferred into a sterile, RNase-free, 1.5ml eppendorf
tubes, mixed completely to ensure that no cell clumps were visible. 600µl of ethanol
in DEPC water were added to each tube, mixed well by pipetting. Subsequently, the
mixture was pipetted to an RNeasy mini column placed in a 2 ml collection tube, and
centrifuged for 15s at 10,000 rpm. The flow-through was discarded and the resultant
RNA was bound to the RNeasy silica-gel membrane. Six hundred µl of Buffer RW1
was added to each sample to wash the column and then centrifuged for 15s at 10,000
rpm. The flow-through was discarded and the washing procedure was repeated twice.
Thereafter, the RNeasy column was transferred into a new 2 ml collection tube, added
500µl of Buffer RPE and centrifuged for another 15s at 10,000 rpm to wash the column.
After the flow-through discarded, another 500µl of Buffer RPE was added to the
RNeasy column and centrifuged for 2 min at 10,000 rpm to dry the RNeasy silica-gel
membrane, ensuring that on ethanol was carried over to interfere with downstream
elution reaction. Finally, the column was transferred to new sterile, RNase-free, 1.5 ml
eppendorf tube, added 30µl RNeasy-free water and centrifuged for 1 min at 10,000 rpm
to elute the RNA. The 1.5 ml tube, which contained the desired RNA, was labeled and
stored at -70C before further experiment.
1.2.3 Reverse transcription for cDNA preparation
RNA was thawed on ice prior to use. A reaction mixture (as shown below) was prepared
in a 0.5ml tube. The following is for one reaction tube.
Mixture 1
Template RNA 2µl
dNTP(5mM) 2µl
Oligo dT 0.25µl
>>Appendix A. Materials and Methods<<
113
ddH2O 9.75µl
——————
Total volume/tube 14µl
The reaction mixture was gently mixed and centrifuged briefly, and then heated to 75°C
for 3 mins to melt any secondary structure within the template. The tubes were
centrifuged again after heated and placed on ice to prevent secondary structure
reforming. The following mixture was then added to each reaction sample.
Mixture 2
5 × RT Buffer (M-MLV) 4µl
Rnasin inhibitor 1µl
M-MLV RT 1µl
——————
Total volume/tube 20µl (+14µl from above)
Again, the mixture was gently mixed and centrifuged briefly, and then incubated at
42°C for 1hr in a thermal cycler (Perkins-Elmer Gene Amp 2400). Finally, the tube
was heated for a further 10 minutes at 92°C to inactivate the reverse transcriptase.
Upon completion, the reaction sample was purified and stored at -20°C.
1.2.4 Standard PCR
The cDNA was further amplified by PCR reaction. In a 0.5 ml PCR reaction tube, the
master mixture was prepared on ice as follows.
Master mixture (per reaction):
cDNA 2 µl
Primer (forward) 0.5 µl
Primer (reverse) 0.5µl
Tap Polymerase 0.5 µl
>>Appendix A. Materials and Methods<<
MgCl2 (25mM) 2.0 µl
dNTP (5mM) 2.0 µl
10 × PCR Buffer 2.5 µl
ddH2O 15 µl
——————
Total volume/reaction 25µl
The mixture was mixed and centrifuged briefly, then placed in the GeneAmp PCR
system 2400 machine and subjected to the following program. The annealing
temperature may vary from the primers used. For the Real-time PCR performing, the
annealing temperature of the primers used in this study was consistent as 60°C. Primer
sequences are listed in table 3.
Program
94°C (activating Hot start polymerase) 3 mins
94°C (denaturing double-stranded DNA) 40 s 35 cycles 60°C (primer annealing) 40 s
72°C (DNA synthesis) 40 s
72°C 10 mins
4°C
114
>>Appendix A. Materials and Methods<<
115
Appendix Table A. 1 Oligonucleotide primers used in PCR amplification
Gene Accession No. Primer A.T.
*
Bp**
Aggrecan M55172 *fw: TCCCCTGCTATTTCATCGAC
**rv: CCAGCAGCACTACCTCCTTC
60 117
Type I
Collagen
NM_000088 fw: GGCCCAGAAGAACTGGTACA
rv: AATCCATCGGTCATGCTCTC
60 81
Type II
Collagen
NM_001844 fw: CTGGAAAAGCTGGTGAAAGG
rv: GGCCTGGATAACCTCTGTGA
60 105
Type X
Collagen
NM_000493 fw: AATGCCCACAGGCATAAAAG
rv: AGGACTTCCGTAGCCTGGTT
60 187
Fibromo- dulin NM_002023 fw: CCACTTCACCCACTCCACTT
rv: CTGGTGACCTCCAATCTGGT
60 201
Fibronectin NM_212482 fw: GAAGAGCGAGCCCCTGAT
rv: GGGGTCTTTTGAACTGTGGA
60 123
Link Protein
(HAPLN1)
NM_001884 fw: TGCTCAGATTGCAAAAGTGG
rv: TATCTGGGAAACCCACGAAG
60 170
MMP-1 NM_002421
fw: AGGTCTCTGAGGGTCAAGCA
rv: CCAGGTCCATCAAAAGGAGA
60 82
MMP-3 NM_002422
fw: GAAGCTGGACTCCGACACTC
rv: GATCCAGGAAAGGTTCTGA
60 88
MMP-9 NM_004994
fw: GATGCGTGGAGAGTCGAAAT
rv: CTATCCAGCTCACCGGTCTC
60 87
MMP-13 NM_002427 fw: TTGAGCTGGACTCATTGTCG
rv: TGCAAACTGGAGGTCTTCCT
60 84
ADAMTS-4 NM_005099 fw: AGGGAAGGGGACAAGGACTA
rv: TATCACCACCACCCTGGATT
60 95
ADAMTS-5 NM_007038 fw: TACTTGGCCTCTCCCATGAC
rv: TTTGGACCAGGGCTTAGATG
60 119
>>Appendix A. Materials and Methods<<
116
TIMP-1 NM_003254 fw: CTTCTGGCATCCTGTTGTTG
rv: AGAAGGCCGTCTGTGGGT
60 84
TIMP-2 NM_003255 fw: AAGCGGTCAGTGAGAAGGAA
rv: TCTCAGGCCCTTTGAACATC
60 108
TIMP-3 NM_000362 fw: ACCTGCCTTGCTTTGTGACT
rv: GGCGTAGTGTTTGGACTGGT
60 95
SOX9 NM_000346 fw: GGAATGTTTCAGCAGCCAAT
rv: TGGTGTTCTGAGAGGCACAG
60 115
c-fos K00650 fw: GGAGGTGATGGCAGACACTT
rv: CCCTTCGGATTCTCCTTTTC
60 96
c-jun U35005 fw: CAGTCAGGCAAGGGATTTGT
rv: ATGTACGGGTGTTGGAGAGC
60 90
IL-1β NM_000576 fw: ACAGATGAAGTGCTCCTTCCA
rv: GTCGGAGATTCGTAGCTGGAT
60 73
TNFα NM_000594 fw: ACGGAGAAGAAGCAGACCAA
rv: CGCAGTTCAAAGGTCTCCTC
60 173
TGFβ NM_000660 fw:
TTGCAGTGTGTTATCCGTGCTGTC
rv:
CAGAAATACAGCAACAATTCCTG
G
60 187
IGF1 NM_000618
fw: GAGTCTGGCCAAAACGGTAA
rv: CCCAAATGGATGGTGTTTTC
60 102
COX-2 NM_000963 fw: CCTTCCTCCTGTGCCTGATG
rv:
ACAATCTCATTTGAATCAGGAAG
CT
60 81
MAPk1 NM_002745 fw: CAAGACCATGATCACACAGG
rv: CTGGAAAGATGGGCCTGTTA
60 163
NOS2 NM_153292 fw: ACAAGCCTACCCCTCCAGAT 60 158
>>Appendix A. Materials and Methods<<
117
rv: TCCCATCAGTTGGTAGGTTC
HSP70 NM_005346 fw: CGACCTGAACAAGAGCATCA
rv: AAGATCTGCGTCTGCTTGGT
60 213
Ribosomal K03432 fw: AAACGGCTACCACATCCAAG
rv: CAATACAGGGCCTCGAAAG
60 112
GAPDH BC029340
fw: GAAGGTGAAGGTCGGAGTC
rv: GAAGATGGTGATGGGATTTC
60 226
18S X03205 fw: CCTGCGGCTTAATTTGACTC
rv: AACTAAGAACGGCCATGCAC
60 119
* annealing temperature; **product size
>>Appendix A. Materials and Methods<<
118
After the reaction was completed, the PCR products were then detected by running an
agarose gel as described in section 4.2.7 or stored in -20°C.
1.2.5. Agarose gel electrophoresis
Agarose gel electrophoresis is a routine technique in molecular biology for the
separation of molecules based on their size. Initially, a slab of agarose gel material was
cast by melting a quantity of agarose powder in a volume of 1×TAE in a microwave
oven at a medium-high setting for 3 mins. Care was taken not to boil the solution for
excessive periods as this would cause evaporation of water thus an increased gel
concentration. The quantity of agarose dissolved and the volume of 1×TAE used is
summarized as below. The gel percentage used in this thesis was 1.2%.
7 × 19 cm2 Gel 10 × 10 cm2 Gel
Agarose Powder (g) 0.6 1.2
1 × TAE Buffer (ml) 50 100
Ethidium Bromide (µl) 1.5 3.0
Upon dissolution of the agarose in 1×TAE, the molten gel was allowed to cool at a room
temperature for 2-3 minutes. An aliquot of ethidium bromide was then added to the
solution (varied with gel size 1-3µl) to give a final concentration of 0.5µl/ml. the
solution was then poured into a Perspex gel tray taped at the open ends.
Approximately 50ml was poured into a small tray (7cm×10cm) and 100 ml into a large
gel tray (10cm×15cm). A well forming comb was positioned at one end of the gel tray.
The gel was allowed to solidify at room temperature for about 20 minute. Once the
agarose gel hardened, the adhesive tape on the open ends was removed. The agarose
forms a matrix of long thin molecules forming submicroscopic pores. The size of the
pores can be controlled by varying the concentration of the agarose.
>>Appendix A. Materials and Methods<<
119
The well comb was removed and the gel slab was placed in a horizontal electrophoresis
tank (Bio-Rad) and submerged in 1×TAE buffer. Individual PCR products and DNA to
be electrophoresed were then combined with spots of gel loading buffer on a sheet of
paraffin wax and pipetted into the wells. A mixture of 2µl of gel loading buffer and 3µl
100bp DNA Ladder or 1kb DNA ladder (Promega), selected by the molecular size of the
DNA products, was loaded at the first well as molecular weight standard.
Electrophoresis was then achieved by applying an electric field across the gel. The DNA,
which is negatively charged due to its phosphate backbone, migrates toward the positive
electrode. Then the gel ran for 30 minutes to separate the PCR samples by different
sizes.
1.2.6 Real-time PCR
1.2.6.1 Apparatus and Reagents
Relative quantitative real-time PCR was performed on the ABI Prism® 7000 Sequence
Detection System (Applied Biosystems) using the Sybr Green PCR Master Mix
(Applied Biosystens) according to the manufacturer’s instructions. The total volume of
the PCR reaction was 10μl, which consisted of 2.5μl Sybr Green PCR Master Mix,
2.5μl ddH2O, 1.5μl cDNA and 5µM of the each of the forward and reverse primers.
The real-time PCR reaction was carried out at 95°C for 10 min (activation), for 40
cycles of 95°C for 15 s, 60°C for 20 s and 72°C for 20 s (amplication) and 72°C for 1
min (final extension).
1.2.6.2 Standard Creation for Real-time PCR
Primers used in this project were described as above. Once primers have been designed,
their performance should be tested. Adjusting MgCl2 and primer concentrations to
optimize primer performance was suggested in other protocol. The Applied Biosystems
SybrGreen Master Mix employed in this thesis works sufficiently without optimization
of MgCl2 concentration.
In relative quantitative Real-time PCR, a standard curve is generated from a dilution
>>Appendix A. Materials and Methods<<
120
series of known concentration purified DNA. A purified stock of the primer product is
necessary. cDNA prepared as described in Section 1.2.3 was amplified using regular
PCR, with the primers used for Real-time PCR, around 40 cycles. The amplified cDNA
containing the PCR product in high abundance should be free of dNTPs, other
unspecific cDNA, nucleotides, and perhaps primer dimmers or salt. The PCR products
(dsDNA) were purified using Micro Bio-Spin P-30 Tris Chromatography Columns
(Bio-Rad) according to the manufacturer’s instructions. Briefly, the Micro Bio-Spin
Column was pre centrifuged at 1,000×g for 2 min in a microcentrifuge to remove the
packing buffer. Following this, the column was placed into a new 1.5ml eppendorf.
The PCR product (20-70μl) was added carefully into the center of the column,
centrifuged for 4 min at 1,000×g. Additionally, to calculate the speed (rpm) required
to reach a gravitational force of 1,000×g, the following equation was used.
RCF (g) = (1.12×10-5) (rpm)2 r
Where r is the radius in centimeters measured from the center of the rotor to the middle
of the Micro Bio-Spin column, and rpm is the speed of the rotor in revolution per
minute.
And then, the purified PCR product was harvested into the 1.5ml eppendorf. The
quantity of the PCR product was determined by the ratio of A260/A280 on SmartSpec
Spectrophotometer (Bio-Rad). According to this result, the PCR product was diluted
into 1×10-4ng/μl and then further serially diluted in two- fold increments (1, 0.5, 0.25,
0.125, and 0.0625). The diluted stock was stored at -20°C until use.
The serially diluted PCR products were then further amplified by using the ABI Prism®
7000 Sequence Detection Systems (Applied Biosystems). A non-template control (NTC)
which contains SybrGreen Master Mix, primers, and water, but not primer product was
run in the same plate. The Real-time PCR reaction was carried out, according to the
reaction program as shown. During amplification, fluorescence emission resulting from
the Sybr Green dye binds to double-stranded DNA is recorded by the ABI Prism® 7000
Sequence Detection software (Applied Biosystems), which processed and analyzed raw
>>Appendix A. Materials and Methods<<
121
fluorescence data to produce threshold cycle (Ct) values for each sample. Ct values
indicate intersection of the exponential phase of an amplification curve and a set
threshold. It is a quantitative measurement of the input target amount because Ct
decreases linearly with increased amplified products (Heid et al., 1996). Any
amplication in the NTC indicated primer dimering. Log-linear standard curves were
created by plotting relative copy number on the Y-axis and the Ct value on the X-axis.
All reactions were carried out in duplicate. The standard curves of genes studied in this
thesis were shown in Figure B.1-4.
1.2.6.3 Standard Creation for Real-time PCR
Melting Curve Analysis was performed after Real-time PCR. The plate temperature was
increased from 60°C to 92°C with a fluorescence reading performed at each temperature.
As the temperature increased, dsDNA denatured and the fluroscence level decreased.
Denaturation temperature (Tm) is related to sequence length, hence a single Tm peak
observed in the melting curve plot indicated the specificity of the PCR product and
good primer performance (Figure B.5). The presence of a lower Tm peak indicates
non-specific binding or primer dimmers (Figure B.6). As the fluorescence level is a
relative measurement, the larger the peak at the lower Tm the greater the amount of
primer dimmers compared to primer product. Hence, primer dimmers for more
abundant transcripts are out-competed and not be a problem. However, the presence of
large low Tm peaks indicates poor primer performance, hence the primers need to be
redesigned.
1.2.6.4 Relative Quantification
Relative quantitative Real-time PCR was preformed on a 384-well/plate ABI Prism
7000 Sequence Detection machine (Applied Biosystems) using Sybr Green PCR Master
Mix (Applied Biosystems). The total volume of each PCR reaction was 10µl, which
contains 5µl Sybr Green PCR Master Mixture, 2.5µl ddH2O, 1.5µl cDNA and 5µM of
each of the forward and reverse primers. The Real-time PCR reaction was carried out at
95°C for 10 minutes (activation), 40 cycles of 95°C for 15s, 60°C for 20s and 72°C for
>>Appendix A. Materials and Methods<<
122
20s (amplicaition) and 72°C for 1 minute (final extension). Housekeeping gene GAPDH
used as endogenous control was always run on the same plate.
1.2.6.5 Data analysis
1.2.6.5.1 Data normalization for relative quantitation
Ct values collected by the ABI Prism® 7000 Sequence Detection software (Applied
Biosystems) was conversed into relative copy number of examined gene by using the
formula:
Relative copy number= 2^(-Ct×slope+offset)
where offset was normalized as 40; slope is obtained from the standard curve of each
interesting gene. The relative copy number of each detected gene was then normalized
by the expression level of housekeeping genes. Expression levels further than three
standard deviations from the mean were considered outliners and removed.
1.2.6.5.2 Comparative 2− ΔΔCt method
The comparative 2− ΔΔCt method was used to calculate the relative expression of each
target gene as previously described (Livak et al., 2001). Briefly, mean Ct value of target
genes in experimental group was normalized to it averaged Ct value of housekeeping
gene to give a ΔCt value, which was further normalized to control samples to obtain a
ΔΔCt value. The value of 2− ΔΔCt was then calculated and compared among the
experiment groups.
1.2.6.5.3 Cluster analysis
Cluster analysis is termed as a number of different algorithms and methods for grouping
objects of similar kind into respective categories. It is used to sort observe data into
groups in a way that the degree of association between two objects is maximal if they
belong to the same group and minimal otherwise.
Clustering analysis done in the Massachusetts Institute of Technology (MIT), U.S. was
performed to further analyze the expression patterns associated with passage number.
>>Appendix A. Materials and Methods<<
123
Clustering analysis with different group number was done to obtain the best clustered
pattern.
1.2.7 Immunostaining
1.2.7.1 Cartilage Tissue preparation
The normal human cartilage tissue section used in the study of calcitonin receptor was
kindly donated by Genzyme/Verigen Australia. The articular cartilage was taken from
patient who has undergone total knee joint replacement after failed autologous
chondrocyte implantation surgery. The tissue biopsy was dissected from the disease-free
region, fixed, decalcified, processed and sectioned for immunostaining.
Sample was fixed by placed into a sterile biopsy jar containing 4% paraformaldehyde
for 7 days at room temperature with no movement. Sample was then decalcified in 10%
Formic acid for 14 days at room temperature. Formic acid solution was changed every
3-4 days to maximize calcium extraction.
After decalcification, cartilage sample was processed in a Miles Scientific Tissue Tek®
vacuum infiltration processor over 24 hours using the automatically controlled protocol
detailed by Table 4.2.
>>Appendix A. Materials and Methods<<
124
Appendix Table A.2 Automated processing run (24-hour) for cartilage biopsies
Reagent Time Temperature
Station 1 70% Alcohol 45 37
Station 2 70% Alcohol 45 37
Station 3 95% Alcohol 45 37
Station 4 95% Alcohol 45 37
Station 5 Alcohol 120 37
Station 6 Alcohol 120 37
Station 7 Alcohol 120 37
Station 8 Alcohol/Chloroform 120 37
Station 9 Chloroform 120 37
Station 10 Chloroform 120 37
Station 11 Paraffin Wax 120 60
Station 12 Paraffin Wax 180 60
Station 13 Paraffin Wax 240 60
Total time 24hours
>>Appendix A. Materials and Methods<<
125
Standard microscopy slides were washed in 2% Decon 90 (v/v) with gentle agitation for
30 minutes, rinsed in running tap water, then DDW. Slides were then immersed in
acetone for 1-2 minutes, 3- aminopropyltriethoxy-saline in acetone for 5 minutes,
washed in DDW for 1 minute, and left to dry overnight at room temperature. After
processing, paraffin embedded samples were sectioned to 5μm using a Reichert- Jung®
2030 microtome. Sections were floated in a 40°C waterbath and adhered to salinated
microscopy slides. Sections were dried overnight at 37°C.
1.2.7.2 Giant Cell Tumor imprint
Giant cell tumor (GCT) imprint slices were used in the study of calcitonin receptor as
positive control to investigate the expression of calcitonin receptor in osteoclast and
chondrocyte. GCT tissue was wetted in PBS buffer and evenly stamped on standard
microscopy slides. The section was then air dried at room temperature and stored at -20
ºC. Previous to immunostaining, the sections was fixed in 4% paraformaldehyde for 20
minutes.
1.2.7.3 Immunostaining Methodology
Parafin embedded sections were then dewaxed, hydrated and stained for analysis by
immunostaining as follow described.
Sections were deparaffinised by treatment in 3 changes of fresh xylene (RNAse-free) at
3 minutes each. Sections were then rehydrated by the following steps: 3 changes of
100% ethanol for 3 minutes each, 1 change of 95% ethanol for 3 minutes, 1 change of
70% ethanol for 3 minutes, and finally rinsed for 10 minutes in running tap water.
Subsequently, each slide was blocked for endogenous peroxidase activity by incubation
in 3% H2O2 for 10 minutes, washed with 3 changes of water and last time in 1 × tris
buffered saline (TBS), and then blocked non-specific binding with 20% FBS for 30
minutes followed by incubation with 1 in 100 dilution of the primary anti-CTR antibody
(Abcam, ab11042) at 4ºC in a humidified chamber overnight. The next day, slides were
incubated with anti-rabbit Ig antibody in the link solution from the LSAB TM2kit (Dako
>>Appendix A. Materials and Methods<<
126
Pty Ltd., Australia) for 20 min at room temperature followed by 3×5 minutes washes
in 1×TBS, and incubated by the Streptavidin-HRP solution from the LSAB TM2kit in a
moist chamber at room temperature using for another 20 min (Dako Pty Ltd., Australia).
Following 3×5 minutes washes in 1×TBS, the sections were color developed by using
a liquid 3,3’-diaminobenzidine (DAB+) substrate-chromogen solution kit (Dako Pty
Ltd., Australia). The reaction was stopped by washing in several change of water.
Preparation in which the primary antibody was omitted served as a negative control.
Slides were counterstained in hematoxulin and mounted in Depex. Light microscope
was used to detect the staining.
1.2.10 Western-blotting
Western Blotting is commonly used in molecular biology to determine the relative
amounts of the protein present in different samples with a specific primary antibody.
Briefly, 1) Samples are prepared from tissues or cell lysate with protease inhibitor to
protect the protein of interest from degradation; 2) The sample is separated using
SDS-PAGE gel and then transferred onto a membrane for detection; 3) The membrane
is incubated with a generic protein (such as milk proteins) to bind to any remaining
sticky places on the membrane. A primary antibody is then added to the solution which
is able to bind to its specific protein; 4) A secondary antibody-enzyme conjugate, which
recognizes the primary antibody is added to find locations where the primary antibody
bound.
1.2.10.1 Preparation of cell/tissue lysate
Ripa Ripa Buffer supplied with 1:100 protease inhibitor was used to lysate cell pellets
or tissue samples for western blot analysis. Tissue samples were first powdered in liquid
nitrogen. Cell pellets were thawed on ice and incubated with the lysis buffer on ice for
20mins before cells were sonicated and centrifuged at 13,000rpm for 30mins at 4ºC.
The supernatant was then transferred into a clear labeled eppendorf and stored at -80ºC
for further experiment.
>>Appendix A. Materials and Methods<<
127
1.2.10.2 Preparation of SDS-PAGE gel
A polyacrylamide gel was prepared to separate the protein by electrophoresis. The 10%
SDS-PAGE was made as follows:
Separating gel solution:
Reagents Volume
30% Stock Acrylamide 5ml
Separating Buffer 3.75ml
MilliQ ddH2O 6.185ml
10% APS 50µl
TEMED 15µl
Firstly, the appropriate volume of 30% stock acrylamide, separating buffer and MilliQ
ddH2Q was added into a beaker. The solutions were swirled to ensure that it was well
combined. Then, APS and TEMED were added. TEMED was last added to prevent
pre-mature solidification of the gel in the flask.
After that, the solution was carefully transferred into a Mini-PROTEAN® 3 cell
Electrophoresis System (Bio-Rad, USA) according to the manufacturer’s instruction.
The separating solution was cast into a preassembled glass plate sandwich aligned with
the bottom of the casting frame assembly and allowed 15~20 minutes to set at room
temperature. Twenty percent of ethanol was overlayed onto the separating gel to remove
bubbles and to smooth the surface. While setting the separating gel, a stacking gel
solution was prepared as follows:
Stacking gel solution:
Reagents Volume
30% Stock Acrylamide 0.65ml
Stacking Buffer 1.25ml
MilliQ water 3.05ml
>>Appendix A. Materials and Methods<<
128
10% APS 50µl
TEMED 5µl
A distinct separation was formed between the gel solution and the ethanol indicating the
gel was set. The ethanol was removed and the stacking solution was overlayed on top of
the separating gel. A 1.5mm 10-well comb was lodged into the stacking gel which was
left to set for another 20 minutes at room temperature. Once set, the comb was carefully
removed, and the gel was placed into a SDS-PAGE apparatus.
Briefly, the SDS-PAGE gel was assembled on one side into the slots at the bottom of the
Electrode Assembly with the short plate of the gel sandwich facing inward towards the
notches of the U-shaped gaskets. On the other side, an assemble cassette was inserted
onto the slots to form the inner chamber of the Electrode Assembly. The insertion
cassette was done to balance assembly when there was only gel to be run. The slide gel
sandwich and Electrode Assembly was loaded into the claming frame and the two cam
levers were closed to tightly seal the inner chamber. The inner chamber was then
lowered into the Mini Tank and filled with 1×SDS-PAGE running buffer to the top to
cover the inner chamber and the electrode wired of the Mini Tank.
Protein extractions were thawed and mixed well. Before loaded, the samples were
denatured to enable the antibody to access to the portion of the protein within the 3-D
conformation. Thirty microlitres of each sample was mixed with 10µl of SDS-PAGE
loading buffer and boiled at 95ºC for 5 minutes. The samples were then spinned at
13,000rpm for 10 to 15 seconds to allow all the solution to settle at the bottom. Five
microlitres of prestained protein molecular weight marker was gently loaded into the
well, along with 35µl of each sample side by side. The gel was electrophoresed at 170V
for approximately 45 minutes to 1 hour or until the bands of the loading buffer ran off
the bottom of the gels.
1.2.10.3 Western blot analysis
>>Appendix A. Materials and Methods<<
129
1.2.10.3a Tranferring of proteins into nitrocellulose membrane
After the gel electrophoresis has done, the gel was ready to be transferred onto nitro
cellulose membrane. The western blot sandwich was opened and placed onto a tray
containing transfer buffer with the black side on top. Six pieces of 3MM filter paper, 2
absorbent sponges (one thick and one thin) and a nylon membrane was sumerged in the
transfer buffer. A thin absorbance sponger, followed by 3 piece of 3MM filter paper,
was placed on the clear side of the sandwich. HybondC® cellulose membrane was
placed on top of the 3MM filter paper.
The gel in the electrophoresis system was now removed. The stacking gel was removed,
and the separating gel was rinsed with running tap water. The gel was carefully
removed from the glass plates and placed onto the membrane on the western blot
sandwich, followed by another 3 pieces of 3MM filter paper, ensuring that there was no
air bubbles between each level. The thick absorbance sponge was then placed on top,
and the frame was gently closed to complete the sandwich. The sandwich was placed
horizontally with the black side of it facing the black side of the frame and the whit
latch facing upwards. An ice pack was placed inside the tank to ensure that the
temperature was kept cool. The lid of the tank was applied, and the system was
electrophoresed at 15V over night to ensure complete transfer of proteins onto the
membrane.
1.2.10.3b Probing the membrane with Primary Antibody
After the overnight transfer, the membrane was removed from the western blot
sandwich and washed once with 1×TBS Tween buffer for 5 minutes on the rocking
machine.
During the wash, the blocking agent was prepared. A 5% skim milk solution was
prepared by dissolving 0.5g of skim milk powder in 10ml of 1×TBS Tween buffer.
The solution was mixed completely by vortex. Then, the blocking solution was poured
gently over the membrane. The membrane was incubated for 1 hour at room
>>Appendix A. Materials and Methods<<
130
temperature on the rocking machine.
Subsequently, the blocking agent was removed, and the membrane was washed once
with 1×TBS Tween buffer for 5 minutes on rocking machine. The primary antibody
agent was prepared during the wash. Firstly, skim milk powder (0.1g) was weight and
dissolved in 10ml of 1×TBS Tween buffer to achieve a 1% blocking solution. The
primary antibody (anti-CTR antibody, 1:500; anti-α tubulin antibody, 1:1000) was
added to the 1% skim milk solution. The solution was mixed completely by vortex, and
then poured gently over the membrane, which was then incubated for another 2 hours at
room temperature on the rocking machine.
1.2.10.3c Probing the membrane with Secondary Antibody
After incubated with the primary antibody, the membrane was washed with 1×TBS
Tween buffer for 3×5 minutes on the rocking machine at room temperature. The
secondary antibody solution was prepared. The secondary antibody was added into
10ml of 1% skim milk solution, and mixed completely. After 3 times wash, the prepared
secondary antibody solution was gently poured over the membrane. The membrane was
incubated for 45 minutes at room temperature on the rocking machine.
After the incubation, the solution was poured away, and the membrane was subjected
for 2×5 minutes wash with 1×TBS Tween buffer followed by another 2×5 minutes
wash with 1×TBS buffer.
1.2.10.3d Visualization of Bands
During the washing steps, the ECL plus Western Blotting Detection Kit I (Amersham,
UK) was brought to room temperature for 20 minutes. Two microliter of Lumigen
Detection Solution A (Amersham, UK) was mixed with 50µl of Lumigen Dectection
Solution B (Amersham, UK). The mixture was carefully added drop by drop onto the
membrane ensuring that the whole membrane was covered. The membrane was then
visualized by exposure in a LAS-3000 imaging system (Fuji Photo Film Co., Ltd,
>>Appendix A. Materials and Methods<<
131
Japan).
1.2.10.3e Stripping and reprobing membrane
The used membrane can be reprobed with different antibodies by stripping it.
Membrane was incubated at 60ºC with stripping buffer for 30 minutes with regular
agitation, the membrane was then thoroughly washed 3×5 minutes with 1×TBS with
on the rocking machine. The membrane was reprobed with second primary antibody as
described in section 4.2.10.3b-d.
1.2.11 RAW264.7 cell culture and osteoclastgenesis
RAW264.7 cells were cultured in general tissue culture flasks. The cells were maintained
in 5ml (T25)/10ml (T75) α-MEM medium supplemented with pre-warmed 10% FBS,
2mmol/L-glutamine, 100U/ml penicillin and 100µg/ml streptomycin. The cultures were
incubated in a humidified atmosphere of 5% CO2 and 95% air at 37ºC in an incubator
(Forma Scientific). The cells were subcultured every 3 days and were seeded to
appropriated cell densities when required for the various experiments upon reaching
confluence.
When cells reached about 90% confluence, the media was removed and cells were
washed with about 3ml of autoclaved 1×PBS. The RAW264.7 cells were subjected to
trypsin treatment for 15 minutes at 37ºC to release adherent cells. The trypsin reaction
was stopped by fresh medium. The cell suspension was then transferred to a clean tube
and centrifuged for 5 minutes at 4,500U/min. The supernatant was removed and cell
pellet was resuspended in fresh culture medium. The cell number was determined
before seeded into new culture flask or cell culture plate for osteoclastgenesis.
To generate osteoclast from RAW264.7 cells, the cells were seeded at a density of 1.3×
103 cells/well with 100µl of culture medium in a 96 well plate. Receptor activator of
NFkappaB ligand (RANKL, 150ng/ml) was added to the cells at the first day of culture.
The plate was then incubated in a humidified atmosphere of 5% CO2 and 95% air at 37º
>>Appendix A. Materials and Methods<<
132
C. The culture medium supplemented with RANKL was changed every two days.
Osteoclast was expected to be visualized after 5-day of culture under light microscope.
1.2.12 Cyclic AMP ELISA assay
A Direct Cyclic AMP Enzyme Immunoassay Kit (Assay Designs, Inc. U.S.) was
employed to detect the intercellular content of cAMP in human chondrocytes upon
salmon calcitonin (CT) stimulation. Human chondrocytes (0.8×105 cells/well) were
cultured in 24-well plate and maintained in serum-free culture medium for 12 hours.
After that, they were treated with salmon CT (10^-7M, 10^-8M, 10^-9M) for 20
minutes. Osteoclasts generated from RAW246.7 cells were served as positive control.
Cells were pretreated with 100µM endogenous phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich, UK) for 20 minutes, and
subsequently stimulated with salmon CT (10^-7M, 10^-8M, 10^-9M), or 1mM cAMP
activator forskolin (Sigma-Aldrich, UK) for another 30minutes. The cAMP contents
from supernatant and cell lysis were measured by ELISA assay.
Briefly, after the culture mediums were collected, 5μl of HCl was added to each 500ul
medium to stop endogenous phosphodiesterase activity. The mixtures were then
centrifuged at 600×g at room temperature, and the supernatants were collected and
used directly in the assay. Cells were washed with 1×PBS twice and incubated with
300µl of 0.1M HCl containing 0.1% Triton X-100 at room temperature for 10 minutes.
The lysis was collected in a 1.5ml eppendorf and centrifuged at 600×g for 5 minutes,
and the supernatants were used to detect the cAMP level. A serial of cAMP standards
was diluted in 0.1M HCl (200, 50, 12.5, 3.12 and 0.78 pmol/mL). The samples were
acetylated by 10μl of Acetylating Reagent and incubated with secondary antibody in a
precoated anti-Rabbit IgG Microtiter Plate (Assay Designs, Inc., U.S.) for 2 hours on a
plate shaker at ~500rpm. After that, the plate was emptied and washed with the wash
buffer 3 times. The plate was then incubated with the Conjugate and pNpp Substrate
solution at room temperature for another hour to develop color. The reaction was
terminated by the Stop Buffer (Assay Designs, Inc., U.S.) and the plate was read at
>>Appendix A. Materials and Methods<<
133
405nm in a POLARstar® OPTIMA multifunction microplate reader (BMG LABTECH,
Germany).
1.2.13 Nitric oxide assay
Nitric oxide (NO) production is highly correlated to the severity of inflammatory
processes. In this study, modified Griess reagent (Sigma-Aldrich, UK) was used to
detect the NO production in chondrocyte culture. Supernatant samples were collected
from 48 hours of cell culture and incubated with equal volumn of 1×Griess reagent in a
96-well plate at room temperature for 15 minutes. A serial dilution of NaNO2 (100µM,
50µM, 25µM, 12.5µM, 6.25μM, 3.12µM, 1.56µM, 0.78µM, 0.39µM, and 0.195µM)
was generated in aqueous solution. The optical density was measured at 550nm in a
POLARstar® OPTIMA multifunction microplate reader (BMG LABTECH, Germany).
Nitrite levels were calculated from the standard curve of NO2-.
1.2.14 Colorimetric Dimethylmethylene Blue Dye-binding Assay
The content and release of proteoglycan were determined in cultured human
chondrocytes and their culture medium as sulfated glycosaminoglycan (GAG), which is
primarily a measure of proteoglycan aggrecan content, using a modified colorimetric
dimethylmethylene blue dye-binding (DMMB) assay (Farndale et al., 1986). A standard
curve was constructed using shark chondroitin sulphate (Sigma-Aldrich, UK) (0, 3, 5,
10, 20, 30, and 40g/mL in 0.15M NaCl), and samples were also diluted appropriate as
required in 0.15M NaCl. The DMMB solution (400μl) was added to all samples and the
absorbance of the mixture was measured at 525nm immediately on a spectrophotometer.
1.2.15 Tartarte Resistant Acid Phosphatase Staining
Tartrate-resistant acid phosphatase (TRAP) is an isoenzyme of acid phosphatase which
is found mainly in osteoclast and some blood cells. TRAP staining was used to identify
osteoclast in cell culture or bone tissue.
The staining was conducted using a method adapted from Sigma Diagnostics Acid
>>Appendix A. Materials and Methods<<
134
Phosphatase Kit (Sigma-Aldrich, UK) according to the manufacturer’s instruction. Briefly,
MilliQ ddH2O was prewarmed at 37ºC in a water bath. In a separate tube, 111µl of fast
garnet and 111µl of sodium nitrite solution were combined (Solution A). Solution A was
subsequently added to 10 ml of pre-warmed MilliQ ddH2O followed by the appropriate
volume of 111µl of Naphthol, 444µl of Acetate and 222µl of Tartrate. Then, the mixture
was warmed to 37ºC before adding 100µl of it to the fixed cells, which were incubated
in the dark at 37ºC for about 40 minutes or until color has developed. TRAP positive
cells stained purple while TRAP negative cells stained yellow. The reaction was
terminated by rinsing cells with with MilliQ ddH2Q.
APPENDIX B
SUPPLEMENTARY FIGURES
>>Appendix B. Supplementary figures<<
Figure B.1 Standard curves for Real-time PCR (aggrecan, type I collagen, type II
collagen, type X collagen, fibromodulin, fibronectin, MMP-1, and MMP-3). A
standard curve is generated from a dilution series of known concentration purifed DNA.
The DNA samples were amplified by Real-time PCR, and a Ct value was collected.
Log-linear standard curves were created by plotting relative copy number on the Y-axis
and the Ct value on the X-axis.
135
>>Appendix B. Supplementary figures<<
Figure B.2 Standard curves for Real-time PCR (MMP-9, MMP-13, ADAMTS-4,
ADAMTS-5, TIMP-1, TIMP-2, TIMP-3, and Sox-9).
136
>>Appendix B. Supplementary figures<<
Figure B.3 Standard curves for Real-time PCR (IL-1β, TGF-β, TNF-α, IGF-1,
HSP-70, Ribosomal, c-fos, and c-jun).
137
>>Appendix B. Supplementary figures<<
Figure B.4 Standard curves for Real-time PCR (COX-2, NOS2, MAPk-1, Link
protein, and GAPDH).
138
>>Appendix B. Supplementary figures<<
Figure B.5 Type I collagen melting curve from 10 samples. As the temperature
increased, dsDNA denatured and the fluroscence level decreased. Denaturation
temperature (Tm) is related to sequence length, hence a single Tm peak in the melting
curve plot indicates the specificity of the PCR product and good primer performance.
139
>>Appendix B. Supplementary figures<<
Figure B.6 Melting curve containing slight primer dimmer interference. A lower Tm
peak indicates non-specific binding or primer dimmers. As the fluorescence level is a
relative measurement, the larger the peak at the lower Tm the greater the amount of
primer dimmers compared to primer product. Hence, primer dimmers for more
abundant transcripts are out-competed and not be a problem. However, the presence of
large low Tm peaks indicates poor primer performance, hence the primers need to be
redesigned.
140
APPENDIX C
SAMPLE OF INFORMED CONSENT FORM
Prof Ming-Hao Zheng MBBS PhD MD FRCPath DIRECTOR OF RESEARCH
School of Surgery & Pathology
Department of Orthopaedic Surgery
M508 QEII Medical Centre 2nd Floor M Block, Nedlands WA 6009
Phone +61 8 9346 4050 Fax +61 8 9346 3210
Email [email protected]
INFORMATION SHEET AND CONSENT FORM
The Use of Cartilage Tissue Sample for Gene Expression Profile Study
Chief Investigator: Minghao Zheng Master Student: Zhen Lin
Contact Phone No.: (08)9346 3213 Contact Phone No.: (08)9346 1963
Email: [email protected] Email: [email protected]
Information sheet
Thank you for agreeing to participate in this research, which will be part of a Master by Research project
for Dr. Zhen Lin. In short, we will be utilizing cartilage debris normally discarded after your joint
replacement surgery to analyze genetic changes in osteoarthritis.
Osteoarthritis is a degenerative disorder of articular cartilage, which has a high incident among aged
people. It is supposed that gene expression profile may be different between osteoarthritic and normal
cartilage, and may be a vital cause of the disease process. To better understand and treat the disease, a
study of differential gene expression profiles between diseased and healthy human cartilage is necessary.
An aim of this study is to identify gene expression profiles of osteoarthritis and discover the important
genes involved in the disease process.
139
140
Once the cartilage tissue sample is obtained, chondrocytes, the cartilage-specific cell, will be isolated
from the tissue and cultivated using standard biological method. After that, RNA and DNA will be
extracted from the cells and detected by a PCR machine. The data will be collected, analyzed and
published in a Master thesis.
This research is essentially laboratory-based, therefore you will not be asked to participate in anything
other than contributing the discarded tissue of your knee joint, which will be removed while undergoing
the knee joint replacement surgery. It will not cause any adverse consequence or further surgical
intervention. Only data showing your age and sex will be collected for variable statistic. All information
provided will be treated as strictly confidential and will not be released by the investigator unless required
by law. Your participation in this study does not prejudice any right to compensation, which you may
have under statute or common law. Also, you will be free to withdraw at any time without reason and
without prejudice
By signing the consent form and returning it to us you have consented for us to use your discarded tissue.
Please feel free to ask any further questions you may have about this research by contacting Prof.
Minghao Zheng or Dr. Zhen Lin at the details provided above.
141
Consent Form
I (the participant) have read the information provided and any questions I have asked have been
answered to my satisfaction. I agree to participate in this activity, realising that I may withdraw at
any time without reason and without prejudice. (Or where applicable - without prejudice to my
future medical treatment).
I understand that all information provided is treated as strictly confidential and will not be released
by the investigator unless required to by law. I have been advised as to what data is being collected,
what the purpose is, and what will be done with the data upon completion of the research.
I agree that research data gathered for the study may be published provided my name or other
identifying information is not used.
___________________ __________________
Participant Date
_________________________
Witness
The Human Research Ethics Committee at the University of Western Australia requires that all participants
are informed that, if they have any complaint regarding the manner in which a research project in conducted it
may be given to the researchers or alternatively to the secretary of the Human Research Ethics committee
(Registrar’s Office, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, telephone
number 6488 3703). All study participants will be provided with a copy of this Information Sheet and Consent
Form for their personal records.
APPENDIX D
REFERENCES
>>Appendix D. References<<
ADACHI, N., OCHI, M., UCHIO, Y., IWASA, J., FURUKAWA, S. & DEIE, M. (2004) Osteochondral lesion located at the lateral femoral condyle reconstructed by the transplantation of tissue-engineered cartilage in combination with a periosteum with bone block: a case report. Knee Surg Sports Traumatol Arthrosc, 12, 444-7.
AIDA, Y., MAENO, M., SUZUKI, N., SHIRATSUCHI, H., MOTOHASHI, M. & MATSUMURA, H. (2005) The effect of IL-1beta on the expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in human chondrocytes. Life Sci, 77, 3210-21.
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APPENDIX E
BONE MORPHOGENESIS AND NORMAL STRUCTURE
Manuscript in Chinese
An invited chapter for book Pathology of Bone and Joint (in Chinese)
>>Appendix E. Bone<<
178
第一章 骨的结构与发生
第一节 骨的组织结构
骨骼系统是由不同组织共同构成的具有多种功能和不同结构的连接组织。骨具有三个基
本功能：1. 机械作用：形成四肢骨，构成了机体内部支持的骨架，提供肌肉和韧带的附着；
与肌肉协同对抗地球引力，保持机体直立行走，构成关节参与运动功能。2. 保护作用：形成
头颅和胸廓，保护机体重要脏器，骨髓腔内储存具有造血功能的骨髓。3. 代谢作用：骨骼对
各种离子的储存具有重要作用，特别是钙离子和磷酸盐离子，体内 99%以上的钙和 85%以上
的磷储存在骨组织中，在机体需要的时候，它们可以从骨骼中释放出来，从而保持血液中各
种离子成分的动态平衡。
一、骨的分类
1、按在体内的部位分类：骨骼分为两大类：（1）中央纵轴性骨：如头颅骨、椎骨、肋
骨、胸骨和舌骨。（2）外周肢体骨，包括四肢骨和骨盆。
2、根据骨的形状分类：称为长骨、短骨、扁平骨与不规则骨。
然而，这种传统的分类方法只能对骨有一般的肤浅的认识，对骨的研究没有太大价值。
因此，对于骨骼应该从表及里认识其结构，从宏观到微观研究骨的构成。并结合其所在部位，
进行具体研究。
二、骨的构成
骨由骨质（骨密质，骨松质）、骨膜（骨外膜，骨内膜）、骨髓等构成。
1、骨密质（compact bone）又称骨皮质，由坚实致密的骨板构成，钙化程度为 80-90%。
主要分布于骨干和骨骺的外侧， 占全身骨的 70%。骨板排列规律，按其排列方式不同分为环
骨板、骨单位和间质板 （图 1-1）。
（1） 环骨板：是环绕骨干外表面与内表面的骨板，分别称为外环骨板（outer cirumferential）
和内环骨板（inner circumferential）。外环骨板较厚，约 10～40 层，整齐平行排列于骨干外侧，
表面衬有骨外膜；内环骨板较薄，仅由一至几层骨板组成，附于骨髓腔内侧面，表面衬有骨
内膜。外、内环骨板内均有穿通管（perforating canal）横穿， 又称为 Volkmann 管。穿通管
与纵行的中央管相通，它们都是小血管和神经的通道，管内含有组织液。
>>Appendix E. Bone<<
179
（2） 骨单位（osteon）：又称哈佛系统（Harversian system），位于外、内环骨板之间，
数量 多，是起支持作用的主要单位。骨单位是由胶原、基质与矿物质形成的结构，具有 大
强度，是骨密质的主要结构单位。胶原排列基本是平行的，而在束间常有相互连接，存在于骨
板内，且见于骨板间，对力学应力的抗力，得以加强。 初数周，胶原内矿物质沉淀积于 60～
70％骨单位，以后继续增多，使骨变为成熟。骨单位呈筒状，直径 30～70µm，长 0.6～2.5mm，
沿骨纵轴排列。骨单位中轴有一纵行小管，称中央管，又称哈佛管 （Haversian canal），内含
血管及其他结构，并与穿通管相连。穿通管是含有小血管、神经及骨膜成分的通道，内有组织
液。因此，骨板内骨细胞的突起经骨小管穿越骨板互相连接，骨板内的骨小管均开口于中央管，
构成骨单位中骨细胞的营养物质和气体交换的通路。骨单位周围为 4~20 层的骨单位骨板
（osteon lamella），又称哈佛骨板（Haversian lamella）， 其内的胶原纤维呈螺旋型，相邻两
层骨板的胶原纤维相互交叉。骨单位表面有一层黏合质，为含骨盐较多而骨胶纤维很少的骨基
质，在横断面的骨磨片上呈折光较强的轮廓线，称为黏合线（cement line）。骨单位 外层的
骨小管在黏合线以内返折，不与相邻骨单位的骨小管相通。
（3） 间质板（interstitial lamella）：间质板是一些不规则形的平行骨板，即是原有的环
骨板或骨单位被吸收后残留填充于骨单位之间或骨单位与环骨板之间的骨板。从三维观察，
间质板和骨单位或周缘性骨板有直接连接，位于两个骨单位之间的吻合处，保持和骨单位的
连续性。部分骨单位外层的骨小管可与间骨板内的骨小管相通连，形成骨单位与间质板之间
的物质交换通道。
2、骨松质（spongy bone），是由大量针状或片状的称为骨小梁（bone trabecular）相互连
接而成的多孔网架结构，骨小梁由几层平行排列的骨板和骨细胞构成。网孔即为骨髓腔，其
中充满红骨髓。骨松质主要分布于骨干和骨骺的内侧。骨小梁在长骨骨干中量少，但充满于
干骺端；在短骨与不整形骨中量较多。骨小梁和骨皮质相似，能承受负荷传导。骨小梁和骨
皮质相比，疏松程度与哈佛系统数量不同。其基本结构模式为定向之密质骨片或骨板，和与
之垂直的杆状骨连接的松质骨，骨板并不分层，厚度不同，宽度也不同。厚度约为 100～200µm。
骨板中有许多小孔，相邻骨板的小孔互相连接而形成界限不清的圆柱，不同部位的骨小梁形
状有所不同。表层骨板的骨小管开口于骨髓腔，骨细胞从中获得营养并排出代谢产物。
3、骨膜：骨膜分为骨外膜（periosteum）和骨内膜（endosteum）。
骨外膜分为内、外二层，外层是由胶原纤维束构成致密结缔组织，粗大的纤维束进入骨
质起固定骨膜和韧带作用，结构相对厚而坚实；内层较薄，疏松，含小血管和神经，还有多
种骨细胞成分，如骨原细胞、成骨细胞及破骨细胞等，这些细胞在幼年时功能活跃，直接参
与骨的生长和构建，使骨不断加粗增长，到了成年期转为静止状态。一旦发生骨折等骨损伤，
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又可重新恢复其功能，参与骨组织损伤后的修复。此外，位于骨外膜内表面的还有周细胞
（pericyte），它是具有间质细胞源性的多形态性细胞，通常与上皮细胞共同参与构成毛细血
管和毛细血管后微血管的管腔结构。近年来认为，骨外膜下的周细胞可能是一种成骨细胞的
前体细胞，具有参与骨形成及血管再生的功能。
骨内膜是一层薄层结缔组织，分布骨髓腔面、骨小梁的表面、中央管和穿通管的内表面。
内含有成骨细胞和破骨细胞，不同条件下能分裂分化为骨细胞或分解破坏骨细胞。同时具有
离子屏障功能，分隔骨细胞周液和骨髓腔内的组织液，使骨细胞周液维持一定的钙、磷浓度，
有利于骨盐晶体形成。
4、骨髓 （bone marrow） 位于骨髓腔和骨松质间隙内，是人体 大的造血器官。主要由
网状结缔组织和造血细胞组成。骨髓分为红骨髓（red bone marrow）和黄骨髓（yellow bone
marrow）。胎儿和婴幼儿时期的骨髓均为红骨髓，含有大量不同发育阶段的红细胞及其他幼
稚型的血细胞，呈红色，故名红骨髓。红骨髓具有造血功能，可以生成红细胞、粒细胞、单
核细胞和血小板等。大约从 5 岁开始，长骨骨髓腔中的红骨髓逐渐被脂肪组织所代替，呈黄
色，成为黄骨髓。黄骨髓失去造血能力，但是存在少量幼稚血细胞，保持一定的造血潜能。
当机体出现慢性失血过多或重度贫血时，可刺激黄骨髓逐渐转化为红骨髓，恢复造血功能。
红骨髓在人体短骨、扁骨、不规则骨和长骨骨骺终身存在，因此，临床上通常选择髂骨或胸
骨进行骨髓穿刺，检查骨髓像，帮助疾病诊断。
5、长骨的构造：长骨沿着纵轴方向可分为三部分， 分别为：骨骺，骨干，干骺端。骨
密质和骨松质的比例在长骨中的不同部分变化明显。长骨的骺端位于两端，含有大量的骨松
质和薄层骨密质。骺端通常与其他骨连接形成关节， 因此一般被关节软骨所覆盖。骨干位于
长骨的中间，主要成分为骨密质，其内为骨髓腔。干骺端介于骨干和骺端之间，由大量的骨
松质和骨密质组成。在处于发育阶段的小儿长骨中，骺端与干骺端有一层骨骺软骨板，也称
为生长板（growth plate），是长骨发育的生长中心。
三．骨的微观结构
显微镜下可分为两型骨结构：胶原纤维分布紊乱的编织骨（woven bone）和胶原与细胞排
列高度规则的板层骨（lamellar bone）
1、编织骨由不规则未定向的胶原纤维与有陷窝分布之骨组织构成，即纤维性骨、非板状
骨或原始骨。见于胚胎发生时软骨内骨化部位，以及肌键或韧带抵止之部位。病理情况下，
可见于骨折修复时所形成的骨痂、炎症病变之部位，也可见于骨肿瘤及瘤样病变如骨肉瘤、
纤维性结构不良、畸形性骨炎等部位。编织骨缺乏象板状骨所具有分化良好之密度斜坡，染
色时组织因含水分高而易于渗透。骨细胞大小形状不等，且数量较多，约为 80000/mm3，板状
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骨的骨细胞数量为 20000/nm3。骨小管排列方向紊乱，直径不同。在普通染色下不能看到胶原
之走向。机化程度有限，可见短来状定向性胶原。同时还可见相互成直角的连接，呈经纬表
现（warp and woof）。
2、板层骨是成熟骨，无论在皮质骨或松质骨中均呈板状结构。每个板层中的胶原纤维走
向均不同，很像胶合板。用未脱钙薄切片行显微放射摄影，可见矿物质呈板状表现。偏光显
微镜下每个板层表现为双向性，由胶原纤维之方向决定，一层为同向性（isotrophic），另一
层为异向性（anisotrophic），或为双折反射。根椐相联骨板胶原束的方向不同，可区分为三型
骨单位。第一型为骨单位之内外周边，沿骨单位纵轴走向之纤维，只有少数同心板与骨单位
纵轴呈横向方向；第二型为大部胶原束以同心性环绕骨单位之纵轴；第三型骨单位是纵向骨
板变成为横向之同心性骨板。由于骨板的形状可改变骨单位力学的性能，纵向骨单位之张力
强度较丈，而横向骨单位之压力强度较大。
四．骨的血管、淋巴和神经
骨的附件包括血管和神经，营养支持骨的生长。骨的血液供应非常丰富，骨骼系统接收
心脏血液输出的 10-20%。 骨的动脉有广泛吻合，且互相连接，部分血管端口开放，以便骨髓
腔的血液滤过。静脉网的直径较大，以适应动脉特点，以便血液迅速排出。
以长骨为例，血液供应来自三方面：①骨端动脉，分别进入骨端和干骺端的不同部位，
营养这些部位及关节；②营养动脉，常有 1～2 条，经骨干的营养管入骨髓腔，营养骨干内层；
③骨膜动脉，营养骨干表层。
进入骨干的营养动脉分为两个大的分支，即升支和降支，每一支又有许多细小的分支，
大部分直接进入皮质骨，另一些分支进入髓内血管窦。升支和降支的终末血管供给长骨两端
的血运，并与骨髓和干骺端血管形成吻合。
起源于髓内营养动脉的皮质小动脉，呈放射状直接进入皮质骨，或以 2～6 支小动脉为一
束的形式进入皮质骨。在皮质骨内的小动脉，又形成许多分支，某些顺骨的长轴纵向延伸，
而另一些呈放射状走行。这些血管分支， 终在哈佛系统形成毛细血管。有一些小动脉入皮
质骨后又穿出皮质骨与骨膜的小动脉吻合，形成动脉网。在髓内，某些小动脉较短，形成骨
髓的毛细血管，给骨髓提供血运。
哈佛斯管内常有管壁很薄的两条血管，一条较细，而另一条稍粗，表明较细的一条为动
脉，而稍粗的一条为静脉，形成进出两个方向的血流。这些小血管壁由单层内皮细胞组成，
在少数情况下，其中一条血管可显示小动脉的组织学特征，哈佛管内有时只含一条毛细血管。
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长骨具有一个较大的中央静脉窦，接受横向分布的静脉管道的血液，这些血液来自骨髓
的毛细血管床，即血管窦。横向管道内也含有进入骨内膜的小动脉。这些静脉管道可将血液
直接引流入中央静脉窦，也可先引流至大的静脉分支内，然后再汇入中央静脉窦，中央静脉
窦进入骨干营养孔，把静脉血引流出骨。长骨的静脉血，主要经骨膜静脉丛回流。仅有 5%～
10%的静脉血经营养静脉回流。许多静脉血经骨端的干骺端血管回流。骨端血管是骨膜静脉系
统的一部分。从骨膜表面骨干皮质骨出现的内皮管（endothelial tube），称作小静脉（venule）。
尽管近来有人认为，皮质骨血液很少回流至内骨膜静脉，但体内研究表明，毛细血管离开哈
佛管后有分支进入骨髓，并进入骨髓血管窦。
淋巴管伴随着神经血管从分布于骨膜，但未能证明存在于在骨质内。
在长骨的关节端，椎骨和多数的扁平骨分布有大量的神经。骨组织具有复合的自主感觉
运动神经体系。骨的细胞具有多种神经肽受体，包括神经肽 Y （neuropepite Y, NY），降钙素
基因相关肽（calcitonin gene-related peptide, CGRP），血管活性肠肽 （vasoactive intestinal peptide,
VIP），P 物质 （substance P， SP）都出现在骨的神经中。骨膜具有丰富的神经分布， 有髓
神经和无髓神经伴随着营养血管进入骨和骨髓中，位于哈佛管的血管周间隙。
第二节 骨的细胞结构
骨组织是由多种细胞和大量钙化的细胞外基质（extracellular matrix）构成。骨组织的细胞
包括骨原细胞，成骨细胞，破骨细胞和骨细胞 4 种。钙化的细胞外基质称为骨基质。是人体
坚硬的组织之一。
一．骨原细胞 （osteoprogenitor cell）
骨原细胞又称为骨祖细胞，是一种具有多分化潜能的干细胞，在不同环境和不同类型和
程度的刺激下，可以分化为软骨细胞或成骨细胞。骨祖细胞小，呈梭形，与内皮细胞和成纤
维细胞形态相似，细胞核呈椭圆形，胞质内仅含少量核糖体和线粒体，呈弱嗜碱性。骨原细
胞位于骨膜下或骨表面，在骨组织生长和改建或骨折愈合时，细胞分裂活跃，分化为成骨细
胞。
二．成骨细胞（osteoblast）
成骨细胞是专门从事骨形成的细胞，位于成骨活跃的骨组织表面。胞体较大，呈矮柱状
或椭圆形。直径由 20～50µm，胞浆丰富，因含大量核糖核蛋白，故呈嗜碱性。于细胞之中央
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与核相邻之处，可见透明区。核较大，呈圆形或椭圆形，核染色质少而较淡，有 1～3 个核仁。
核常位于新生骨表面相对之一侧，而胞浆靠近新生骨。成骨细胞常呈单行排列，似上皮样覆
盖于新生骨之表西。细胞之间有时是以裂隙连接（gap junction）。于成骨细胞表面可见多数
短绒毛突起，和邻近的成骨细胞或骨细胞的突起形成缝隙连接，以协调细胞的功能活动。
电镜下可见大量粗面内质网和线粒体、丰富的游离核糖体和发达的高尔基复合体。成骨
细胞具有活跃的分泌功能，合成和分泌骨胶纤维和有机基质，形成类骨质（osteoid），故可见
大量细胞器。
胶原的前身在粗面内质网内合成，转移到高尔基器而合成原胶原纤维丝。经分泌性空泡
之外放作用，而排出细胞体外。原胶原在细胞外转变成为胶原。一旦形成有机的骨网，立即
开始矿化作用（mineralization）。
线粒体中含有一些小的矿化颗粒，沉积并附着于线粒体嵴外面。这些颗粒有较高浓度的钙
、磷和镁，还存在其他一些有机成分。线粒体在细胞内的能量循环中是氧化加磷氧基作用的
部位，其后产生ATP。线粒体的第二个重要功能是从细胞浆中清除钙离子。线粒体的钙通过和
磷的共同沉积，形成线粒体颗粒，调节细胞浆内的钙水平。此外，成骨细胞线粒体可移到细
胞表层，将电子致密颗粒排出到胞浆，然后排出细胞外，对矿物质的运输起到重要作用。
成骨细胞以细胞膜出膜方式向类骨质中释放基质小泡（matrix vesicle）。基质小泡直径约
为 0.1µm，膜上有钙结合蛋白、碱性磷酸酶、焦磷酸酶和 ATP 酶等，并含有酸性磷脂，小泡
内含钙和钙盐结晶等。基质小泡在类骨质的钙化过程中起重要作用。
成骨细胞起源于骨髓内的多能干细胞（pluripotential stem cell），也称间充质细胞
（mesenchymal stem cell）。间充质细胞还可以分化为成纤维细胞、软骨细胞和脂肪细胞。在
细胞培养中，成骨细胞几乎无法与成纤维细胞相鉴别。在形态学上，成骨细胞唯一的特性是
在细胞的外面形成矿化的细胞间基质。但是没有证据显示，骨基质的矿化是由成骨细胞选择
性表达的特异性基因造成的。所有在成纤维细胞中表达的基因也同时在成骨细胞中表达。到
目前为止只确定了两种成骨细胞特异基因：转录因子 Cbfa1 和抑制成骨细胞功能的分泌性分子
骨钙蛋白（osteocalcin） 。因此，从基因上的角度上讲，成骨细胞可以看作是进化的成纤维
细胞（sophisticated fibroblast）。幼稚的成骨细胞（preosteoblast）是介于骨髓干细胞和成熟成
骨细胞之间的细胞分化状态，该状态一般维持 2 到 3 天。前成骨细胞同样很难与其他基质干
细胞相鉴别，胞质呈弱嗜酸性，细胞核伸长，染色淡。碱性磷酸盐在细胞内的存在，可用于
鉴别幼稚的成骨细胞。分化完全的成骨细胞和幼稚的成骨细胞表面都具有 1, 25-羟化维生素 D
受体和甲状旁腺激素（parathyroid hormone, PTH）受体。与成熟成骨细胞不同的是，幼稚的成
骨细胞具备有丝分裂能力。
>>Appendix E. Bone<<
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成骨细胞的分化受到多种因子的调节控制，主要有转录因子 MSX2, Runx2、Osterix、
LEF/ECF、Dlx5、Twist、C/EBP、Fra-1、Krox20、Sp3、Alx5 和 Bapx1 等，和生长因子骨形
成蛋白（bone morphogenetic proteins, BMPs）、转化生长因子（transforming growth factor β,
TGF-β）和血小板源生长因子（Platelet-derived growth factors, PDGFs）等。
三．骨细胞（osteocyte）
骨细胞是骨中数量 多的细胞。为成骨细胞的终末分化细胞形态。成骨细胞的平均寿命
为 6 个月，在活跃的骨形成活动中，10-15%的成骨细胞位于类骨质中成为幼稚的骨细胞。幼
稚的骨细胞仍是具有成骨细胞形态，同样具有分泌类骨质的能力，随着类骨质的钙化，幼稚
的骨细胞逐渐成熟，胞体包埋于骨基质中，形成骨陷窝（bone lacunae）。其余的不被类骨质
包埋的成骨细胞变成扁平状，排列于骨组织表面，成为静止的成骨细胞。他们不具有分泌类
骨质的能力，但是仍然对 1, 25-羟化维生素 D 和 PTH 敏感。
成熟的骨细胞单个分散于骨板内或骨板之间，长轴与骨板平行。胞体较小，呈扁椭圆形，
包埋于骨陷窝中，具有许多细长突起，伸进周围基质的骨小管（bone canaliculus）内。胞质呈
弱嗜碱性或嗜酸性，细胞器相对较少，粗面内质网少，Golgi 器不发达，线粒体少，提示蛋白
合成能力低下。相邻骨细胞的突起形成缝隙连接，传递细胞间信息和沟通细胞间的代谢活动。
相邻骨陷窝通过骨小管互相连通，内含组织液，可营养骨细胞并带走代谢产物。
同时，骨细胞还通过骨小管与静止的成骨细胞相联系，构成一个广泛的细胞间联系网络，
称为骨细胞膜系统（osteocytic membrane system）。骨细胞膜系统具有两个主要的功能：1、
参与 PTH 刺激骨质吸收的快速效应，动员骨的钙、磷入血。骨细胞膜系统覆盖除破骨细胞邻
近的区域以外的全部骨表面和腔隙表面，从而在骨质与细胞外液之间形成可通透性屏障。这
层膜与骨质之间有少量滑液，细胞外液侧的骨细胞膜有钙泵。PTH 提高滑液侧的骨细胞膜的
通透性，使滑液中的钙、磷进入骨细胞，进而加强钙泵活动，将钙、磷转运至细胞外液。当
钙泵活动增强时，骨液钙浓度下降，便从骨基质吸收骨盐，即骨细胞附近的骨盐溶解释放，
称骨细胞性溶血。2、作为力学感受器，传递电子信号。骨细胞膜系统直接感受力负荷和骨应
变产生的液体流动的剪切力，将力学信号转换为生物信号并迅速传递到整个骨组织，从而调
节骨适应性再建，使骨保持稳定的应变能力和稳定的骨小管内液流环境。
四．破骨细胞（osteoclast）
破骨细胞是从事骨吸收的细胞，故名破骨细胞。位于骨表面被吸收的浅腔内，称为 Howship
陷窝（Howship’s lacunae）。直径约为 20～100µm，含有 1～2 个核或多达 100 个核，平均有
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20～30 个核。多为不规则的圆形、卵圆形，核膜光滑，染色质颗粒微细，分布均匀，故染色
淡。有 1～2 个核仁。胞浆内含有大小形态不同的空泡和大量线粒体，线粒体多位于远离皱折
缘之处，可见 Golgi 器和量少而分布广泛的中央小体、核糖体和内浆网。光镜下，破骨细胞的
胞质呈泡沫状，多为嗜酸性。
功能活跃的破骨细胞具有明显的极性，于贴附骨基质处胞浆边界不清，并见纤细指状突
起纵纹，称皱褶缘（ruffled border）。皱褶缘侧有许多不规则形并分支的指状突起，皱褶缘
细胞膜上有酸性磷酸酶和 ATP 酶。皱褶缘周围的环形胞质区稍隆起，细胞膜平整，胞质内含
有许多微丝，而缺乏其他细胞器，电子密度低，故称为透明带（clear zone）。透明带的微丝
含肌纤维蛋白，垂直于细胞表面，伸入到骨基质内。其功能是使皱褶缘表面移动，或封闭破
骨细胞与骨相连接的部位。破骨细胞离开骨组织表面时，皱褶缘与透明带消失。
破骨细胞另外一个结构特点是含有大量的溶酶体，内含抗酒石酸酸性磷酸酶
（tratrate-resistant acid phosphatase）。抗酒石酸酸性磷酸酶是破骨细胞特异性的酶。该酶的特
异染色可以用于鉴别组织中的破骨细胞以及其他原始破骨细胞。这些酶可能具有分泌与消化
功能。
破骨细胞有溶解和吸收骨基质的作用。其功能活跃时，透明带紧贴骨基质表面，形成一
道环状围堤，封闭其所包围的皱褶缘区，形成溶骨微环境。破骨细胞向微环境中释放有机酸，
如柠檬酸和乳酸等，还有多种蛋白酶，如组织蛋白酶（cathepsin）和基质金属蛋白酶（matrix
metalloproteinases, MMPs）等。有机酸溶解骨盐，蛋白酶降解胶原纤维和其它基质蛋白。溶解
的骨盐和被降解的有机物经皱褶缘吸收，在溶酶体中进行消化。破骨细胞还同时产生氧自由
基，参与溶骨。
破骨细胞的来源是由单核细胞源性的单核/巨噬细胞，通过互相融合而形成的多核细胞。
破骨细胞的祖先也可同时分化为单核细胞、巨噬细胞和其它外周血白细胞，它们同时表达单
核细胞或巨石细胞的表面标记物：CD13，CD15 和 CD54。破骨细胞的祖先分化为单核的破骨
细胞前体， 后完全分化为成熟的多核破骨细胞。
在 90 年代，人们认为破骨细胞的形成必须有骨髓造血微环境中基质干细胞和成骨细胞前
体的参与。经过十几年的研究后，我们终于清楚破骨细胞必需并且只需要两种分子的存在：
巨噬细胞集落刺激因子（macrophage colony-stimulating factor, M-CSF）和细胞核因子kB受体活
化因子配基（receptor for activation of nuclear factor kappa B [RANK] ligand, RANKL），或称为
护骨素配基（the ligand for osteoprotegerin, OPGL）和肿瘤坏死因子相关激活诱导因子（Tumor
Necrosis Factor [TNF]-related Activation-induced Cytokine，TRANCE）。美国骨矿研究学会
（ASBMR）将其标准化命名为RANKL，以避免不同命名在学术上引起的不便。M-CSF是巨噬
>>Appendix E. Bone<<
186
细胞成熟所必需的，为一种分泌性分子，它结合到早期的原始破骨细胞上的受体C-Fms， 传
递破骨细胞生存和繁殖的信号。基质细胞或成骨细胞在其细胞表面产生M-CFS分子基团，与
破骨细胞相互接触，作用于破骨细胞的生成。另外，RANK和RANKL在基质细胞和成骨细胞
与破骨细胞间的通讯中起着至关重要的作用，大多数细胞因子和激素均通过调控基质细胞或
成骨细胞RANKL和RANK的表达，RANKL与原始破骨细胞上的受体结合，从而间接刺激原始
破骨细胞的分化、融合和成熟，抑制破骨细凋亡。
RANKL/RANK/OPG 调节轴的发现是对破骨细胞研究上的突破性意义。基质细胞和成骨
细胞都能表达和产生大量的 RANKL。RANKL 能和破骨细胞前体细胞表面上的特异性受体
RNAK 相结合， 从而诱导前体细胞的融合成多核的细胞破骨细胞， 这其中需要激活一系列
的信号传导通路。与此同时基质细胞和成骨细胞能产生可溶性的ＴＮＦ受体相关蛋白
（TNFR），或称为骨保护素（osteoprotegerin, OPG），它是 RANKL 的假性受体（decoy receptor），
通过竞争性的与 RANKL 结合来抑制 RANKL 和 RANK 的结合，从而达到抑制破骨细胞的形
成。
体外实验表明，在骨髓干细胞培养中添加纯化的 M-CFS 和 RANKL 可以获得分化完全的
破骨细胞。而且破骨细胞生成的数量随添加的 M-CFS 和 RANKL 浓度改变而改变。此外，诱
导 M-CFS 和 RANKL 表达的物质，可以引起原始破骨细胞繁殖。这可能是人体骨质疏松症
主要的发病机制。我们已经知道过量的 PTH 可以造成加速的骨质疏松，但是破骨细胞上并不
具有高亲和力的甲状旁腺激素受体。现在清楚，PTH 通过其在成骨细胞和部分基质细胞上的
受体作用，产生诱发破骨细胞生成的因子 RANKL，从而引起破骨细胞数量增多，加速骨吸收。
同样，维生素 D3 的活化形式 1，25-羟化维生素 D3，可以通过诱导基质细胞和成骨细胞表达
RANKL 从而直接刺激原始破骨细胞的分化。
在细胞内信号传递途径上，破骨细胞表面的 RANKL 受体激活后，引起分子结构的变化，
从而聚集了细胞内肿瘤坏死因子受体相关因子（necrosis factor receptor-associated factors,
TRAF）和 c-src， 随后激活下游信号途径，分别由 NF-kB 传导通路、c-Jun 氮端信号传导通
路（c-Jun N-terminal kinase or JNK）、负荷激化蛋白激酶（stress-activated protein kinase）p38、
细胞外信号调剂激酶信号传导通路（extracellular signal-regulated kinase or ERK） 和 Src 传导
通路。RANK 与胞浆上的特异性功能域与肿瘤坏死因子受体相关胞浆因子（TNFF-associated
cytoplasmic factors，TRAFs）结合是激活这一系列的信号传导通路中 初步的和关键的一个环
节。TRAF 家族中 2,-5 和-6 中有着不同区域可以和 RANK 相互结合。在 TRAF6 敲除老鼠中研
究中发现，由于 TRAF6 缺失引起破骨细胞功能的丢失，从而导致骨硬化症（osteopetrosis）。
另外缺少TRAF6结合位点的RANK突变体不能恢复起缘于RANK-/-造血的前体细胞的破骨细
>>Appendix E. Bone<<
187
胞形成的潜能。这些都提示了 TRAF6 作为主要整合信号蛋白的接合体，指导着调控破骨细胞
分化和活性的特异性基因表达。
在 TRAF6-RANKL 的结合之后，NF-kB 和活性蛋白-1（Activator protein-1，AP-1）这两
条传导通路很快被激活。在 NF-kB 组成分子 p52/p50 双敲除老鼠和 AP-1 组成分子 c-Fos 单敲
除老鼠模型中，破骨细胞缺失，从而引起骨硬化症。在细菌性感染引发的骨溶解和卵巢切除
的小鼠模型中，运用特异性的 NF-kB 活性抑制短肽药物，发现阻断 NF-kB 的活性可抑制破骨
细胞的形成和骨吸收，可以防治细菌性相关的骨溶解和雌激素缺乏引起的骨质疏松。另外，
p38 在 RANK 信号传导通路中起也起着重要作用。p38 被 MMK6 磷酸化激活，导致下游转录
调节因子 Mif 的活化。Mif 转录调节因子的活化能诱发与成熟破骨细胞相关的 TRAP 和 CATK
等蛋白的表达。另外小分子量的 ERK 的抑制剂 PD98059 和 U0126 能诱导由 RANKL 引发的破
骨细胞形成，表明 ERK 信号传导通路在破骨细胞形成中有负性调节的作用。
除此之外，还有 Src 信号传导通路和破骨细胞功能紧密相关。在 Src 敲除老鼠中也有骨硬
化症，虽然骨组织中有 TRAP 阳性的破骨细胞的形成，但这些破骨细胞不能进行骨吸收。Src
的破骨细胞中的具体调节机制还不是很明确。磷脂酰肌醇三羟基激酶 （phoshptidylinositol
3-OH kinase or PI(3)K）和丝氨酸/苏氨酸蛋白激酶（serine/threonine protein kinase or AKT）是
Src 的下游激活因子，与破骨细胞细胞骨架的重组，细胞的生存和新技术的出移动性有关。
进年来随着基因芯片的出现和应用，更多破骨细胞和其前体细胞基因的相异性被发现。
比如：发现Ｔ细胞活化的核因子-1 和-2 （nuclear factor of activated T cells -1,-2，NFAT1 and
NFAT2）在破骨细胞形成过程中起着关键性的作用。在缺少 RANKL 的情况下，过度表达
NFAT1 的小鼠单核细胞源性巨噬细胞 RAW264.7 细胞具有自主性诱导破骨细胞样形成。 更有
意义的是，从 p52/p50 双敲除老鼠和 c-Fos 单敲除老鼠中提取的骨髓细胞，即使在有 RANKL
的情况下也是不能形成破骨细胞。但是，在这些骨髓细胞过度表达 NFAT 能恢复它们的破骨
细胞形成。经研究表明，NFAT 是钙调磷酸酶（calcineurin） 和钙调节的转录因子。钙调磷酸
酶是依赖于钙调蛋白的丝氨酸-苏氨酸磷酸酶，它的抑制剂可以阻断 NFAT 的激活来抑制破骨
细胞。 近研究又发现 NFAT 在成骨细胞中也起着重要作用。
破骨细胞作为骨吸收的主要成分，对骨骼的形成和体积的调节具有重要的作用，破骨细
胞的活动可导致骨质疏松症的进展，造成骨体积下降，使得骨结构不稳定，从而使患者容易
发生继发性骨折。无论任何原因诱发的成人骨质疏松，都与大大加强的骨吸收能力有关。对
于破骨细胞生物学行为的研究，可以让我们加深对骨质疏松类疾病发病机制的理解，帮助治
疗。
>>Appendix E. Bone<<
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五．骨基质的化学成分
骨基质的化学成分包括有机物（占 30-40%）和无机物（占 60-70%）。有机物主要是骨胶
原纤维和少量的非胶原蛋白。胶原蛋白与非胶原蛋白的比例在骨组织中具有独特性，通常在
其他组织中胶原蛋白的含量占基质有机物的 10-20%左右，而在骨组织中占 90%。胶原蛋白和
非胶原蛋白在骨中的比例决定了骨的韧度。而无机物主要是各种钙盐，包括碱性磷酸盐、碳
酸盐、氟化钙、氯化钙等，它们沉积在胶原纤维内。钙盐在骨中所占的比例决定了骨的硬度。
在骨中，有机物质与无机物质相互结合，刚柔并蓄，使骨坚实强硬同时又具有一定的弹性和
韧性。水分在骨基质只占 10-20%。
（一）胶原
细胞间质中的胶原是一种结晶纤维蛋白原纤维。骨组织中 主要的胶原为 I 型胶原，由成
骨细胞产生，在骨基质有机物的 90%，另外还有极少量的 III 型，V 型和 IX 胶原。 正如所有
的结缔组织，胶原纤维主要发挥机械功能，提供韧性和弹性。I 型胶原的分子结构为三条多肽
链，包括两种类型，即两个 α1 链和一个 α2 链。这三条肽链交织呈绳状，故又称三联螺旋结构，
有强大的共价键横向交联，排列整齐，结构紧密。每一条链含有一千多个氨基酸，如丙氨酸、
亮氨酸、甘氨酸、精氨酸、谷氨酸、脯氨酸和羟脯氨酸等，其总氮量为 18.45%，而甘氨酸、
脯氨酸和羟脯氨酸组成胶原总量的 60%。胶原纤维平行排列，分子间的空隙较大，有利于骨
盐沉积。
（二）非胶原蛋白
骨的非胶原蛋白是由一系列复合分子组成，分为外来的（主要是血清来源蛋白）和局部
来源的（由成骨细胞产生）分子。它们包括：骨钙蛋白（osteocalcin）、骨粘连蛋白（osteonectin）、
骨桥蛋白（osteopontin）、骨涎蛋白（bone sialoprotein）等，其中一些非胶原蛋白通过共价键
与碳水化合物联结，形成糖蛋白。骨钙蛋白常作为骨形成的一种标志，与骨盐高亲和力结合，
参与骨的钙化并调节骨的吸收。其他几种糖蛋白主要与 I 型胶原蛋白和骨盐结合，与细胞和骨
基质的黏合有关，也参与骨钙化的调节，或是调控成骨细胞和破骨细胞的代谢。非胶原蛋白
的具体作用机制还未清楚。
（三）无机质
骨组织中的无机质又称为骨盐（bone mineral），约占骨组织干重的 65%， 主要成分有钙、
磷和镁等。羟基磷灰石 [Ca10(OH)2(PO4)6] 是骨盐的主要成分，以结晶体（hydroxyapatite
crystal）的形式存在。电镜下，结晶体为细针状，长约 40nm，厚约 1.5-3nm，沿着胶原纤维的
长轴平行排列，间隔 60-70nm，也可存在于胶原原纤维内胶原分子的空隙中。骨盐与有机质结
合，形成坚硬的骨基质，以适应骨组织的支持功能。
>>Appendix E. Bone<<
189
人在不同的年龄，骨的有机物与无机物比例不同。在儿童和少年时，骨中的有机物含量
比无机物为多，因此他们的骨柔韧性比较高，但是硬度较小，受到外力的作用时，不容易发
生骨折，或可发生不完全性骨折。如果小儿在机体缺钙或是长期保持不良姿势的情况下，骨
容易变形，严重时可造成永久性畸形。而老年人骨的无机物的含量比有机物高，因而骨脆性
大而弹性小，在外力的作用下容易发生骨折，且多为完全性骨折。
第三节 骨的发生、生长和再生
骨由胚胎时期的间充质发生，出生后继续生长发育，加长和加粗，直到成年才停止。此
外，骨的内部改建（remodeling）持续终身，随年龄增长而逐渐减慢。
一、 胚胎内骨发育
骨骼在胚胎内的发育时间与顺序可以根据胚胎的分级系统确定，以排卵后日数或周数代
表年龄。中胚层 早是出现于胚胎外（5a 级，7～8 天），于一周内进入胚胎体内（6b 级，13
天）。各种器官（包括骨在内）开始形成时，间充质的数量与细胞间纤维均有所增加，此即
间充质凝聚（mesenchymal condension）。胚胎时期全身各骨的发生均开始于间充质凝聚。
在胚胎期，于排卵后第 8 周（9 级）出现体节。1 周内（12 级，26 天），脊索的腹侧与
背侧（形成椎体）以及背侧（形成神经突）的细胞延长。继之，肋骨的前身（13 级 28 天）与
关节突（14 级 32 天）出现。此后，椎体开始软骨化（17 级 41 天），一周内相继出现于神经
突、椎板、肋骨与关节突，软骨性颅骨可出现于枕部（17 级 41 天）。骨化 先出现于下颌骨
（18～20 级 6～7 周）与上颌骨（19 或 20 级 7 周）。上下肢芽表现为体壁隆突是在 26 天（12
级）与 28 天（13 级）。上肢骨的间充质凝聚是在 33 天（15 级），而下肢是在 37 天（16 级）。
数日内，长骨的间充质，然后是软骨相继出现。胚胎末期，开始骨化。手足各骨的软骨化大
约是在 6 周开始至 8 周间，软骨化中心数量有所增加。近年研究表明，在排卵后 11 周，男性
胚胎手骨发育比女性胚胎快。
二、 骨组织的发生
骨组织的发生（osteogenesis）有两种不同的方式，即膜内成骨（intramembranous ossification）
和软骨内成骨（endochondrol ossification）。但是其发生基本过程一致，主要包括软骨形成、
骨形成、钙化、基质形成与吸收等。一开始，骨原细胞分裂分化为成骨细胞，成骨细胞分泌
类骨质，并被包埋其中，成为骨细胞。继而类骨质钙化成骨基质，与各种细胞类型共同形成
骨组织。破骨细胞在骨组织中起吸收的作用，它们黏附于骨组织的表面，分泌有机酸和溶酶
>>Appendix E. Bone<<
190
体酶，溶解骨盐和降解有机质。骨组织发生的过程中既有骨组织的形成，也有骨组织的吸收，
两者同时存在，相互协调，形成动态的平衡。
1、 膜内骨化
膜内骨化是由间充质先分化为胚胎性结缔组织膜，然后在此膜内成骨，这个过程不涉及
任何软骨样模板。人体的锁骨，下颚骨，和头颅骨的某些骨块均以此方式发生。在将要形成
骨的部位，血管增生，间充质细胞增生密集成膜状，某个部位的间充质细胞增殖分化为骨原
细胞，进而分化为成骨细胞，成骨细胞分裂成群开始成骨，形成 早的骨组织，该部位称为
骨化中心（ossification center）。以后，成骨过程由骨化中心向四周扩展，使骨不断加大。
初的骨组织为针状，即初级骨小梁，连接成网，构成初级骨松质，新生骨质表面的间充质分
化为骨膜，骨膜内的成骨细胞又在这些骨质表面造骨，使骨逐渐增厚增大。
同时，破骨细胞将已形成的骨质破坏吸收，成骨细胞再将其改造和重建，如此不断地改
变着骨的外形和内部结构，使骨达到成体时的形状，适应机体生长的结构需要。以顶骨为例，
破骨细胞吸收初级骨松质，骨膜内的成骨细胞进行改建，在顶骨内、外表面形成骨密质，即
内板和外板，中间为骨松质构成的板障。顶骨外表面以骨形成为主，使顶骨不断生长，内表
面以骨吸收为主，使顶骨弯曲成形。通过不断的改造重建，使颅腔增大，以适应脑的发育。
2、 软骨内骨化
软骨内成骨是由间充质先分化为软骨，然后软骨逐渐被骨组织取代。人体的四肢骨，躯
干骨和部分颅底骨等均以此方式发生。
以长骨为例，在将要形成长骨的部位，间充质细胞密集，分化为骨原细胞，进而分化为
软骨细胞，分泌软骨基质，周围的间充质分化为软骨膜，形成透明软骨，此时为软骨雏形
（cartilage model），无血管侵入。
软骨雏形达到一定的体积之后，在中段周围部进行软骨周骨化（perichondral ossification），
其过程类似膜内骨化。血管进入软骨膜，软骨膜改称为骨外膜，膜内骨原细胞增殖分化为成
骨细胞，成骨细胞在软骨表面形成薄层初级骨松质，包绕软骨雏形的中段，像领圈，故名骨
领（bone collar）。成骨细胞向骨领表面和两端不断添加新的骨小梁，使骨领逐渐增厚增长。
以后骨领逐渐改建成骨干的骨密质。
在骨领形成的同时，软骨雏形中央的软骨细胞肥大并分泌碱性磷酸酶，软骨基质开始钙
化；随之软骨细胞退化死亡，留下较大的软骨陷窝。骨外膜的血管侵入，形成初级骨化中心
（primary ossification center），为软骨内首先骨化的区域。破骨细胞随血管进入初级骨化中心，
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溶解吸收钙化的软骨基质，形成许多不规则的隧道，称为初级骨髓腔，腔内为初级骨髓。随
后，成骨细胞在被溶解残留的钙化软骨基质表面生成骨组织，以之为中轴，形成过渡型骨小
梁。
过渡性骨小梁不久又被破骨细胞溶解吸收，形成为一个较大的次级骨髓腔。由于骨领的
外表面不断成骨，而骨领的内表面又逐渐被破骨细胞分解吸收，使骨干增粗的同时又保持骨
组织的厚度，骨髓腔不断增大。初级骨化中心两端的软骨不断生长，成骨过程也不断由骨干
向两端推移，使长骨不断增长。
在胎儿长骨的纵切面上，从软骨到骨髓腔之间，可依次分为代表成骨活动的 5 个区域：
（1）软骨贮备区（zone of reserve cartilage），又称静止区，此区软骨细胞较小，分散存在，
软骨基质呈弱嗜碱性。（2）软骨增生区（zone of proliferating cartilage）：软骨细胞快速分裂，
纵向排列成行。（3）软骨成熟区（zone of maturing cartilage）：软骨细胞肥大，胞质内糖原
含量增多，细胞柱之间的软骨基质变薄。（4）软骨钙化区（zone of calcifying cartilage）：软
骨细胞逐渐变成空泡状，核固缩， 后退化死亡，软骨基质钙化，呈强嗜碱性。（5）成骨区
（zone of ossification）：可见过渡性骨小梁，骨小梁之间为初级骨髓腔。
出生前后，在长骨的两端出现新的骨化中心，称为次级骨化中心（secondary ossification
center），形成过程与初级骨化中心相似，但骨化从中间向四周呈辐射状进行， 后大部分软
骨被初级骨松质取代，使骨干两端变成骨骺。骨骺表面不断骨化，但是始终残留薄层透明软
骨，即关节软骨（articular cartilage）。骨骺和骨干之间也保留一层软骨，称骺板（epiphyseal plate）
或生长板（growth plate），是长骨继续增长的基础。在成年以前，骺板的软骨细胞不断增值，
生成新的软骨，依照软骨内成骨的方法进行成骨，使骨不断增长。到了青春期末，骺板增长
减慢之停止增长， 后骨化，形成骺线（epiphyseal line），长骨因而不再增长。骨骺通过改
建，表面为薄层骨密质，内部为骨松质。全身各骨骨化点出现和干骺愈合的时间由一定的规
律，临床上可据此通过 X 线检测骨的年龄，称骨龄。正常是骨龄与本人的实际年龄相符合，
在疾病情况下，骨龄可大于实际年龄，表示发育过快，或小于实际年龄，表示发育缓慢。
初形成的骨干主要由初级骨松质形成，但未形成骨单位。大约在 1 岁左右，骨单位才
开始形成。破骨细胞在骨干外表面分解吸收陈旧骨组织，形成一条纵沟，骨外膜的血管及骨
原细胞等随之进入沟内，形成骨组织，将纵沟封闭为管道。成骨细胞贴附于管道的表面，从
外而内形成同心圆排列的骨单位骨板。原先的管道缩小，成为中央管，其中含血管。中央管
与骨干表面之间留下的通道即为穿通管。随后，旧的骨单位逐渐被分解吸收，新一代骨单位
不断形成，残余的旧骨单位即为间骨板。同时，骨膜下的成骨细胞形成环骨板，并不断改建。
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由于骨单位的相继形成和外环骨板的增厚，骨干逐渐增粗。成年后骨干不再生长，但其内部
的骨单位改建仍持续进行。
三、 影响骨生长发育的其他因素
骨的生长发育与各种因素对成骨细胞和破骨细胞的调控息息相关，同时受到其他多种因
素的控制，包括激素，维生素和应力作用。
1、 激素
生长激素和甲状腺激素可促进骺板软骨的生长和成熟，若人体在成年前这两种激素水平
过低，可导致身材短小；若生长激素分泌过多，可导致巨人症。甲状旁腺激素和降钙素通过
反馈机制调节血钙水平：血钙降低时，甲状旁腺激素激活骨细胞和破骨细胞的溶骨作用，分
解骨盐，释放钙离子入血；降钙素则抑制骨盐溶解， 并刺激骨原细胞分化为成骨细胞，增强
成骨活动，使血钙入骨形成骨盐。甲状旁腺激素分泌过多，骨盐大量分解可导致纤维性骨炎。
雌激素和雄技术能增强成骨细胞的活动，参与骨的生长和成熟。更年期后的妇女雌激素分泌
不足，成骨细胞处于不活跃状态，而破骨细胞的活动相对增强，引起更年期后骨质疏松症。
此外，糖皮质激素抑制骨形成。
2、 维生素
维生素 A 及其衍生物维 A 酸（retinoic acid）能协调成骨细胞和破骨细胞的活动， 维持骨
的正常生长和改建。维生素 A 严重缺乏是可导致骨的发育畸形， 也可引起骺板生长缓慢，骨
生长迟缓或停止。维生素 A 过多使破骨细胞活动增强，骨吸收过度而容易发生骨折。维生素
C 与成骨细胞合成骨胶纤维和基质有关，严重缺乏时骨干变薄变脆，骨折后愈合缓慢。维生素
D 能促进肠道对钙、磷的吸收，提高血钙和血磷水平，有利于软骨基质和类骨质的钙化。 儿
童期缺乏维生素 D 可导致佝偻病，成人缺乏则引起骨软化症。过量服用维生素 D 可引起骨的
钙化。
3、 生长因子和细胞因子
近年来发现一些生长因子和细胞因子与骨的发生、生长发育、骨重建，维持骨体积和结
构有密切关系。这些生长因子和细胞因子包括：成纤维细胞生长因子（FGF）、转化生长因子-β
（transformation growth factor, TGF-β）、胰岛素样生长因子（insulin-like growth factor, IGF）、
血小板衍化生长因子（platelet-derived growth factor, PDGF）、骨形态发生蛋白（bone
morphogenetic protein, BMP）。 它们可由成骨细胞分泌，也可来自骨外组织，通过旁分泌或自
分泌作用，激活或抑制成骨细胞或破骨细胞。这些因子或是通过与细胞表面的受体结合起作
用，或是调控细胞间的相互作用。
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成纤维细胞生长因子（FGF）及其受体（FGFR）与骨的正常发生有密切关系，FGFR-1
和 FGFR-2 共同表达与软骨形成和骨形成发生的区域，FGFR-3 表达于长骨的软骨省长扳，这
些受体的突变与骨骼发育不良如颅缝过早闭合和侏儒等有关。
胰岛素样生长因子（IGF）是骨细胞总含量 为丰富的生长因子，由骨细胞生成并存储与
骨中，在骨吸收时从骨组织中释放出来。成骨细胞在骨吸收活跃时分泌 IGF 增多，以自分泌
方式作用于成骨细胞，促进骨形成。同时破骨细胞也产生 IGF 以旁分泌方式作用于成骨细胞。
因此，IGF 在骨吸收活跃时诱发骨吸收和骨形成之间的偶联作用，使骨吸收增加的同时骨形成
也增加，有利于骨重建的正常进行。
转化生长因子 β-1（TGFβ-1）是骨基质中含量丰富的细胞因子，它刺激干细胞生长并使之
分化为成骨细胞。成骨细胞也可分泌 TGFβ-1，它可能通过 TGFβ-1 进行自我调节。同时 TGFβ-1
又是破骨细胞分化的一个重要自分泌因素。TGFβ-1 通过调节成骨细胞和破骨细胞的功能而调
节骨形成与骨吸收的平衡。TGFβ-1 基因突变与代谢性骨病的发生有关，其基因型的不同影响
青少年时峰值骨量以及成年后的骨丢失率。
血小板衍化生长因子(PDGF)可以由血小板产生，或是由内皮细胞、平滑肌细胞、巨噬细
胞、成纤维细胞及转化细胞产生，存在于血清中，能诱发骨髓间质细胞增值分化。当骨折发
生时，血小板释放出大量的 PDGF，增强单核细胞和巨噬细胞的游走性，促进骨折局部成纤维
细胞的大量增殖和分化，协同其它生长因子促进骨折愈合。
骨形态发生蛋白（BMPs）是 TGFβ超家族成员，具有刺激成骨的作用。BMP 可由成骨
细胞产生，与骨基质结合，诱发未分化的间质细胞分化形成软骨和新生骨，与其他生长因子
互相作用，共同诱导新骨形成。
4、 应力作用
应力为结构对外部加载负荷的反应，骨的发生和生长于骨的受力状态密切相关。实验表
明，骨在处于生理范围内的应力作用下，以骨形成为主，而在低应力下以骨吸收为主，周期
性应力作用可同时刺激骨形成和骨吸收。如运动员骨密度与下肢运动量成正比；因椎间盘突
出而卧床休息的患者，骨盐平均每周下降 0.9%。
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致谢：作者感谢中山大学病理教研室王连唐教授为本文提供图片资料。
所需图片
图 1-1 骨板排列方式示意图
图 1-2 骨密质 HE 染色
图 1-3 骨松质 HE 染色
图 1-4 骨膜 HE 染色
图 1-5 长骨的构造示意图
图 1-6 编织骨 HE 染色
图 1-7 板层骨 HE 染色
图 1-8 骨原细胞 HE 染色
图 1-9 成骨细胞 HE 染色
图 1-10 成骨细胞电镜图
图 1-11 成骨细胞分化及细胞内信号传递示意图（已提供）
图 1-12 骨细胞 HE 染色
图 1-13 破骨细胞 HE 染色
图 1-14 破骨细胞电镜图
图 1-15 破骨细胞分化示意图（已提供）
图 1-16 破骨细胞细胞内信号传递示意图（已提供）
图 1-17 胶原纤维结构示意图
图 1-18 膜内成骨示意图
>>Appendix E. Bone<<
199
图 1-19 软骨内成骨示意图
图 1-20 胎儿长骨成骨活动分区示意图
>>Appendix E. Bone<<
200
图 1-11 成骨细胞分化及细胞内信号传递示意图
（图片来源：修改自 Miyazono K, Maeda S, Imamura T: Coordinate regulation of cell growth and
differentiation by TGF-beta superfamily and Runx proteins. Oncogene 23:4232-4237, 2004.）
>>Appendix E. Bone<<
201
图 1-15 破骨细胞分化示意图
（图片来源：修改自 Katagiri T, Takahashi N: Regulatory mechanisms of osteoblast and osteoclast
differentiation. Oral Dis 8:147-159, 2002.）
>>Appendix E. Bone<<
202
图 1-16 破骨细胞细胞内信号传递示意图
（图片来源：修改自 Katagiri T, Takahashi N: Regulatory mechanisms of osteoblast and osteoclast
differentiation. Oral Dis 8:147-159, 2002.）