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MACROPHAGE PHAGOCYTOSIS OF POLYETHYLENE PARTICULATE IN VITRO

Irina Voronov

A thesis submitted in confonnity with the requirements

for the de- of Master of Applied Science

Graduate Department of Chemical Engineering and Applied Chemistry

University of Toronto

O Copyright by Inna Voronov 1997

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Macrophage phagocytosis of polyethylene prticulate in vitro

Master of Applied Science, 1997

Irina Vofonov

Graduate Department of Chernical Engineering and Applied Chemistry

University of Toronto

ABSTRACT

Polyethylene particles are the major constituents of the material debris fonned as a result of

orthopaedic implant Wear. The purpose of this study was to develop an in vitro mode! which

would allow the introduction of polyethylene paniculate into macrophages. UHMWPE ( 18-20 p

m) and HDPE (4-10 pm) were characterized by XPS and FT-IR and suspendrd in soluble

collagen typ I which was subsequently solidified on glass covenlips. Mouse crll line

macrophages (IC-2 1) were established on the collagen-particle substrats and maintained for up

to 96 hours. The response of the cells to the pnicles was examineci by light microscopy. TEM,

and SEM. Histological analysis of the samples rcvealed that the macrophages surrounded larger

prticlcs (18-20 pm) and the cells appeared to be attached to the surface of the panicles, while

the smaller particles (4- 10 pm) had been phagocytosed within two hours. lnflammatory

cytokines. lysosomal enzymes, and prostaglandin E2 were released into the medium, and IL-la,

IL- I P, PGE2, P-plactosidase and hexosaminidase levels wcre signifimtly increased over

control values. These results validate the mode1 as means o f audying the specific in vitro

interactions of polyethylene with cells.

Dr. E.L. Boynton, Women's College Hospital

Dr. J.W. Callahan, Department of Neuroscience, Hospital for Sick Children

Mr. R. Chemecky, Department of Dentistry, University of Toronto

Dr. J.E. Davies, Centre for Biomaterials, University of Toronto

Dr. A. Hinek, Department of Cardiovascular Research, Hospital for Sick Children

Ms. J . Hwang, Department of Pathology, Hospital for Sick Children

Mr. S. Lugowsky, Centre for Biomsterials, University of Toronto

Ms. M. Mandes, Department of Pathology, Mount Sinai Hospital

Dr. A.B. Nathens. Toronto General Hospital

Dr. J.P. Santerre, Department of Chemical Engineering, University of Toronto

Dr. R.N.S. Sodhi, Centre for Biomaterials, University of Toronto

Dr. J . Sundhu, Samuel Lunenfeld R e m c h Institute, Mount Sinai Hospital

TABLE OF CONTENTS

ABSTRACT

ACKNOWLEDGMENTS

TABLE OF CONTENTS

LlST OF TABLES

LIST OF FIGURES AND ILLUSTRATIONS

LIST OF APPENDICES

1 . INTRODUCTION

1.1. Clinical problem

1 2. Physical/chemicaI factors contri buting to aseptic loosening

1 2 . 1 . General design of total hip prostheses

1 .2.2. Materials used in total hi p prostheses

1.2.3. Factors contributing to the mechanical properties of UHMWPE

1.2.3.1. Synthesis

1.2.3 2. Contaminants/residues

1.2.3.3. Processing

1.2.3.4. Steniization

1.2.4. Fornation o f wear particles

1 24 .1 . Hyphesis of Wear formation

1 .S.4.2. Particdate characterization

1.3. Biological factors contributing to aseptic loosening

1.3.1 . Characterization of the infiammatory membrane

1.3.2. Macrophages anci their role in s p i c loosening

1.3.3. Lysosomal enzymes

1.3.4. Inflammatory mediaton released in r e s p ~ w to wear particles

1.3.4.1. Cytokines

1.3.4.2. Prostagluidin E2

1.3.5. In vivo and in vitro studies of Wear particulate

Page

t i

111

IV

vi1

VllI

1X

1.3.6. In vitro studies of polyethylene perticulate 19

1.4. The role of extracellular matrix proteins in cellular response to biomaterials

1.4.1. Collagen type 1

1.4 2. The effect of collagen type I on macrophages in vitro

1 S. Scope of thesis

1.5.1. Objective

1.5 2. Experimental approach and rationale

2. MATERIALS AND METHODS

2 1 . Particles

2.2. Panicle surface chrmical charactenation by X P S

2.3. Puticle bulkchemical chamterkation by FT-IR

2.4. Endotoxin test

2.5. Particle suspension preparation for triton x-100 model

2.6. Experiments usine mouse peri toneal macrophages

2.7. Particlr suspension prepmation for col lagen mode1

2.8. Coverslip preparation

1.9. Experiments using the m o u macrophage cell line

2.10. Viability test

2.1 1 . Hernatoxylin and eosin staining

2.12. Cell/area count

2.13. SEM

2.14. TEM

2. I S. Media analysis

2.15.1. IL-la, IL49 assay

2.15.2. I L 4 assay

2.15.3. TNFs assay

2.15.4. p-GALand HEXassay

2.15.5. PGEr assay

2.16. Statistical analysis

3. ESULTS

3.1. Particle characterization

3.2. Model development

3.3. Model characterization

3.3.1. Histology

3.3.2. Cell counts

3.3.3. Cytokine and lysosomal enzyme analysis

3.3.4. Viability test

4. DlSCUSSlON

4.1. Histological characterization

4.2. Biochemical characterization

4 2 . 1 . Cytokines and prostaglandin E?

4.2.2. Lysosornal enzymes

4.3. Summary

5 . CONCLUSIONS

6 . RECOMMENDATIONS

7. REFERENCES

8. APPENDK

LIST OF TABLES

Table 1 .1 . Summary of particulate characterization studies.

Table 1.2. Sectetory products of macrophages.

Table 1.3. Summary of macrophage surface receptors.

Table 1.4. Targets and activities of major inflammatory cytokines.

Table 1 S. Main types o f collagen.

Table 2.1. Physical properties of UHMWPE and HDPE.

Table 3.1. XPS analysis of UHMWPE and HDPE.

Table 3.2. Swnmaty of the absorbante peaks in FT-IR spectra.

Table 3.3. Summary o f the perfonned experiments.

LIST OF FIGURES AND LLLUSTRATIONS

Figure 3.1.

Figure 3.2.

Figure 3.3.

Figure 3.4.

Figure 3.5.

Figure 3.6.

Figure 3.7.

Figure 3.8.

Figure 3.9.

Figure 3.10.

Figure 3.1 1 .

Figure 3.13.

Figwe 3.14.

Figure 3.15.

Figure 3.16.

XPS spectra of polyethy lene particles.

FT-IR spectra of polyethylene micles

Scanning rlectron micrograph of HDPE particle distribution in

collogen matrix on a coverslip.

Media analysis of the mouse peritoneal macrophages exposed to

UHMWPE particles, coaled with triton x- 1 0 , for 24 and 48 houn.

H&E staining of the IC-21 cells exposed to UHMWPE particles for 20

hours.

H&E staining of the IC-21 cells exposed to HDPE puticles for 24

hours.

Scanning electron rnicrograph of IC-21 cells exposed to HDPE

particles for up to 24 hours.

Transmission electron micropph of IC-21 cells expowd to HDPE

particles for 20 houn.

Changes in cell number and cell area % with time (Experiment 3).

Changes in cell number and cell area % with time (Experiment 2).

Media analysis of IC-21 cells exposed to 18-20 pm UHMWPE

particles.

Cytokine release by IC-21 cells exposed to HDPE particles; 34 hou

time point.

Lysosomal enzyme and PGE2 release by IC-21 cclls exposed to HDPE

particles; 24 hour time point.

Sumrnuy of L I results ôaseâ on Experiments 3-6.

Summary of TNFa results based on experiments 3-6.

IC-2 1 cells exposed to HDPE perticles for 24,48,72, and % hours.

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Tabie 9

Summary of the performed expenments

Summary of cell and area % counts

Summary of T N F s data

Summary of 1L- 19 data

Summary o f extracellular P-GAL data

Summary of extracellular HEX data

Summary of PGE2 data

Summary of IL- la data

Summary of IL-6 data

1. INTRODUCTION

1.1, CLINICAL PROBLEM

i t has been estimated that 300,000 to 400,000 total joint replacements are implanted each

year in North America alone.' Aseptic loosening o f total joint prosthesis has been established as

the number one cause of implant failure.In It is defined as a loosening of the prosthesis in the

ahsence o f an infection.'.'" Numerous facton, b t h hiological and mechanical in nature, are

responsible for prosthesis failure. The patient's general health, predisposition to allergies,

patient's lifestyle, infection, and osteolysis represent the biological facton, while surgical

problems, prosthesis design. mechonicol Wear, and stress shielding are the mechanical facton

that contribute to implant failure.

Ravision surgeries of the failed implants have demonstmted the presence of a membrane

at the bone-implant interface.' This membrane forms ri thick inflammntory tissue in some

patients, and remains thin and non-progressive in othen.' Histological evaluations of the

membranes retrieved at revision surgeries reveal that thin, non-progressive membranes contain

mainly fibroblasts and a frw macrophages, with almost no particulate debris. while thicker

inflammatory membranes have abwidant macrophages, fibroblasts, ond foreign body gant cells

which were sunounding and engulfing particulate debris.'.' The characterization of these

membranes have drmonstrated the presence of various types of particulate Wear debris

consisting of metal. polyethy lene and pol ymethyl methacry late, with sizes ranging frorn

submicron values up to hundreds of rnicrond' Immunohistochemical evaluation revealed the

presence of a number of inflammatory mediaton which are known to take part in inflammation,

wound heal ing, and bonr remdel

Aseptic loosening of orthopaedic implants is oflen accompnied by severe osteolysis."*"."

Histological evaluation o f the osteolytic lesion has demonstrateâ active bone resorption6 It hm

bKn hypothesized that the inflammatory mediators, including Ma, IL-1. IL-6, PGE,, which

have been releaseâ by activated macrophages are capable o f stimulating osteoclasts to resorb

borie.'"*' These mediators are thought to inhibit bone &position by osteoblasts, thenfore,

breaking the balance between osteoclasts and osteoblasts in bone turnover. 46J.9 Another factor

contributing further to bone lysis could be the presence of lysosornal enzymes which are released

as a result of ccll death or/and fnistrated phagocytosis. These enzymes can cause tissue damage

as well as lower the pH of the surrounding tissue. Furthemore, it has been shown chat activated

macrophages themselves are capable of direct bone resorption chus conttibuting to bone loss at

the bone-implant interfàce. 7,10.11 Therefore, three biological mrxhanisms contribute to bone

resorpiion at the bne-implant interface and a!! of these processes are in p n rclatcd to

particulate açt ivation of macrophages. 3,6,8,12

Polyethylene particulate i s considered to be the most abundant particle of the Wear debris

and i s believed to be causing a stronger inflammatory response than any other constituent of the

Wear debris." It is not fully understd why numerous Wear debris are producad in some cases,

while there is no accumulation of Wear particles at the bone-implant interface in othen. It i s

also not clear how the particles migrate fiom the high friction areas of the implant to the

inflammatory membrane at the bone-implant interface, however, several mechanisms have been

proposed. Schmalzried et al." introduced a concept of the effective joint space in order to

explain the migrrition of the Wear debris around the implant. According to Schmal~ed's

hypothesis, Wear debris i s dispersed in joint fluid. Depending on contact between the bone and

the prosthesis, particulate debris is camed by joint fluid to and along the bone-implant interface.

and through the sot? tissues and bone. " Thus, the particles are delivered to distant sites along

the bone-implant interface and stimulate bone resorption far from the place of Wear generation.

Therefore, the integnty of the bonc-implant interface is crucial to limiting the transport of

particles and hence ensuring the survival of the prosthesis.'

Since the inflammatory membrane is located at the bone-implant interface, and the

inflammatory mediators are believed to have an efTect on bone rern~delin~,"~'~ a continuous

cycle of bone fesotption/wear formation cm ensue in the following manner. The bone

resorption ultimately provokes mechanical instability which accelerates implant loosening by

increasing the production o f more wear debris. This, in tuni, increases the flux of inflammatory

cells to the injured site, which is then followed by an increased secretion of inflarnmato~

mediators. This propsgates the subsequent bone resorption around the implant

Even though numerous clinical, in vivo. and in vitro studies have k e n perfomed. the

exact mechanism o f aseptic loosening is not well underst~od.~ In order to treat (or prevent)

aseptic loosening by means other than revision surgery, it i s crucial to undentand the processes

i nvolved. The potential therapies could inc l uûe blocking the inflammation, bloc king bone

resorption. or enhancino hnne heding and formation around the implants. However, pnor to

achieving these goals a better understanding of mechanism of aseptic loosening is clearly

required. Pan of this undentanding will corne from the study of particulate interactions with

cells and specifically macmpags. The purpose of this thesis was to develop a mode1 that would

allow for the study of polyethylene particulate phagocytosis. Polyethylene panicles are of

specific interest since they are the least studied component of prticulate Wear debris, but yet the

most abundant particle type found in the inflammatory membrane at the bone-implant interface.

This model may be further used to contribute to a better understanding of the mechanism of

aseptic looxning and to investigate the process of bone lysis by cells which have k e n activated

via exposure to polyethylene and other particulate matter.

2 . PHYS~CAL/CHËMICAL FACTORS COhiTRIBCTINÇ TO ASEPTiC LOOSERING

1.2.1. Generat dcsign of total hip prorthga

Total hi p replacements were f i nt introduced in England by t wo inde pendent

investigators, McKee and ~ h a m l e ~ . " Ektween 1958 and 1961 three hundred total hip

prostheses were implanted by Chamley. Cunently about 400,000 total joint replacements an

perfonned each year in North Amenca alone. ' In the last 35 yean the general design of joint

implants has remained fairly constant. Total hip implants usually consist of a metal stem with a

metal bal1 and a metal socket covered with a plymer (usually UHMWPE).'~~'~ The metallic

compnents of the prostheses are attached to the bone with polyrnethyl methacrylate cernent in

older and less active patients, while cemendess piostheses are wiolly recornmended for younger

recipients. The association of bone cement particles (PMMA) with the observation of aseptic

loosening had originally led scientists to refer to the clinical problem as "cernent diseaseY6 As a

result, different methods of fixation were developed and cementless prostheses were introduced.

Fixation of uncemented components was enhanced by making the surface of the metallic stem

pcrous. e.g. by coating the surface with beads. This system facilitatecl implant fixation by

allowing bone ingrowth to occw within the porous structure." This close contact between bone

and implant is vcry important kcause it provides stability to the implant and prevents the

development o f a conduit for particles an4 therefore. the development o f osteolysis.

1.2.2. Materials uscd in total hip prostheses

The choice o f materials for total hip prostheses has k e n narrowed down to three popular

polymrrs (acrylate cernent, polyethylene. and Silastic@) and fourteen metal alioys (three iron-

based. xv rn cobalt-base4 and four titanium-based)." Composite structures. such as the

combination o f cobalt alloy femoral heads and titanium alloy stems have also been used in order

to take odvantage of high Wear resistance of cobalt alloys and the high fatigue strength of

titanium ûlloys. '"

1.23. Factors contributiag to the mechanical properties of UHMWPE

UHMWPE has been used as the matenal of choice for the sockets of total joint implants

since 1%2.'4 This material has a higher rcsistance to wear and creep than does TeflonB, and

since the 1970s the polyethylendmetal piring has become the most common choice for joint

prosthesis of the hip. knee, ankte, shouldrr. elbow. and wrist.14 UHMWPE has a molecular

weight in the order of millions as compared to 10,000 to 500,000 for other ptyethylenes.14

IJHMWPE i s characterized by high notched bar impact strength, high abrasion resistance,

chernical resistance, and a low friction coefficient. '" 1.2.3.1. SyntbMr UHMWPE i s synthesized by choin polymerization when ethylene

molecules are added to catalyst particles.14 The catalysts usually employed in the process aie

made from titanium chloride and organoaluminwn compounds such as triethylaluminium or

diethylaluminium chloride. These catalyst particles are usually 5-10 pm in size, white newly

synthesized UHMWPE particles are not uniform and Vary in size from 63 to 250 pm."

1 *2*3.2. ContamiaanWresidues. Different types of residues are encountered in

UHMWPE powder. Titanium and aluminum salts, which cm be present as hydroxides,

chlorides. and hydroxychlorides. as well as chlorine ions have been detected.'" Small amounts

of the calcium sali of an organic acid are addrd as a corrosion inhibitor and therefore are also

present in the powder '" 1,2.3,3. Processimg. A k r synthesis, the powder of UHMWPE is processed into implant

parts using two processing methods: the compression sintering technique and the injection

rnoulding technique." UHMWPE has a very hi@ molecular weight which does not pennit the

material to be transformed into a free-tlowing mass by heating. " Using the compression

sintering technique, UHMWPE powder i s inserted into the moulds and then air 1s forced out of

the moulds by an oil-hydraulic mechanism." During the plasticizing phase, heat is applied to

the moulds (between 200°C and 250°C) until the powder is plasticized.'' Then the heat i s slowly

and uniformly removed in order to minimize the shrinkagr stress from the cooling process and to

achieve optimal physical proprrties.14 The injection moulding technique requires high injection

pressures, processi ng temperat ures between 200°C and 230°C, and mould temperat ures between

40°C and 70°C. ' 1.2J.4. Steriliution. Implants made of UHMWPE are stenlized by y-inadiation.'i Even

though this method of sterilization has numerous advantages, such as a high microbicidal eftèct,

it changes the characteristics of the matenal." Formation of radicals, polymerization,

depolymeriuition, and oxidation take place aAer the polymer has been exposed to y-rays It has

been show that these changes in matenal structure c m lead to changes in the polymer's

ultirnate mechanical propnies. which, in tum, may affect the functian of the prosthesis and

cause the formation of wear debris. ''

1.2.4. Formation of wear particles.

1.2.4. 1. Hypotbab of w a r formatioa. It has been proposai by Clarke et al.'' that in

stable, well- fixed implants, the main trigger mechanism for osteolysis and accompnying

loosening is the release of UHMWPE and metal puticulates from the articulating surfaces.

Wear of the joint's bearing surface is an inevitable conseqwnce of activity. The more the

patient uses the joint and the more active he or she is, the greater the amount of Wear debris that

will be releosed into the tissues and the geeter the risk of hone loss " Clarke's rtudies

rstimated that 20 million to 40 billion UHMWPE poriicles shed into the joint space every year.

Clarke et al." hypothesized that even in wdl-fixed implants there i s micromotion between ( 1 )

the stem of the implant and the bone cernent and (2) between the polyethylene socket and the

metal ball. This micromotion produces some metal and polyethylene debris. Any debris smaller

ihan 5 Fm i s phagocytosed by macrophages, which in tum secretr inflammatory mediators as a

result of their activation.'' Thetefore as more particdate is proâuced at these interfaces, more

particles are phagocytosed by macrophages, and more in flammatory cytokines are released into

surrounding tissues. " Hence. extensive bone loss and loosening of the implant are the inevitable

results of this process.

However. Clarke's scenario has not been the oniy hypothesis put fonvard. Pienkowski et

al? postulated a hypothesis bosed on the study o f virgin UHMWPE powder. This goup

obsewed that submicron size spheres, which exist within the processed material, retain their

morphological identity throughout the pmcessing, sterilization. storage, and Wear." Pienkowski

et al.'* hypothesized that submicron UHMWPE Wear debris may oridnate not only from Wear

processes and fnction rn VIVO, but also from relrase o f these submicron particles fwnd in virgin

UHMWPE. These particles wil l then contribute to macrophage activation and bone resorption.

1.2.4.2. Prriiculrte cbamcterizrtioo. Since it has been suggested that, in the

presence of debris, macrophages can release vanous cytokines and factors that induce bone loss,

it has been hypothesized that the composition, the size, the amount, and possibly the shape o f the

particles may be important variables thrt influence macrophage nsponse and subsequent

ostco~~sis.~ As a result of these thoughts the characteri~ption of wear debris has stimulated an

Digcsting rgeat

NaOH

KOH UHMWPE 0.1-2.0 pm spheroiâs

Partick type

UHMWPE

concentrated nitric

1

2 1

1 1 NoOH, KOH 1 1 crude papain,

Table 1.1. Summaiy of particdate characterization studies.

Particle a i z t

0.07-6.3 pm

0.57-12.2 pm

Ti

pol yethy lene,

acid

Soluene 350

extensive area of research. A summary of particulate characterization studies is presented in

Particle sbrpc

rounded

elongated

("fibri 1s" and

metal

HDPE, Ti

Table 1.1. There are some variations in the methods for particulate isolation. In order to digest

1.1-10.0

1 - 2 0 pm

1 0-400 ~ I I

0.63 pm

metal

pol yethy lene

retrieved inflammatory membranes Campbell et al.12 and Shanbhag et al! used strong bases,

fibrils s hards

flakes of wire

no consistent par-

0.8-1.8 pm ,

did not evaluate

Margevicius et a1.10 employed concentrated nitric acid, while Lee et al.?' utilized a commercial

2.0-1 3.0 pm

shard-shaped

did not evaluate

tissue solubiiizer. Campbell et al.?' fowid that crude ppain offired more complete digestion

ticle morphology

did not evaluate

than either purified papain or sodium hydroxide. but they aiIl noted apparently undigested

cellular debris in filirates. The meihods of ~ l l ~ l y s i s and the results obtained varied from group to

group. Lee et al.,-" on the basis of light microscopy. suggested that the mean size o f the

polyethylene panicles was two to four by eight to thirteen micrometers; other authon have

recognized thot many particles are smaller than the resolution of the light micro~cope.~2

Margevicius et measureâ the puticles with an elecbical mistance puticle-size analy~er

(Coulter Multisizer) and studied the particle morphology with scanning electron microscopy.

They estimated that the majority of the puticles naieved Eiom the synovial membranes are

submicron in size; other invest iga tors~~~9~2~~ have reportcd similsr results. However, it was

reported by Margevicius et a1.I0 that this particular Coulter Multisizer prt icle analyzer has finite

detection windows and therefore cannot analyze particles below 0.58 pm and above 20 It

has been observed that the particles were irregularly shapecî, and did not have rough edges. This

could be due to the e ffect of strong bases and acids during the extraction processes. ' 2 ~ 1 Y ~ ? l ~ ? 2 Al l

of the above s t u d i e ~ ~ ~ J ~ ~ ~ ~ ~ 2 ~ have concentratcd on the isolation of UHMWPE particles, since it is

believed that polyethylene is a key particle and plays an important role in macrophage activation

and bone rcsorption. Nevert heless, metal and PMMA particles were also prewnt in the wrnples.

However, it has ken observed that PMMA particles melted and clurnped topther as a result of

tissue digestion used for the particle reco~er~ . '~

1.3. BIOLOCICAL FACTORS CORTRIBUTINC TO ASEPTIC LOOSENINC

13.1. CharacterU.tioa of the intlamntatory membmne.

It has been consistently observed that an inflammatory membrane is fomed at the bone-

implant interface and is associated with particulate d e b d Histological evaluation of this tissue

has dcmonstrated that i t consists o f Iibroblasts, macrophages, foreip body gant cells, and

occasional T cells and B cells. 44.n.9.23 Characterization of this tissue has also shown that the

inflammatory membrane has al1 the characteristics of a synovial membrane and, therefore. is

alw referred to as a pseudosynovial membrane. Just like che synovial membrane. the

periprosthetic tissue contains collagen type 1 and type III as the predominant extracellular matrix

proteins.24 Cloxr analysis o f the inflammatory membrane hm revealeâ the prewnce of

particulate Wear debris. 44iN.9.23 It was observed that al1 (4 pm) srnall particles were associated

with mcicropheges. while large ( M O pm) particles were surrounded by foreign body gant cells."

It has been reported that the most noticeable cellular reactions, such as inflammation anâ Foreign

body granulome formation. are associated with the response to polymeric ( polyrnethy l

rnethacrylate m&or polyethylene) pnrtic~late.~

Immunohistochemical evaluation of the inflarnmatory membranes confinned that

macrophages were the most abundpnt cells in the tissue and that they were actively involved in

phagocytosis of the particulate. 2,5.6.13 It was also demonstrated that T cells made up about 10%

of the inflarnmatory cells, while B cells were o~casional.~ The presence of T cells could imply

their involvement in the amplification of the inflarnmatory response, or in a specific delayed

hypersensitivity reaction io particulate debris.' Thus. macrophages are the crll type responsible

for particle phapcytosis and the development of a mode1 to study the macrophage-pariiclr

interactions would be useful in understanding the mechanism of aseptic loosening.

1.3.2e Macrophage and their rok in aseptic looscning.

Macrophages (or mononucleir phagocytes) are the scrvenger cells of the immune

~~stern.'~''' They play an important role in host defense. bacteria and foreign body destruction.

and in wound healing and tissue repair. Macrophages are mature and fully differcntiated cells

that arc located in different parts of the body and, therefore, have different pmperties.2' These

cells belong to the hemopoietic system in the bone rnarr~w.'"'~ The progenitor of macrophages

is the granulocyte-monocyte colony forminy unit (CFU-GM) stem cell: i t &es rise to the

monoblnst, which divides once to fonn two promonocytes; their division leads to the formation

of two monocytes. Monocytes then migrate from the bone marrow into the circulation. where

aAer about 24 hours they migmte into tissues to become macrophages.'h" Depending on the

type of tissue, different types of macrophages are fonned. These cells do not divide and their

number depends on the influx of fresh monocytes into the tissues.26

Macrophages perforrn numerous functions. The classical function of these cells is

phagocytosis, killing, and digestion of micmorganisms. Macrophages xcretr a geai variety of

biologically active molecules, such as enzymes, components o f the complement system and

coagulation cascade. growth foctors, cytokines, reactive oxygen and nitrogen intermediates (see

Table 1.2). 25-29

Poivoe~tide bormonca and cvtolrincs:

Interleukin-l a and P (IL-la, CI), interleukin-6 (iL4), tumour necrosis factor a (Wu),

interferon y (IM-y), platelet-derived growth factor (PDGF), fibroblast gowth factor,

transfonning growth factor f3 (TGF-P), colony stimulating factor-1 (CSF-I ), g~ulocyte colony

stimulating factor (GCSF), granulocyte-macrophage colony stimulating factor (GM-CSF),

rnonocytederived neutrophil chemotactic factor

Biorctive lioid~:

6-ketoprostaglandin FI; 12-hydroxyeicosatetranoic acid; leukotrienes BI, C. D, E; platelet

activating factor (PAF); prostacycline; prostaglandins Er, Fr, Il; thromboxane Bz

Coagulation commnents:

facton V, VII, W. X; prothrombin; prothrombinase; plasminogen activator inhibitors; tissue

thromboplastin

Com~lement cornnonenta:

C 1, C2, C3, C4, CS; facton B, D, H, I (C3b inactivator); propdin

Enmmes:

Acid lysosorna1 hydrolases:

aryl sulfatase: cathepsins B and D; cholesterol esterase; deoxyribonucleases; glycosidases;

phosphatases; proteinases; ribonucleases; trygl yceride lipase

Neutra1 proteinases:

angiotensin convertase; collagenases: elastase; plasminogen activator; stromel ysin

Others:

arginase; l i poprotein lipase; lysozyme; phosphol ipase A2; transglutaminase

Reactive orvsen rad a i tmea metabolitu:

01, HZ02, OH-; NO-, ~ 0 ~ -

Stcroid bormoaa:

I ,2 5dihydroxyvi tamine D3

Table 1.2. Secretory products of macrophages. AQpted from Austyn et al.'' and aha an.''

Macrophages possess a variety of surface recepton that allow these cells to react quickly

to different types of stimuli.25 Some receptors enable the cell to recognize the biochemical

features of the pathogen, while other receptors distinguish host proteins attached to the surfacc

of the foreign bodyJ0 Major surface receptors of macrophages are summarized in Table 1.3.

Receptor Abbreviation .- CD Ligand

I

1 FcvRi i CD61 Fc recrpton i monomeric igGZa 1 1

cornpiement recepton 1 CR3 1

CR 1

Chemotactic recmtor I

CD I I b/CD 1 8

Carbohydnitc: receptor

FMLPR

complement C3 bi, others

CD 35

/ f-met-leu-phe

- cornplement C3b

MFR high-mannose polysaccharides

Extracellular matrix receptors

Table 1 .3. Surnrnary of macrophage surface re~e~tors. '~. '~

VLA proteins

lrnmunoglobulin Fc recepton (FcR) and complement recepton (CR) enable macrophages

Fi bronectin R

various

to bind to opsonized bacteria andior foreign bodies. Opsonization of foreign material by

fibronectin

antibodies and complernent compomnts enhances the process of phagocytosis by

macrophages.25 The mannosyl-fucosyl receptor (MFR) is utilized by macrophages to bind

glycoproteins thst contain fucose-mannose midues, such as zymosan and LPS. and, therefore, is

responsible for removal of nonspsonized bacteriaV2' Chemotactic peptide recepton tngger

macrophages to move towards the site of inflammation by following the gradient of chemotactic

peptide, such as f - ~ e t - l e u - ~ h e . ~ ~ Extracellular mairix receptors allow macrophages to bind to

the proteins of extracellular matrix, which become exposed during tissue injury, and therefore, to

rernain ot the site o f inflammation."

It has been observed that phagocytosis via complement receptors requires a "second

signal".25 Furthemore, it has been demonstrated that the function of complement recepton i s

sornehow linked to extraceIl iilrr matix receptors and that, in order for phagwytosis via

cornplement receptors to be triggered, the cell has to lx attached to some component of

extracel l u lu r n a t d 5 This phenornenon can be linked to the developmental or activation stages

of the cell, since i t has been observed that freshly isolated human monocytes and resident mouse

macrophages are unable to phagocytose via complernent recepton, while activated and

inflammatory celis can."

Since monocytes and macrophages have surface recepton for serwn proteins, the

rngulfment occurs accordiny to the "zipper mechanism" (cellular membrane extends around the

particldrnicroorganism, and ligand-receptor interaction resembles a zipper a~tion). '~ The

foreign object becomes surrounded by the cell membrane thus foming a phagosorne. Later, the

phagosome fuses with lysosomes (cytoplasmic yanules), containing numerous enzymes (Table

1 .Z), cceating a phagolysosorne. where the killing and digestion of microorganism occ~rs .~'

These enzymes act together with reactive oxygen intemediates (ROI) and reactive nitropn

intemediates (RNI) for better efficiency of foreign body digestion.'5."

The phagocytosis of particulate Wear debris Rom orthopaedic hip implants is believed to

proceed via similar mcchanism as those described above, however, the fate of the particle

following phagocytosis by a macrophage is not clear. It can be conceived that as a result of

phagwytosis, macrophages could try to digest the i ngested particles, however, fol lowing their

inability to digest these foreign bodies, the cells could release the undigestcd particles as well as

products of degradation. As well, macrophages are known to clear the debis (cellular and

particulai) from the inflammation site by migrating to lymph nodes as well as to other tissues.

Therefore, in aseptic loosening macrophages could be hgnsferring the Wear debris to other

oryans of the body. possibly creating a systemic response in addition to the loosened implant.6

1.3. Lysoaomal enzymes.

As mention4 above, the engulfment and destruction of fonign maner (bacteria and

other foreign bodies) is one of the primary functions of macrophages. AAer the foreign body is

phagocytosed, i t hecomes incorporateci into the phagmrne, which later fiises with another

cellular body, the lysosome, in order to form a phagolysosomr where the digestion of the

engulfed material oc~urs.'~ Lysosomes (also known as cytoplasmic ganulrs) are packaged with

numrrous hydrolytic enzymes (sec Table 1.2). which act at low pH and are capable of depding

'5.3 1.32 proteins. lipids, polysaccharides. peptidoglycans. and nucleic acids., It has been observed

that distinct populations of granules with different constituent hydrolases are present within

macrophages." ARcr k i n g synthesized the fate of tach lysosomal enzyme can be either

associated with the degradation of the engulfed foreign body or secretion out of the cell vin

exocytosis.".'' A knowledg of the levels o f lysosomal enzymes secreted out of the ccll can

provide valuable information about cell activation and about the toxicity of the phagocytosed

rnateria~.'~.''

As well. the release of lysosomal enzymes by macrophages at the bone-implant interface

could play an important role in aseptic loosening. For example. the synthesis and release of an

enzyme like collagenase could lead to digestion of collagen present in the bone, while the

resulting low pH could cause dissolution of the bone mineral. thus enhancing the process of bone

lysis.

1.3.4. Iiflimmitory mediatom m k r d in ruparc to w u r prrtkks.

Activation of the macrophage by the process o f phagocytosis iack to increased

production and secretion of various inflammetory mcdiators (Table 1.2). These mediators have

nwnerous effecis and can be chernoamactive to other types of cells, thus causing inflw of fiesh

cells (neutrophils, monocytes, T cells. B cells, fibroblasts) to the site of infiammation. The

presence o f inflammatory cytokines in the retrieved inflammatory membranes has k e n

illustrated by nwnerous investigaton. 'J,I3,l7,21 Specifically, tumour necrosis factor a. (TNFa),

interleukin 1 (IL-l) , prostaglandin E2 (PGE:). and platelet-derived groowth factor (PDGF) were

shown to be present in the inflarnmatory membranes as demonstrated by in situ hybridization as

well as by tissue culture.'*"

1 A4.1. Cytokines. "Cytokines" is a name used for a b~oup o f proteins that are produced

and secreted by numerous tells in the responsc to a stimulus.""' This grwp or protrins is

involved in irnmunity and inflammation where they regulate the amplification and duration of

the resPnx." Even though cytokines are a diverse group of proteins. they share a number of

common properties. These proteins have low molecutar weight (€80 kDa) and are ofien

glycosylated. Cytokines arc: extremely potent agents and usudly act in an autocrine and a

paracrine manner at picomolar concentrations.'"'"' Since thrir role is to be a messenpr between

the cells. cytokines are rarely produced at a constant rate and, instead, are induced or suppressed

in response to a stimulus. Their half-life is usually shon thus limiting their action. Cytokines

are involved in a widr range of processes including control of cell proliferation and

ditTerentiation, rcgulatiun of the immune response, bone remdeling, wound healing and other 11-15 processes.

Three cytokines (IL-1. IL-6, and W u ) are considered to be the most important

inflammatory cytokines since they mediate a number of both local and systcmic inflammatory

responses. IL4 and T N F a ate produced primarily by monocytes and macrophages in response

to exposure to stimuli such as bacteriel endotoxin. and immune complexes." IL-6 is secreted by

a range of cells in response to stimulation by a number of factors. including other cytokines such

as IL4 and T N F ~ . ' ~ The detniled summary of the targets and activities of major inflammatory

cytokines is shown in Table 1.4.

Cytokines are believed to play a crucial role in aseptic loosening. 56.8- I O IL- l and TNFix

are produced primarily by macrophages in response to phagocytosis of cell debris and Wear

particulate.6 These cytokines have the potential to remit k s h monocytes to the site o f

inflammation and induce the formation of new b l d vessels in the inflammatory membrane. At

Cytokine

IL4 a and P Pducer cells

monocytes,

microphagu, other cells

monocytes,

macrophaga,

ostwblasts, T- cells, mast cells,

endothelium,

fibroblasts,

manow stroma1

cells

- - - - - - -. - -

Tamet cells

immune cells,

osteoblwb, cateocI8sQ,

fibroblasts, hepatocytes.

endothelium

ojteobiasts, osteoclarb,

B-cells, T-ce l ls, hcpatocytes

15

Activitits

-promotes brealrdown of

cartilage rad boae

-rtimulrtes wteoclwt-like cell

formation in vitro

-induces cytokine syntbaia

-cofactor for T-cell activation

-cofactor for B-cell activation

-promotes hemaiopoeisis

-activates neutrophils

-activates vascular endothelial

cells

-rnitopcnic for fibroblasts

-stimula tea mtceoclast lormition

-stimulites growtb and

diflerentiatioi of pmunors

for osteadrats

-stimulites bonc raorption

-CO-stimulates T-cells and t hymocytes -induces B-cell differentiation

-cornpetence factor to confer

responsivrness to hematopoietic

growth factors

Table 1.4. continued on next page

monocytes,

macrophages. T-cells. fibroblasts

osteobbsb, osteoclrsts, T-cells, B-cells, other ceIl types

-activates granulocytes and

macrophigcs

-promotes bone resorptioa by osteoclasts

-st imulates osteoclast-like cell

formation in vitro

-stimulates both proliferation

and differentiation of

precurson for ostcoclast-like

tells to osteoclasts

-indura cytokine sceretion cg.

IL4 and IL6 qtolytic and cytostaiic for transfonned and virus infected cells -protects cells from viral infection -CO-stimulates T-ce 1 l pro1 i ferntion -activates vascular endothelial cells

Table 1.4. Tarpts anci activities of major inflammatory cytokines. 33.35-37

the same time, they cause an increase in secretion of other cytokines. such as IL4 and IL-6,

therefore, increasing the inflammatory response. Aside from these activities, IL- 1, TNF-a, and

IL-6 have the potential to interfere with cell ditrerentiation in bone marrow, thus affecting the

balance between osteoc lasts and osteoblasts towards increased bnc resorption. fi.lt.9. I 1.36

1.3.4.2. Prostaglandin E2. Prostaglandins belong to a family of arachidonate metabolites

(eicosonoids) and play an important role in inflammation. They are synthesizeâ by various

enzymes ftom f k e arachidonic acid released From membrane phospholipids during the ptocess

of inflammati~n.'~~ Piostaglandin E2 is synthesized via the cyclooxygenase pathwoy by

conversion of arachidonic acid to PGH2 by the action of an enzyme called PGH synthase."

PGH? i s a common precursor for many prostanoids (PGD?, PGE2, PGFla. KI2. and TM2), i t is

rapidly converted into prostanoids. and its fate is cell specifi~.~'~'~ PGE synthesis can be induced

by LI. TNF-a. lipopolysaccharides, and components of the complement cascade." It has been

show in vitro that PGEs affect the secretion of IL-2, IL-4. and IFN-y by T cells, upregulate the

expression of IL4 recrptors on fibroblasts and peripheral blood monocytes. as well as enhance

the svnthesis of GM-CSF? the cytokine which promotes the pmduction of macrophages h m

bone marrow."'

It has been obxrved in in vilru e ~ ~ r i r n e n t s ' ~ that even though macrophages exposed to

Wear part ides produce low lcvels of PGE?. the osteoblasts. exposed to macrophage-conditioned

medium, secretr: large amounts of PGE?. Horowitz et aLJh have hypothesized that the

phagocytosis of PMMA particles by macrophages leads to the production of TNFa, which acts

on osteoblasts to produce other mediaton including PGEl. This prwess leads to the recruitment

of more macrophages, osteoclasts. and other inflammatory cells, which ultimatcly results in bone

resorption and, therefore, aseptic loosening o f the prosthesis. It is believed that Horowitz's

hymthesis'" also applies to polyethylene particulate."

1.3.5. In vivo and in vitro studia of W e a r partieulate.

In vivo models have k e n uxd to illustrate the forrip-body reaction to Wear particdate.

study synovial tissue formation, and detennine cytokine levels from retrieved tissues. There are

a few commonly used animal models. Howie et al.'' developed a rat mdel in which a non-

we igh t -hng sterile plug of acrylic cernent was placed in the distal part o f the femur, in

continuity with the knee joint, afier which particles of polyethylene were injected into the knee.

In this study Howie et al. was able to demonstrate resorption of bone and the formation of

connective tissue at the interface between acrylic cernent and bone. Th i s goup associated the

findings with a macrophage and giant-cell mponse to parcicles of polyethylcne that had been

injected into an adjacent joint. However, 200 pm particles were used in ihis study and it was

noted that the foreig-body response was higher than is usuall y ObSe~ed in hman samples.

A robbit mode1 has also been used to study aseptic loosening. Goodman ct al? put a

bulk pellet of UHMWPE directly into a 6 mm drill hole in the proximal tibia. This research

group demonstrated synovial membrane formation, bone resorption in the experimental animals,

and elevated lrvels o f PGE?. This was an interesting fmding since PGE, was known to play a

major role in bone nmodeling and therefore it was concluded that PGE2 could k an important

factor in the case of aseptic loosening and bone resoiption. However, since there was no direct

contact of the particles with joint synoviocytes. this mode1 cm not he directly related to the

process of aseptic loosening in humans.

A canine mode1 was developed by Spector et al.4? ln this mde l total hip replacements

were implanted into dogs and then bone cement paniculate was placed into the femoral canal

just before insertion of the stem. Spector et al.4? demonstrated synovial membrane formation

identical tu the membranes found in humans; biochemical analysis showed elevated levels of

PGE, in these dogs. Treatment of cells fiom the synovium-like tissue in v r t w with naproxen,

reduced the levels of PGE,. Spector and his group used 500 Pm particles and assumed thai the

particles would become smaller as a result of abrasion in the joint; histological evaluation

revealed the presence of particles of size 400 Pm, which is still considerably larger than the

sizc of particles recovered from retrieved human membmnes. ILI^,^ 1.22 All of the above animal

mudels consistently demonstrated the formation o f an inflammatory membrane at the bone-

implant interface, a certain degree of bone resorption and elevated levels of inflammatory

mediatoa in responx to Wear particulate.

While in vivo models can illustrate the body's reaction to paniculate and other foreign

objects, rn viiro expriments can provide the opportunity to study the response to different

stimuli at o cellular level. Then are cunently no in vitro "models" which have described the

process of phagwytosis and bone resorption in a similar manner as to the animal rnodels

summorized above. Most in vitro studies tu &te have used primary cells (e.g. monocytes, 1 1 .d?-tS

f~broblasts~"~) and ce1 1 l i nes (e.g. macrophages,'1 l0 fibroblasts, l0 o~teoblasts~*'~), and have

exposed them to different types o f particulate (titanium, 10.1 1.44.45 bone cernent," ' ' polystyrene, lu* l '

polyethylene4346) with various sizes (subrnicr~n~~*~ '*4'45~47 to 100 pm9). Some invcstigators9*

1 1.36.43-45.47 have studied the direct effect of the particulate on cell function, proliferation. and Y. 10.36 secretion of intlamrnatory mediaton. Others have used the conditioned medium from cells.

exposed to the particulate, to analyze its effect on cell proliferation, differentiation, and cytokine

secretion. Almost ail reported investigations demonstrate elevated levels of IL-1. IL-6, TNF-a.

PGE2, and other inflammatory cytokines. A common shortcoming of these studies is the absence

of histological confirmation of particulate phagocytosis. Histological evaluation of the cells

could provide information not only about internalimtion o f the particelate, but alw cniild show

the effect of phagocytosis on cellular morpholoby, activation, and viability.

1 .Mi. In vitro studies of polyetbyknc partirulate

Despite the fact that UHMWPE is one of the major constituents of Wear debris. detectrd

in intlammatory membranes retrieved at revision s~rgerirs,?J~~~* few studies have rxamined the

phagwytosis of polyethylene particles by macrophages rn vrîro.434t~.~n This may be due in pan to

trchnical difficulties associatrd with handling the polyethylene particdates. The material's

hydrophobic character render it particulad y non-compatible with aqueous media. As well, it has

a relatively lower density than water and resides at the surface of the aqueous medium rather

than within the medium or at the bonom of the tissue culture wells. This hinden the ability to

study cell-polyethylene interactions by methods established for PMMA and polystyrene

prt i~les.~- ' ' J~ Some models which have been employed for the investigation o f

polyethylendmacrophage interactions have included work by Haracia,13 Chiba,& S h a ~ ~ b h a g , ~ ~ ~ J

and hir rata.^ Handa et al." used agarose to keep the polyethylene particles (purchased from

Polysciences, 1-10 pm) attached to the sdace, but the level of macrophage activation in the

presence of agarose alone was sufticiently elevated to make it particularly difllcult to assess the

specific biochemical responses induced by the particles. Shanbhag et al."-'5 uscd serum to

suspend retrieved and fabricated submicron particles of polyethylene. Both goups noted

incteases in cytokine pmduction (IL- I P. IL-6, TNFa, PGE,), but there was little discrimination

in cytokine releose compared to control groups. Shirata et al.18 utilized an invcried culture

system in order to enable cells from a mouse macrophage ceIl line IC-21 to phagocytose the

fabricated polyethylene pniculate. This group demonstrated a statistically significant increase

in L l p reiease. Of al1 the studies encountered in the c m n t literature, only Chiba et al? have

show histological evidence (an SEM) of retrieved üHMWPE phagocytosis by human

monocytes. Unfortunately, the method of introduction of polyethylene into the cells was not

clearly described in the abstract.

1.4. THE ROLE OF EXTRACELLULAR MATI\IX PROTEINS IN CELLULAR RESPONSE TO

BIOMATERIALS

In response to the implantation of any type of prosthesis. the M y reacts with an initial

phase o f inflammation. The processes involved in this reaction are similar to those in the tissue

repir mechanism, exhibited as r normal response to tissue injury" However. since the implant

is also present a i the site of the tissue injury, it can influence the outcome of the repir

mechanism by aupenting the inflammatory response and, possibly causing a state of chronic

in flamrnati~n.~' I t has k e n established that several materiai parameten can in fl ucnce the speed

and the result of such tissue repair. These include but are not limited to surface morphological

characteristics (rough venus smooth), size, chernicd composition, and location of the implant.'"

As was described in Section 1.2.1 o f this thesis. the change in porosity of a prosthesis

surface, achieved by coating the surface with beads, can increase bone ingrowth and, therefore,

augment the healing process. However. changing the surface morpholoby o f the implant is not

the only m e W available for pmmoting tissue healing around implants. It is believed that

coat ing the surface of the implant with biologicall y relevant materials can enhance the fixation

of the implant, promote tissue ingrowth, anâ d u c e capsule formation."" Collagen type I and

hydroxyapatite are the most common matenals used for this purpose. 50-54 It has b e n

demonstrated in vitro, that a mixture of hydroxyapatite and collagen promotes the production of

a calcified collagenous maaix by hurnan osteob~asts.~~'~ The matrix has k e n reportai to be

similar in nature to bone tissue. It has also b e n show that a sponge layer of collagen,

mechanically bonded to silicone sheets and implanted in rats, exhibits a reduction in capsular

formation in cornparison to control silicone sheets without the collagen." ln another study, it

was demonstrated that the immobilization of collagen onto the surface of porous polyethylene

proved to be very effective in forming a finn bond with soft connective tissues and in reducing

the incidence of tumour fomiati~n.'~ All these studies demonstrate the importance of

extracellular matrix protein at the irnplantltissue interface.

I A l . Collagen type 1

Collagen is the most abundant protein in vertebrates. 3m.30,55,56 Due to its unique tensile

strength, collagen is a major protein of the connective tissue. 311.39.55.s6 At least 18 distinct iypes

of collagen have ken discovered; these types exist in a voriety of tissues forming fibers,

filaments. sheet-like structures, and anchoring fibrils.% The main types of collagen found in

connective tissue are types 1, 11, Ill, V, and XI, while the most cummon collagen is type 1, which

is found in ski% lung. bone, and many other tissues (Table 1 3). 3H.39.55.50 The types of collagen

found in the inflamrnatory membrane retrieved at revision surgeries of failed onhopaedic

implants are types 1 and 111 . '~

III

fibrils

fibrils

fibrils

fibrils

fibrils

most connective tissues, except cartilage and basement membranes (skin, bone. tendon, blood vessels, comea) carti l a~e , vitreous humour mostly colocelized with type I col lagen minor components in many

tissues -- - --

cartilage, vitreous humour

Table 1.5. Main types of collagen. Adapted from ~ r ~ ~ a s o n ' ~ and ~oet.' '

Collayen molecules fom triple-stranded helical structures, in which three polypeptide

chains are wrapped around each other.'"" Each chain is composed of a series of repeating

sequences with the general formula (Gly-X-Y),,. While glycine is the most abundont amino acid

in collagen, X is otkn proline and Y is often hydroxyproline. This combination creates very

strong hydrogen bonds between the chains thus making a well-pac ked and rigid structure,

characterized by unusual tensilc strength. Collagen fiben are insoluble at biologiccil pH dur to

the crosslinking of the chains via hydrwen bonds However, i t i s soluhle at acidic pH 'X~''' This

tnnsfonnation between a col lagen solution and an insoluble crossl inked collagn matrix has

been used for c r l l culture studies, since i t allows for the generation of a three dimensional matrix

for the cells in labontory condition^.^' As one o f the major components of the extracellular rnatri~,~' collagen is vital to cell

attachment. division and g ~ o w t h . ~ ~ In cell culture, substrates are commonly coated with

extracellular matrix proteins, such as collapns type 1 and II. îïbronectin, and laminin, depending

on cell type, to prornote anachment of the ce l~s .~ '

1.4.2. The effect of collagen type I on macrophages In vitm

Collagen type I i s the most comrnonly used protein in cell culture and is kmwn to

enhancc growth or differentiation of numerous marnmalian cell types."." It has k e n previously

describecl that exposure to extracellular rnavix proteins can enhance phagocytosis. For

example, Kaplan et al. demonstrated that monocytes cultured on collapn rather than ylass

phenotypically rewmble resident tissue macrophages, while Newman et al? found that the

phagocytic ability of monocytes adhered to type 1 collagen gels increased 2.5-1 2 times. It has

also ken shown that coating plastic substrates with collagen type 1 modifies the cytokine gene

expression of such inflammatory cytokines as T N F ~ , " L I P and 1 ~ 0 6 , ~ ~ thus demonstmting the

importance of extracellular matrix proteins.

It has been demonstrated that collagen is ptesent in the inflammatory membrane at h e

bone-implant interface." The presence of collagen could have numetous effects on macrophage

activation and hction according to these studies. While it remains dificult to extrapolate the

in v i i w experiments discussed a b o ~ e ~ ' ~ ' to the in vivo environment of the inflammatory

membrane, it would be desirable to consider implementation of collagen into in vilro studies in

order to render the system more analogous to the in vivo environment of the inflammatory

membrane, since it could prornote cell differentiation, phagocytosis of the particulate, and

cytokine expression.

1 .S. SCOPE OF THESlS

Aseptic loosening remains the most common reason for implant failure. Polyethylene

particulate has k e n implicatrd as the most abundant component of the Wear debris associated

with aseptic loosening. However, polyethylene remains the least studied particle due to ils

unique physical propenirs; it has a low density, i t i s hydrophobic. and it is transparent, therefore.

making it dificult to study. At this point, there is liale dota availablc& that describes the process

of intemalization of polyethylene paniculatr.

I .S. 1. Objective

The purpose of this study was (1 ) to develop a simple and versatile ln vitro mode1 that

would allow macrophages to phagocytose polyethylene particulate; and (2) to investigate the

effect of the phagocytosis on cellular activation.

1 .Sel . Experimental a pproaeh and rationak

In order to introduce polyethylene into macrophages, this study descnbes the methoâ by

which the panicles are coated with collagen and then adhered to glass coverslips. In these

studies the macrophages are plated on top of the particle/collagen matrix and incubated for

difirent periods of time, ranging from 2 to 96 hours. The use of a coverslip system is

intentional, and permits one to perfom any type of histological analysis (conventional staining,

scanning electron microscopy (SEM), transmission electron microscopy (TEM)) without the

time consuming necessity to cut tissue sections. Post incubation, the cells are analyzed

histologically to confimi the internalization of the polyethylene particles. The media are tested

for the presence of major inflammatory mediaton (IL-l , IL-6, TNF-a, PGE:), as well as

nonspecific lysosomal enzymes (P-GAL and HEX). The selection of the above mediaton was

made in order to confirm the activation of macrophages by polyethylene particulate

phagocytosis.

Macrophages are of parîicular interest to ihis study because ihey are the prirnary tells

associated with phagocytosis of wcar debns in aseptic loosening of the onhopaedic implants.

These cells are believed to play art important role in internalization of the wvear debris and

release of inflammatory mediators that are capable of not only augmenting the inflammatory

response, but also of breaking the blance in bone remodeling, ththerefore, causing bone

resorption, more loosening of the implant. and production of more Wear debris. initiating the

vicious circle leading to implant failwe.

The use of collagen in the mode1 has been rationalized by the following. Type 1 collagn

is an extracellular matrix protcin that is present in inflammatory membranes retrieved from the

bondimplant interface of failed prostheses. Aside from the fact that collagen i s the most

abundant protein in the body. it has some unique proprties that are vital to cell function, both rn

vivo and in vitro. Collagen provides an extracellular matrix for the cells. promotes cell

differentiation. augments phogocytosis by macrophages, and modulates cytokine expression. At

the same time, collagen has the ability to crosslink and fonn an insoluble mntrix that can be used

for anachment of the particles to the bonom of tissue culture well.

In smmary, the development and characterization of a model that allows polyethylene

phagocytosis by macrophages can help towards pining a h e r understanding of the

mechanisms involved in aseptic lousening. It could have an impact on the development of

alternatives to the revision surgery of a loose implant, such as blocking of the inflammatory

cytokines; or stimulate a production of new types of prostheses, either pnmcle-lree or made from

a type of matenal that does not cause an inflammatory response by the macrophages.

2. MATERIALS AND METHODS

2.1. PARTICLES.

Two types of polyethylene partictes (1 8-20 pm ultra high molecular weight polyethylene

(UHMWPE) and 4-10 Hm high density polyethylene (HDPE) particles, obtained in-kind from

Shamrock Technologies, Newark, NJ) were used in the expenments. It was originally desired to

have submicron panides of I JHMWPF: since this would most appropriatrly reflect the type of

particle generated from oithopaedic implant devices. Howevrr, no commercial source of

üHMWPE in submicron size was found. As a substitute, 4-10 prn HDPE was used. While the

chemistry of the polymer chain does not diffet from that of UHMWPE, the two materials differ

in their molecular weight. Physical properties for both types of pl yethylene were provided by

Shamrock in Technical Data Sheets and are summarized in Table 2.1. The particles were

sterilized by y-irradiation in air (2.5 MR) (Chernical Engineering Department, University of

Toronto) and stored at 4°C.

2.2. PARTICLE SURFACE CHEMICAL CHAMCTERlZATlON BY X-RAY PHOTOELECMION

SPECTUOSCOPY (XPS).

X-ray photoelectron spectroscopy is a technique commonly useû for surface analysis of

polymers in the biomaterials field, providing a total elemental analysis of the top 10-200 A of the

surface." M>S spectra of the polyethylene perticles wete obtained by Dr. Rana Sodhi using a

Leybold MAX 200 XPS system, located at the Centre foi Biomaterials, University of Toronto.

Properties r

Size (microns)

Melt puint, %

Spceific grrvity

Table 2.1. Physical properties of UHMWPE and HDPE

UHMWPE

1 8-20

166

0.9

HDPE J

4-10

II8

0.95 h

An unmonochromatized MgK, x-ray source operating at 12 kV and 25 mA was used. The take-

off angle for the analysis was 90".

The energy range was calibrated aainst Cu 2p3/2 and Ag 3d92 at 932.7 eV and 368.3

eV, respectively, and scaled to place the main C peak at 285.0 e ~ . " As no observable

differential charging of the surface was produced with this source. extemal charge compensation

was not employed. Features in the resultant spectra due to excitation from the x-ray satellite

compnents were subtracied using an algorithm suppliecf with the instrument and based upon a

program by van Attekurn and ~ r o o s t e r . ~ Atomic ratios of C and O were denved from spectra

run in a low enerw mode ( p s enerw192 eV) which had k e n nonnalized to correct for the

transmission function of the spectrometer. Both the transmission function and the sensi tiviry

factors for oxygen and corbon ( 04 .78 , C=0.34) were empirically derived by the manufacturer

of the XPS system. Funher information on the nature of the substrate surfaces were obtained

from the C I s region run in a high resolution mode (pass energy=48 eV). Binding energies and

Peak areas were obtained using the curve- fin h g routines provided wi th the spectrometer.

Non-sterile UHMWPE and HDPE particles and stede (y-irrndiated) UHMWPE and

HDPE particles were analyzed (n=3), and differences in particle surface chemistry afler y-

irradiation were assessed. In order to analyze the powden, the samples were attached to the

sample holder by using double sided copper tape.

2,3. PARTICLE BULK CHEMICAL CHARACWRIZATION BY FT-tk

Infrared spectroscopy is a simple and rapid method for identifjing a stmcture of an

organic c ~ r n ~ o u n d . ~ ~ Non-sterile and sterile UHMWPE and HDPE particles were analyzed

(n=3) by infwed spectroscopy with nitrogen purge (iFS85, Bruker, Bruker Spectrospin (Canada)

Ltd., Milton, ON). For the analysis, 1% (by weight) of polyethylene particles in dehydrated KBr

pellets were prepared by pressing (0.2 x IO-' ~lm') and measuring the absorbante spectra ( 100

scans/specmun). The diflercnces in bulk material chemistry between both types of the parcicles.

and the effect of y-irradiation on bulk propxties w m then assessed.

2.4, ENDOTOXIN TEST.

HDPE particles were tested for the presence o f endotoxin (lipopolysaccarides h m the

walls of gram-negaiive bacteria) using a stanhrd E-TOXATE detection kit (Sigma. St. Louis,

MO). This test i s based on the ability of Limuluv amebocyte lysate (LAL) to change viscosity

and opacity when exposed to minute quantities of endotoxin?

In order to obtain a sample, the polyethylene particles were mixed with sterilr ddii?O ovemight

on a shaker, and the watcr was later ccrllected for analysis. The pH was tested and was

approximately 7.0. Samples, dong with positive and negative controls provided with the kit,

were exposed to E-TOXATE working solution, mixed gently, and incubated for 1 hour at 37°C.

ARer incubation, the tubes were examined for evidence o f gelation.

UHMWPE particle were not analyzed for the presence of lipopolysaccharides since they

were found to be too large to be phagocytosed by the cells md were not wd in characterization

of the collogen model.

2.5. PARTICLE SUSPENSION PREPAMTION FOR THE TRITON X-(00 MODEL,

Approximately 1 gram of UHMWPE particles was mined thoroughly with 0.5 mL of

ZO/o(vol.) Triton x-100 (Sigma, St. Louis, MO) in PBS (Mount Sinai Hospital, Media Prepamtion

Department) and the panicle mixture w3s then resuspended in PBS. The number of the particles

was counted using a Coultcr Counter (mode1 ZM, Coulter Electronics, Hialeah, FL) and the final

concentration of the Triton x-100 was then detemined. based on the amount of the panicle

suspension required to provide 10' particles. The concentration of Triton x-100 used in the

following experiments (see Section 2.6) was 0.0083%(vol.). Triton X- 100 was selected for these

experiments since this surfactant is routinely used in cell cul t~re.~'

2.6. EXFERIMENIS USlNC MOUSE PERrrONEAL MACROMCES.

Mouse peritoneal macrophages were obtained From 6- to 8-week-old specific pathogen-

free female Swiss Webster mice as dexribed by Nathens et al? in the facility located at the

Toronto Hospital, General Division, Toronto. Briefly, mice were injected with 2 mL o f

thioglycollate broth (Difco Laboratories, Detroit, MI) into the peritoneal cavity and peritoneal

exudate macrophages were collected by peritoneal lavage with iceîold Hanks' balnnceâ salt

solution (Gibco BRL, Burlington, ON), five days afler the injection. The cells were centrifuged

a i 200g ( 1500 rpm) for 10 minutes, washed twice with HBSS, resuspended in fully supplemented

media (RPMI-1640 (Sigma, St. Louis, MO) supplemented with 10% FCS (Gibco BRL.

Burlington, ON)), and then counted using a hemocytometer. The cells were diluted to a final

concentration of 1 x 10" cel!s/rnl in fully supplemented media and plated in 24-wel! plates

(Falcon, Fisher Scientific, Whitby, ON) ai I mUwell. At this point an oliquot of Triton x- 100-

prticle solution and Triton x- 100-control was added and the cells were incubated for 24 and 48

houn. The media was then collected and analyzed for TNF-a, P-GAL, and HEX release (see

sections 2.1 5.3 and 215.4 for details on the biochemical analysis methods).

2.7, PARTICLE SUSPENSION PREPARATION FOR COLLACEN MODEL.

Approximately 1 gram o f particles was mixed thoroughly with 0.5 mL o f dimcthyl

sulfoxide (DMSO, BDH Laboratones) using a sterile quartz pestle and mortar. The prticle

mimure was then resuspended in 14.5 mL of 0.0 1 % solution o f collagen type 1 monomer, denved

from calf skin (C-8919, Sigma, St. Louis, MO) at 4°C. The number of suspcnded particles was

then counted using a Coulter Counter (mode1 ZM, Coulter Electronics, Hialeah. FL) and the final 7

concentration o f particles adjusted to 10 panicles/mL.

2.8, COVERSLIP PREPARATION.

Microscope covenlips 22x22 mm (Fisher Scientific, Whitby, ON) were sterilized by dry

heat ai 2OOoC for 2 hours in the ovcn. Aliquots of cold collagen solution with suspended

particles ( 1 0 ~ particlesll0 pi.,,) werc spread evcnly on each coverslip and thc collagen was then

allowed to polymerize at room temperature and the solution was then evaporated. A typical

distribution of particles in the collagen matrix is shown in Figure 3.3a Uncoated coverslips.

coverslips coated with DMSO; collagen type 1; and a mixture of DMSO with collagen type 1,

were olso prepared and useô as the controls for these experiments. Al1 samples were then pl&

in 6-well tissue culture plates (Falcon, Fisher Scientific, Whitby, ON) and exposed to W light in

a sterile tissue culture hood for overnight and for I hour immediately before use. Following the

initial ovemight UV sterilization, the plates were wrapped in clean non-sterile polyethylene bags,

covered with aluminurn foil, and stored at 5% until required.

2.9. EXPERIMENTS USINC THE MOUSE MACROPHAGE CELL LINE.

A mouse macrophage cell line was used to characterize the collagen model becaw of

their well characterized biological function as well ûs their commercial avai labil ity. The mouse

macrophages are f'ully differentiated cells. capable of secreting various cytokines and lysosomal

enzymes, and do not require special isolation and purification procedures that ore necessery with

primary cultures.

IC-21 mouse macrophage ceIl line obtained fiom ATCC (TlB-186, Rockville, MD) was

maintained in tissue culture flasks (Falcon, Fisher Scicntific. Whitby, ON) in RPMI-1640

medium (Sigma, St. Louis, MO) supplemented with 10V0 heat inactivated FCS (Gibco BRL,

Burlington, ON), 196 penicillin-strrptomycin (Gibco BRL, Burlington, ON), and ? mM L-

glutamine (Gibco BRL, Burlingon, ON) at 37°C in 5% CO2 atmosphere with 100% humidity.

lmmcxliately pnor to the experiment, cells were collected by incubation with ca2+/Mg?* free PBS

(Mount Sinai Hospital, Media Prepration Department), centrifuged for 10 minutes at 170 g

( 1 100 rpm), resuspended in fully supplemented medium as described above. plated on the

pnviously prepered covenlips at 1 x l$ cclld2 rnUwell and then incubated for up to 96 hours.

At the end of each incubation perd, the cultures were washed twice with PBS (1 mUwell) and

then fixed for histology (LM, SEM, or TEM) according to the protocols dcscribed further in this

chapter (sections 2.1 1, 2.1 3, and 2.14). The media were collected for cytokine and lysosomal

enzyme analysis (methods descnbed in section 2.15). The ce11 viability was checkeâ by a

standard trypan blue excl usion method (see section 2.1 O)?

2 . 0 VIABILITY TEST,

Cell viability was detemiincd by trypan blue exclusion test? The cells were plated on

glass covenlips, glass covenlips coated wi th col legen-DMSO mixture, and glass covenl ips

coated with particle mixture and incubated for 24 hours. At the end of the incubation pend the

media was removed and the coverslips were washed twice with PBS (ImUwell). Then 200 pL

of 20°h (vol.) trypan blue in PBS was added per well and the coverslips analyzed under the

microscope. The number of the cells that uptake the dye was then detennined.

2.1 1. HEMATOXY LIN AND EOSIN STAPNINC.

The cells were fixed using a mixture consisting of 100°'o methanol and 10°,h

fomaldehyde (2: I per vol m e ) for 2 hours (2 mUwell ), then stained wi th hematoxyl in and eosin

using the standard staining procedure'' at Mount Sinai Hospital. Pathology Department. Bne fly ,

the fixed covenlips were n n ~ d with water, stained with hernataxylin for 5 minutes. rinsed with

woter, counterstained with eosin for 2 minutes, and rinsed with water again. The coverslips were

then dehydrated by graded alcohols (70% to 1000/0) and then by xylene ( 100%). The covenlips

were then mounted on microscope-grade glass, dried. and photographed both under regular and

polarized light, using a VANOX AHBT3 Olympus (Carsen Group Inc., Markham, ON)

microscope.

2.1 2, CELL/AREA COl.INT,

Hematoxylin and eosin stained slides were analyzed for cell number and area percent

count using a monochrome image analyzer equipped with QSOOMC software by Leica

Cambridge Ltd. located at Mount Sinai Hospital, Pathology Department, Toronto. The

assessrnent was performed on an 'Dnhoplan" Leitz microscope using the 4 x objective (N. A. O. 1 )

coupleâ with JVC TK- 1 XOU colour video camera. Twenty fields (560 x 5 12 pixels, 1 pixel =

3.22 Pm, as detennined by the microscope calibration built into the program before each use)

per slide were analyzed. Gray levels were detected and cell number, area of the gray and area

percent were calculated. In this software, the cell count was defined as the number of isolated

features cnding within the measured frame, and the area percent was defined as the proportion of

detected pixels in the image, expressed as a percentage from O to 100.

2.13. SEiM.

Scanning electron microscopy is a histological technique that allows one to investigate

the overall morphological features of a specimen.'" The cells incubated for 2. 6. and 24 hours

(Expriment 2 in Table I of the Appendix) were fixed with 2 5 O h glutanldehyde in phosphate

buffer, dehydrated in graded alcohols, critical-point dried from CO2 (Ladd Rrsearch Industries

Inc., Burlington, VT), sputter-coated with plaiinum (-3 nm) (Polaron lnstnunent lnc..

Doylestown, PA) and cxamined by Mr. Robert Chcmecky using an Hitachi (mode1 2500)

scanning electron microscope, located in the Faculty of kntistry, University of Toronto.

2.14. TEM.

Transmission electron rnicroscopy is a histological technique that allows for the

investigation of the intemal structure of the specimen." Following incubation for 20 houn. cells

were scraped off the coverslips into 1 mL of PBS, pelleted in a microcenirifbge at 5309 (2500

rpm), and then tixcd with 2.0°/0 glutaraldehyde in O. 1 M cacodylate buffer. postfixed witb 1%

osmium tetroxide in the same buffer, dehydrated in ethanol, and embedded in Epon, and

sectionrd. The analysis was performed by Ms. Julia Hwang at the Histology Department.

Hospital for Sick Children.

2 . 1 MEDIA ANALYSIS,

AAer each incubation p e n d the media were collected and stored at -80W in 200 pL

aliquots. Mouse interleukin- 1 a (IL- l a), interleukin- 1 f3 (IL4 P) and interleukin-6 (IL-6) ELISA

kits (Endogen, Cambridge, MA) were used to analyze for cytokine release at 24 hours. Tumow (rll

necrosis factor a (TNF-a) release was also analyzed by ELISA as described by Nathens et al.

Prostaglandin E2 (WEI) release was detennined by radioimmuno assay using PGE2 RIA kit

(Dupont, Boston, MA). P-galaciosidase ( P-G AL) and hexoseminiâase (HEX) activity were

7 1 measured as described by Hubbes et al. All cytokine and lysosomal enzyme data were

normalized by a m Yi counts.

2.15.1. I L I a , IL lP assry

The samples and kit supplied standards (50 pL) were incubated at room temperature with

biotinylated antibody reapnt (50 PL) in anti-mouse IL- 1 d L - 1 precoated well plate for ?

hoan Recombinant (:: trdrderived mouse IL- 1 d l L - I P \vas used as a st3n&rd. Al! sarnplcs and

standards were analyzed in duplicates. AAer the incubation, the wells were washed three times

with wash buffer and then 100 pL of streptavidin-HRP (horse radish peroxidase) was added to

cach well and the plate was incubated for another 30 minutes at room temperature. Then the

plate was washed thrce times with wash buffer and 100 @ of TMB substrate solution was added

to each well. The plate was then incubated in the dark at room temperature for 30 minutes. The

reaction was stopped with 100 pL of stop solution (0.18 M sulfuric acid) and the absorbance of

the plate was determined on an ELISA plate reader set at 450 nm. The results were calculated

using Microsofl Excel soflware.

2.15.2. IL6 assay

The samples and kit supplied standards (JO pi.) were incubated at 37"C in a humidified

incubator with plate reagent (50 PL) in anti-mouse IL-6 precoated well plate for 2 houn.

Recombinant I~colidenved mouse I L 4 was used as a standard. All semples and standnrds were

analyzed in duplicates. Afler the incubation. the wells were washed five times with wash buffer

and then 100 pL of anti-mouse I L 4 conjugate repgent was added to each well and the plate was

incubated for another hour at 3i"C in a humidified incubator. Then the plate was washed five

times with wash buffer and 100 pL of TMB substrate solution was added to each well. The plate

was then incubated in the dark at room temperature for 30 minutes. The reaction was stopped

with 100 pL of stop solution (0.18 M sulfunc acid) and the absorbance of the plate was

determined on an ELISA plate reader set at 450 nm. The results were calculated using Microsofl

Excel software.

2.153. TNF-a assay

The cytokine was measured with a double sandwich enzyme-linked immunosorbent assay

technique. The plates wrre coated with monoclonal hamster anti-murine TNF-a antibody

(Genzyme, Cambridge, MA) (100 Wwell, ovemight, at 4°C). The samples/standards (100 pL)

were added to coated wells, incubated for 3 houn at room temperature on a rotating shaker,

washeâ, and ihen incubated overnight at 4°C with a polyclonal rabbit anti-murine ThWu

antihodv (Genzvme: Camhrido' M A ) (100 ul.). Then 100 u1. of alkaline phosphrtaw-

conjugated goat anti-rabbit IyG was added to each well and the plates were incubated for 1 hour

on o rotating shaker ai room temperature. The fluorogenic substrate 5-fluoro-sa1 icyl phosphate

( 100 PL) and developing reagent (terbium-ethylenediamine tetraacetic acid) ( 100 PL) were then

added. Fluorescence was measured with a time-resolved fluorimeter located in the Department

of Biochernistry, Mount Sinai Hospital, Toronto. The concentration of T N F a in the samples

was cûlculated by an automatic immunoanalyzer (Cyber Fluor 6 15; Cyber Fluor, Toronto, ON).

Recombinant Gcoliderived murine TNF-a (Genzyme, Cambridge, MA) was usrd as a standard.

Al l samples and standards were anaiyzed in duplicates.

2.15.4. P-CAL rad HEX auciys

PGAL activity was measured by using 4-methylumbelliferyl-~alactopyranoside

(4MU-gal) (Koch-Ligbt laboraiories). Briefly, 60 pL of sample/standard were rnixed with 190

p.L o f substrate solution (0.56 mM 4MU-gal in O. 1M citrate buffer, pH 4.3) and incubated at

37°C for 30 minutes. All samples and standards were analyzed in duplicates. The reaction was

tenninated with 2 mL of O. 1 M 2-arnino-2-methyl- l -propanol buffer, pH 10 (MAP) and the

tluorerence was determined at an excitation wavelength of 365 nm and an emission wavelength

of 450 nm on the spectrofluonmeter (Perkin-Elmer 650-40 Fluorescence Spectrophotometer). 4-

Methylwnbelliferone solution was used as a standard.

Hexosaminidase activity was detcrmined in a si milar mmner using 4-methy l umbeil i fery 1-

~-N-afetyl-glucosominide4-sulfa~ (CMUGS) as a substtate. The analysis has k e n çarried out

in Dr. J. W. Callahan's laboratory at the Hospital for Sick Children.

2.1S.S. PGE2 assay

The principle of this radioimmunoassay is based on cornpetitive binding of radioactive

and non-radioactive antigen to a f i x d number of antibody binding sites, i.e. as the arnount of

unlabeled antigen is increascd in sampledstandards, the amount of the tracer bound to the

antibody is decreased. Briefly, samples/standards (100 PL) were incubated with 100 pL of tracer

(PGE~["~I]) and 100 pL of antiserurn (rabbit anti-PGE2 antibody) ovemight at 4'C. 100ng/mL

solution of PGE: in acetonitrile was used as a standard. At the end of the incubation period, 1

mL of cold precipitating agent (MOh polyethylene glycol PEG6000 and O.OSO/o sodium azide in

50 m M phosphate buffer. pH 6.8) was added to the tubes, the tubes were vortexed thoroughly,

and then incubated for 30 minutes at 4°C. The tubes were then centrifuged in a microcentrifuge

at 13800 g ( 13000 rpm ) for 30 minutes, the supematants were decanted, and the tubes were thcn

counted in a gamma cowiter for 1 minute. The results were crlculated as described in the

protocol supplied with the kit using Microsofl Excel software. All samples/standards were

analyzed in duplicates.

2.16. STATISTICAL ANALYSIS

The means, standard mon. and standard deviations were cakulated utilizing the

statistical program built into Sigrnaplot 1 .O2 scienti fic gaph system (Jandel Scientific) and

GraphPad InStat sothvare package. The cornpanson between the groups to determine the

significance of the differences was perfomed using student pired T-test utilizing the GraphPad

InStat software package.

3. RESULTS

In this section a summary of the results is presented. The data were organized in a

logical sequence corresponding to the development of this research project starting with

characterization of the particles, and followed by development of an in vitru mode1 for

polyethylene particle phagocytosis and detailed characterization of the model.

XPS analysis (sec Table 3.1) of the y-irradiated particles and non-y-irradiated particles

showed that thrre was no difference in oxygen content on the particle surface following

sierilization for UHMWPE, however, there was a statistically significant increase in oxypn

content ( ~ 0 . 0 5 ) for HDPE, suggcsting oxidation of HDPE due to y-irradiation. The levcls of

silicon were low, thus demonstrating that the samplrs were not contaminated with silicon.

Typical XPS spectra for UHMWPE and HDPE are presentrd in Figure 3.1 a,b.

TYPE OF

POLY ETHYLENE

UHMWPE

ClMMWPE, y-irradiatrd

HDPE

FT-IR analysis demonstrated that there was no difference in bulk chemistry due to y-

irradiation for both types of panicles, however, there was a distinction between UHMWPE and

HDPE specûa (Figures 3.2~-d). This may result fiom differences in the degree of crystallinity

which has been reporteci for the two types of p~lyethylenes.~?

ATOMIC %

O 1s

3.60 * 1 .O6

HDPE, y-irraâiatrxl

3.73 * 0.91

0.83*0.12

ATOMIC '!%

C 1s

95.97 * 1.29

Table 3.1. XPS analysis of UHMWPE and HDPE

1.53 * 0.40

ATOMIC Y.

Si 2p

0.40 * O. 17

95.93 * 0.95

99.03*0.15

0.30 * 0.00

0.13*0.06

98.33 * 0.46 O. 13 i 0.06

HDPE

H DPE-irrad iated

Binding Energy (eV)

Fipre 3.1. XPS spectra of polyethylene particles

Peak

2949 cm-'

Peak strenfib

,7845 cm''

very stron~

1474 cm''

[731cm*' 1 medium 1 740-720 cm-' 1 -(CH2)n- hydrocarbons 1

Range

strong

1464 cm-'

l'able 3.2. Summary of the absorbance peaks in tfl'-LK spectra (Lambert et al.'',

Croup I

2990-2850 cm"

strong

As can be wen fiom the spectra (Figure 3.2) and from the peak assibnment (Table 3.2) al1

-CH3 and CH2- in aliphatic compunds

2850-2650 cm-'

strong

major peaks correspond to the hydrocarbon structure which is typical for polyethylene. At the

-CH3 attached to O or N

1475-1 450 cm-'

1475- 1450 cm-' [ CH2- in aliphatic compowids 1

same time, the presencr o f absorbance peaks at approxirnately 3330 and 1640 cm" suggests the

-CHL- i n aliphatic compounds

presence of trace contaminants in the samples of HDPE (Figure 3.2 a,b). These may have been

introduced during polymer synthesis. Similar peaks were not found in the UHMWPE samples

(Figure 3.7 c.d). Despitr the fact thot the XPS data indicated the presencc of oxygen, the 1720

cm" absorbance peak that represents carbonyl (C=O) groups in ketones and is commonly

associated with polyethylene oxidation7bas not observed in any o f the samples. This suggests

the absence of oxidation in the bulk propenies of both plyethylenes.

The iwo particle characterization mrthods have demonstrated that while the bulk

chemistry of both polyethylenes was not seemingly affected by y-irradiation, the sudace o f

HDPE poriicles was altered as a result o f the same treatment.

SEM was performed on HDPE particles in order to investigate the morphology o f the

phagocytosable particles and the distribution o f the particles within the collagen matrix on the

coverslips (Figure 3.3a-b). Figure 3.3a represents a typical pi t ic le distribution on the covenlip,

while Figure 3.3b demonstrates the surface morphology of the particles.

Endotoxin test. performed on HDPE particles, demonstrated that the particles were free

of endotoxin.

Figure 3.2. FT-IR cpe*n of polyethyiem putides

(a) HDPE puticles, not y-imdirtd; (b) HDPE puCic1e, y-Vndiated

Figure 3.2. F T 4 cpcctn of poiyetiiyicne pariic1es

(c) üHMWPE puticlu, not ylirndirtd, (d) üHMWPE pvticlcs, y-imdirted

Figure 3.3 Scanning electron micrograph of HDPE putide distribution in collagen matnx on

a coverslip. (a) x 100 original mgnification; @) x 5000 onginil mafification

3.2 MODEL DEVELOPMENT

The purpose of this study was to develop a mode1 that would allow the investigation of

interactions between polyethylene prticles and macrophages. The reason for the absence of

such a mode1 in the current scientific literature could be explained by the inherent physical

propenies o f polyethy kne particutatr. These particles are hydrophobic (Le. incompatible with

an aqueous environment) and have a low density (sre Table 3.1 \ and hence thev float on the

surface of aqueous solutions which have a higher density. Therefore, one o f the major

dificulties encountered in this work was to prepare a particle suspension that would enable the

interaction o f individunl particles with the cells in aqueous media and to achieve this with

processing aids that would be non-toxic to the cells.

Several reagents (such as ethanol, cangeenan, senim) were considered to be used for

preparation of the particle suspension in the process of the mode1 development. Triton x-100

was one of the surfactants which was extensively studied. A limitation in the use of this

surfactant was the fact that at hi@ concentrations of the reagent the cells would disintepte. It

was found that the cutsff concentration of triton x-100 which would be tolerated by the cells

was approximately 0.018% (vol.). However, even afier achieving a suspension of the particles in

triton-x at such a low concentration, the panicles did not remain evenly dispersed in the media

for extended petiods of time and would eventually rise to the surface in a matter of a few

minutes. This was undesirable since the cells were attached to the bottom of the tissue culture

plate. Another problem observed with utilizing triton-x was the fact that even at concentrations

of 0.0083%(vol.). the surfactant rendered cellular membranes more permeable and caused

leaching of the cellular contents, such as cytokines and lysosomal enzymes, into the sumunding

environment (see control amples in Figure 3.4). As can be observed in Figure 3.4. the levels of

TNFu, P-GAL, and HEX release are sipificantly higher in the groups with triton-x than in

groups with panicles and triton-x. As a result o f these elevated background values it would be

impossible to study the effect of puticles on the release of lysosmal enzymes and cytokines

from macrophages. Furcherniore, the use of triton x- 100 as a surfactant would rquire

- 24 hours 48 hours

- 24 hours 48 hours

CTRL (triton-x only)

UHMWPE and triton-x

- 24 hours 48 hours

Figure 3.4. Media analysis of mouse peritoneal macrophages exposed to UHMWPE particles, suspended in Triton x-100, for 24 and 48 hours. (a) PGAL release; (b) HEX release; (c) TNF-a release

employment of a non-adherent cell line since the particles would reside in suspension and not

remain at the bottom of the tissue culture plate. Such a system would not be relevant to the in

vivo environment of the inflammatory membrane. In addition, a system with a shaker would be

required to keep the cells and particles agitated and this would not make the m d e l practical or

easy to use.

An altemate approach was taken in aitempt to embed the particles in a matrix which

could simulatr the biological environment R~sed on cnmmvnicat!ons with Dr. .4lex Hinek

(Research tnstitute, Hospital for Sick Children), the use of collagen was suggested. It is a

biological agent which is routinely used in cell culture to prornote ceIl adhesion to different

swfaccs."' At the same time the inhercnt feature of collagen to crosslink" could be utilized to

trap the polyethylenr: particles in a stable matrix. Because collagen is not a surfactant and does

not produce a single particle suspension by itself, DMSO was required in order to wet the

particles befure they were resuspnded in collagen solution. Following subsequent coating on

glass covenlips, the particles were trapped in the crosslinked collagen. In this f o n they

remained attached to the surface for a prolonged per id of time (the longest time period tested

wos 96 hours in culture). As a result of the latter analysis the collagen mode1 was then selected

for further study. The model's characterization is presrntcd in the followiny sections.

3.3.1 H btology

Histological characterization of the cells with hematoxylin and eosin staining permits a

fast but valuable evaluation of the general condition of the cclls. allows for distingwishing dead

and live cells, and provides a prelirninery evaluation of their association with the polyethylene

particles. While SEM analysis provides a three dimensional image of the cell surface, TEM

illustrates an accurate image of the intracellular events and provides evidence for the process of

particle i ntemalization by t hc ce1 1s. Histological evaluation of polyethy lene particles by light

microscopy also pennits easy identification of the pomcles. Since polyethyler is transparent, it

is not easily distinyished in cell culture. however, it becomes readily visible under polarized

light.

Histological analysis of the cells exposed for 20 hours to the larger UHMWPE particles

(18-20 pm) revealed that al1 large prticles were suvounded but not phagocytosed by the

macrophages (Figures 3.54b). In the experiment the cells were evenly distributed at the outset.

but by 20 hours most of the macrophages were associated with panicles. leaving large cell free

areas suggesting that the cells were attmcted to the particles.

Since it was desired to develop the model to specifically look at polyethylene panicle

phagocytosis, further work on the mode1 characterization was perfonned only with the smaller

HDPE (4-10 pm) particles. It was found that afler 24 houn in the wells containing HDPE d l

cul tured crl ls had phagocytosed the particles with most cells having engul fed several particles

(Figures 3.6a.b). When the mouse macrophages wcre exposed to smaller particles. phagocytosis

was progressive over time and engulfment of particles was observcd after 2 h o m (see Figure

3.7).

Upon SEM analysis at 2 hours, even though several prticles had been phagocytosed, the

cells appeared to be very motile (Figures 3.7a.c). and cell pseudopodia could be seen in contact

with particles (Figure 3.7~). Beyond 6 hours and up to 24 hours in culture no unphagocytosed

panicles were found. At these later time points the cells were less motile. with numerous

pseudopdia attached to the substratum (Figure 3.7e,f). The cells did not stop dividing ai any

tirne point indicating ihat the particles did not have an apparent toxic rffect on the cells. Upon

division the phagocytosed particles appeared to be cqually distributed between the two âaughter

cells (Figure 3.7d). The cells in the control cultures (glass. DMSO, and DMSO with collagen)

were similar to those shown in Figures 3.7b,f for ail three categories.

TEM analysis (Figure 3.8a,b) was perfomed at 20 houn of incubation and confirmed the

internalization of the PE particles. An examination of Figure 3.80 showed eight particles located

within a cross-section of a single cell. The pnicles were sunounded by double layer

membranes and numerous vacuoles were distributeâ dong the interface (Figure 3.8b).

Figure 3.5. HdtE stoining of the IC-2 1 cells exposed to UHMWPE particles for 20 houn.

(a) nomai light; @) polarized light; original magnification x 2000

The transparent particles becorne nsibk undcr polarized light.

Figure 3 -6. W E staining of the IC-2 1 ctlls acpored to HDPE particles for 24 hours.

(a) nonnai light; (b) poluiztd üght and Nomurki optics;

original magnification x 1000

The transparent particles becorne visible under polrrUed light.

Figure 3.7. Scanning elcctron micrograph of IC-21 cells exposed to HDPE particles for up to

24 hours. (a) cells with puticles, 2 hours of incubation; (b) control, no particles,

2 hours of incubation.

Figure 3.7. Scanning eltftron micrograph of IC-2 1 ctlls exposed to HDPE putides for up to

24 hours. (c), (d) cells with particles, 2 hours of incubation.

Figure 3.7. Scuuiing electron micrograph of IC-2 1 cells exposed to HDPE particla for up to

24 houn. (e) cells with particles, 24 houn of incubation; ( f ) control, no particles,

24 hours of incubation.

FI pure 3.8. Transmission electron micrognph o f IC-2 1 cells cxposrd to HDPE particies for

20 hours; (a) onginal magni fication x I 1 920; (b) original mapiticrtion x 48600.

* HDPE poniclr: > lysosome.

3.3.2. Cell counts

The analysis of the histological slides for the cells exposed to 4-10 pm particles was

carried out using an image analyzer and demonstrated differences in celllarea counts between the

control and particle containing groups (see Table 2 in Appendix for the cornplete listing of cell

count data). A representative samplc of the cell count data is show in Figure 3.9a-b. The data

shows that after 24 hours of incubation the cell counts for collagen with DMSO groups had

statistically higher values over p u p s where the cells were expnsed to particles. This

observation of cell number differences was repeated in four out of six experiments. whereas the

area merisurement data were significant in threr: out of six experiments (see Table 2 in the

Appendix). At 48 houn uf incubation, cell counts as well as area counts were significantly

higher in collagen with DMSO g~oups than in panicle goups in three out of four experiments,

thus suggesting inhibition of cell proli feration. In one experiment, where the ce1 ls were exposed

to the particles for longer pends of timr (73 and 96 houn) the differences were more

signi ficant ( ~ 0 . 0 0 1 ). Therefore, these data demonstrate chat exposure to particles appears to

have aflected cell ulrr proli feration and this effect was progressive with time (see Figure 3.9).

The cells exposed to the particles for shorter periods of time (2 and 6 hours of incubation) did

noi demonstrate significant differences between the groups for cell number as well as for orea 96

(Figure 3.10a,b), probably, due to shortness of the incubation periods. However, the cell

incubation experiments for the 2 and 6 houn exposure periods was only camed out once.

Since the image analysis work was a computerized process, it was believed that area %

counts provided a more sensitive measure of the cell number than did the cell counts. Because

of the nature of the experiments, the cells wodd group amund the particla making it impossible

for the computer or operator to distinpish how many cells were in the clump. Even though the

computer was set up io pick up the hek staining of the nucleus, it could rnistakenly count a few

cells located in one clump as one single cell, while area counts would remain the same

indepndently of how many cells were in the clump. B a d on this assessment, al! cytokine and

lysosornal enzyme data were normalired by area % counts.

Cell number

Cell area % 30

INCUBATION TIME, HOURS

O GLASS A HDPE

Figure 3.9. Changes in cell number and cell ami % with time (see Table 2 in the Appendix for n values)

Cell number

Cell area %

INCUBATION TIME, HOURS

GLASS 0 COLL+ DMSO A HDPE

Figure 3.10. Changes in cell nwiber and cell area % with time (see Table 2 in the Appendix for n values)

3.3.3. Cytokine and lywomal enzyme analyris

The secretion of the cytokines and lysosornal enzymes is an important parameter that can

be used to estirnote the inflammatoiy effect of the particles when engulfed by the cells. In this

study seven experiments were carried out to analyze the cytokine and lysosornal enzyme release

by the cells at different time points (see Table 3.3.).

I l

I Expcriment Psrtick type Incubation t

#

O *

1

2

H&E, TNF-a, P-GAL, HEX

H E , MF*, P-GAL. HEX

H&E, SEM, TEM, W u ,

P-GAL, HEX, ILI P, PGE?

HBE, TNFa. p-GAL,

HEX, IL- l a, IL- I P, IL-6,

PGE?

3

UHMWPE

HDPE

HDPE

periods

20

20

2.6.24

HDPE

5

1 1 1 HEX, ILI P

24,48,72,

6

# of celYwell

plated

1 x 106

HDPE

Table 3.3. Summary o f the perfomed experiments (* image analysis was not performed on

Expriment O)

HDPE

The cells exposa4 to large UHMWPE particles (1 8-20 p) for 20 houn released TNF-a,

P-GAL, and E X (Figure 3.1 1 a-c), however, this responsc was not statistically significant when

cornparrd to collagen controls (this study corresponds to Expniment O in Tabks 3, 5, and 6 of

the Appendix). It was also obseMd that while the TNFa data for the glass control were

24,48

HEX, IL- I P

H&E, TNF-a, PGAL,

24,48

HEX, L I p

HBE, W-U, P-GAL,

dramatically lower than for collagen groups, the same samples had sipificantly higher values of

lysosomal enzymes than for the collagen groups.

The cells exposed to smaller HDPE particles (4-10 pm) also released cytokines and

lysosomal enzymes and typical profiles (based on the results h m Experiment 3, recorded in

Tables 2-9 of Appendix) for cytokine and lysosomal enzyme release data at the 24 hour

incubation time point are shown in Figure 3.12(ad) and Figure 3.13(a-c). The levels of IL- l a

(Figure y 3 . 1 2 ~ ) and 11.4 p (Figure 3 1Zd) in the media were significant!} highcr ( ~ ~ 0 . 0 5 ) as

compred to non-prticulate controls at the 24 hour incutmtition petid. Since biological

activities o f IL-la are similar to those of IL-IP.'' the supcmatants were analyzed for the

prewnce of IL4 a only once, while IL- I P release was measured in five experimcnts and was

statistically significant over al! control groups in all experiments (Figure 3.14). Even though the

levels of IL-IP production by the cells in response to the particles are different for the four

cxperiments shown in Figure 3.14, the pattern of the cytokine relrasr remained the same. The

diffe~nces in cytokine release could be attributed to the fact that direrent kits from different

lots as well as two different ELISA plate readers were employed during the course of the

analyses.

At 24 hom, RIF* (Fibure 3. I2a) and IL6 (Figure 3.12b) release were not statistically

significant over collagen with DMSO values (pH). 1 ). As well, it can k seen from Table 2 in the

Appendix that R I F a release was consistently not significant (p0.05) in al1 expenments,

including the experiments with 48.72. and 96 hour time points (see Figure 3. !Sa). At these later

time points, a decrease in R J F - a production wes observed.

The release of both f3-GAL and HEX (Figures 3.13a,b) was statistically significant at the

24 hour incubation p r i d @<O.OS) for puticle gmups over collagen with DMSO values. This

w u noted in thtee out o f six expenments (see data in Tables 5 and 6 of the Appendix). At 48

hows of incubation, particle phagocytosis resulted in significant diflerences for P-GAL release

in three out of four expenments (see data in Table 5 of the Appndix), and for HEX release in

two out of four experirnents ( ~ 0 . 0 5 ) (see &ta in Table 6 of the Aplnndix). An increose in

lysosomal enzyme production was observed from 72 to 96 hours of incuôation (Figure 3.16b).

HEX

1500 1

u GLASS

0 COLLAGEN

UHMWPE

Figure 3.1 1 . Media analysis of IC-2 1 cells exposed io 18-20 Mm LRIMWPE particles; 20 hours incubation period; (a) P-GAL; (b) HEX; (c) W-a (*) samples were cornparrd using pid student's 1-test

(see Tables 3,5,6 in the Appendix for n values)

u DMSO

HDPE

Figure 3.12. Cytokine release by IC-2 1 cells exposed to HDPE prticles; 24 hout time point (a) TNF-a; (b) IL-6; (c) IL- 1 a; (d) iL- 1 P; (') samples were compared using poireû mident's t-test (see Tables 3,4. 8-9 in the Appcndix for n values)

PGE,

0 GîASS

0 IMSO

0 COLLAGEN

COLL+DMSO - HDPE

Figure 3.13. Lysosomal enzyme and PGE, release by IC-2 1 cells exposed to HDPE particla 24 hour time point: (a) P-GAL; (b) HEX; (c) PGE, (*) m p l e s were compreâ using pPired shident's t-test

(see Tables 5,6,7 in the Appendix for n values)

1 - glua, 24 hn Uic, 2 - coU+DMSO, 24 hm inc; 3 - HDPE, 24 hm inc,

4 - glua, 48 Iin k, 5 - coil+DMSO, 4û hm inc; 6 - HDPE, 48 hn inc

Tumoui Necmis Factor* piofik

Fipe3.15. S u n m u y o f R S u r u u l b k w d o n ~ 3 - 6 ;

SI - ~ 6 ; S Z - a t p a i a w n t S ; S 3 - C n p a i m c n t 4 ; S 4 - ~ t 3 ;

1 24hninc, 2 -wU+DMSO~ 24hninC; 3 -HDPE, 24hnUic;

4 - glua, 48 !us inc; 5 - coIl+DMSOB 48 htr inc; 6 - HDPE, 48 hn inc

PGE,

INCUBATION TIME, HOURS

O GLASS A HDPE

Figure 3.16. IC-2 1 cells exposed to HDPE pacticles for 24,48,72, and % hours;

(a) m a ; (b) P-GU; (c) PGEz (see Tables 3,5,7 in the Appendix for n values)

Interestingly, the lysosomal enzyme release data for glas and DMSO alone were similar to those

for al1 groups that contained collagen (see Figures 3.13 and 3.16. and Tables 5 and 6 of the

Appendix). This suggests that collagn did not have an effect on cellular activation.

PGE2 (Figure 3 . 1 3 ~ ) release was not significant at 24 hour incubation perd (pHl.05).

nor at 48 and 96 hour time points (Figure 3.16). The only differencc: between the collagen with

DMSO and the particle goups appeared to be at the 72 hour time point ( ~ 0 . 0 5 within samples

of a specific experiment). Howwer, 48.72. and 96 hours o f incubation experimental timc points

were only investigated once. A decrease in PGEL production relative to 24 houn was observed

ai 48, 73, and 96 hours of incubation (Figure 3.16).

3 f 4 . Viability test

Crll viability was assessed by using trypan blue stain and was always above 95% for dl

test b~ooups. Histological evaluation of the samples also confkned that the cells appeared viable

and exhibited features that are usually associated with normal cell morphology.""

4. DISCUSSION

The purpose of this study was to develop a model that would allow phagocytosis o f

polyethylene particulate in vitro. A necd for such an in v i m system exists since polyethylene is

the miijor contnbutor to prosthetic Wear and, at the same time, is the least studied cornponent of

the wear debris that has been isolated from patients with orthopaedic implant failure. Of

prticular cuncern at this time is the fact that despite the rn V I W J findings showing abundance o f

polyrthylenr particulate.f' there is linle 43.Jb.4H in virro data available that has demonstrated

phagocytosis of polyethylene particulate, and no histological findings from such studies that

con fim the engul fmrnt of the particles.

The absencc of histological evidence of particulate phagocytosis in rn vrrro studies (not

only polyethylene, but also PMMA and metal) could be explainrd by the Fact that aseptic

loosening has bcen investigated by two distinct ûpproaches: (1) to determine the role of material

in the process of aseptic loosening by mrasuring the levels of inflammatory mrdiators in

response to the material tn vrtm, and; ( 2 ) to establish thc role o f these mediaton in the aseptic

looseni ng by utilizing rn vrvo models and studying retrieved inflammatory membranes. The first

approach has employed biochemical methods of analysis, such as ELISA, producing large

quantities o f data without histological conlinnntion of either phagocytosis or crl l viiibility. The

second approach has utilized histological methods as well as immunohistochemistry to m l yze

retrieved specimens for cellular rcsponses to paniculate debris, however. these rn vrw models

and retrieved membranes do not provide the detailed information about individual cellular

rcsponses to panicles, at the mechanistic level. Thus, a vacuum in the basic research of

celVparticle interactions exists. producing a necessity for a well characterized rn v~trï) model

studying macrophage-prticle interactions.

The new in vrrro model presented in this work enables us to consistently introduce

micron size polyethylene particles into macrophages. The intemalization of particles has ken

repeatedly demonstrated by SEM (Figure 3.7) and TEM (Figure 3.8). The in vitro system

described herein i s simple and versatile, and, funhermote, allows for the media analysis of

cytokines and lysosomal enzymes. Thus, it incorporates the traditional methods of histological

analysis and permits biochemical characterizahon of the model.

As mentioned before, there are some dificulties associated with handling polyethylenc

particulate: i t i s hydrophobie and has a low densip. and. thrrefore. i s incompatible with aqueous

media. This problem was overcome in the following manner. Type 1 collagen was selected for

use in this system because polymeritation of the collagen was considrred to be a practical tool

for trapping the particles in a solidified matrix fomed in the culture system. In piirt, this mimics

the rn vrvo environment, since collagen type 1 i s the most abunûant protein in the bone. and it i s

present in the inflammatory membrane." At the same time, type 1 collagen is commonly applied

in tissue culture to promote cell attachment onto different surfaces."'

The use of collagen type 1 for incorporation o f the polyethylene panicles into the cells

has biochemical significance. It has been previously desfribed (see Section 1 A) that proteins of

the extracellular matrix have numerous effects on cell differeniiation and behavior. Apparently,

the exposure to collagn can also increesp the phagocytic activity of macrophages. Newman et

al.'" demonstrated that monocytes adhered to type 1 coilngen gels phagocytosed 2.5- 12-fold more

opsonized bacteria than plastic adherent monocytes. This group discovered that the adherence to

collagen gels activated complement receptors, as well as Fc receptors for phapcytosis, thus

causing the increase in phagocytic ability of the cells. Newman et al? hypothesized that the

adherence of monocytes to the extracellular matrix dunng inflammation could activate the cells

for enhanced phagocytic bactericida1 activity. There arc a few observations made in this study

that could be extrapolated to the cunent research project. Polyethylene particles, as well as

other constitwnts of Wear debris, become opnized by senun proteins in vivo and in vitro (since

the culture media contains 10% fetal serum)(Boynton, Sandhu, unpublished dad4). The

prescnce of collagen in the inflammatory membranes as well as in the in vitra model could

activate the complement rece~tors," thus enhancing the pphegoytosis of the panicles. That

phenornenon renders the in vitro mdel even more relevant to the in vivo situation since it not

only mimics the sunounding environment of the inflammatory membrane, but also imitates the

processes involved in phagocytosis of the Wear particulate.

This in vitro model takes advantage of another useful property of collagn. As

mentioned in the introduction (section 1.4), collagen promotes differentiation of human blood

monocytes into macrophages. Kaplan et al." demonstrated that monocytes matured on collagen

matrices nppear to be tissue macrophage-like cells with enhancrd Fc- and Cmediated

phagocytosis. Even though this property of the mdel may not be relevant to the present study

with fully differentiated macrophages, it has been useful for the development of experirnents

with human peripheral b l d monocytes in our laboratory 75

The histological evaluation of the cells exposed to the polyethylene particles

demonstrated the ability to perform any type of histological analysis. Conventional hematoxylin

and eosin staining, SEM. and TEM are easily perfoned with this mode1 and the histological

findings in this study are consistent with that reported in scientific ~iterature.~~ Two different

types of cellular reactions, associated with particle phagocytosis, are observed rn vivo: ( 1 )

foreibm body giant cell formation in response to large particles and; (2) the phagocytosis of small

(< 5 pm) particles. The rn vivo processes involved in response to particulate Wear debris are

mimicked by the model presented in this thesis. Histological evaluation of parttcle phagocytosis

was demonstrated by hemntoxylin and eosin staining (Figures 3.5. 3.6) and by SEM (Fipre 3.7)

analysis of the cultures. The majority of cells have engulfed particulate polyethylene. TEM

analysis funher confirmed that small particles were intemalized by the macrophages, where they

were incorporateci into vacuoles (Figure 3.8) and not just surrounded by the cells. The cells

appeared to be bigger in groups that contained particles as compared to control groups. As well,

the former cells contained numerous lysosomes, which is a feature that is consistent with

macrophage activati~n.'~ It should be noted that lysosome formation may not only be due to

particle digestion but also result frum collagen phagocytosis and digestion.

Despite the fact that there was no apparent cytotoxicity in mponse to phagocytosis of

polyethylene particulate, biisad on histdogidy obxrved specimens, the particles appeareâ to

have had a sipificant effect on cellular function. In order to assess the effect of the particles on

macrophage pro1 i feration, the cell nurnber and area percent counts were detemined (complete

data are given in Table 2 of the Appendix). When evaluating cell counts (Figures 3.9 and 3.1 O),

a consistent inhibition of ccll proliferation by polyethylene p~mcles ( ~ 0 . 1 ) was demonstrated at

24 houn of incubation in four out of six experiments. At 48 hours of incubation three out of

four experiments il1 ustrated si gni ficmt (pcO.05) di fferences between collagen/DMSO contro l s

and the particle groups. Onlv one expriment was designed to look at later tirne points and the

differences between the groups were even more dramatic for 72 and % hours of incubation

(p=0.0007 ai 72 houn and p<0.0001 at 96 hours, see Figure 3.9). The waluation of ccll numbers

using area % counts demonstrated a similar picture. It can be suggesteû from these data that the

inhi bition o f ce1 l proliferation was consistent between experirnents and progressive with time.

Nevertheless, the cell viability, as venfied by trypan blue, was 98% for al1 test groups, an4

therefore. was not affected by part iculate phagocytosis. Thus, pol yethy lene phagocytosis was

not toxic to the cells but affectrd the ceIl proliferation.

The observed decrease in the rate of macrophage proliferation during particle

phagocytosis was consistent wi th the data available in li terature. lu*'' Shanbhag et al. lU

demonstrated that P388Dl moue macrophage cells exposed to polystyrcne and titania particles

genersll y incorporated l e s thymidine than controls, thus illustrating the decrease in DNA

synthesis. At the same time, human fibroblasts exposed to PMMA particles demonstrated an

increasc in tibroblast proliferation,47 while the experiments with bovine synovial fibroblssts

illustrated a correlation between particulate matenal, panicle site, particle concentration and

cell proliferation." An increase in particle concentration usually correspondcd with a decrease

in proliferation rate? It should k noted that even though the results obtained with image

analysis correlate to those performed by other methods of analysis. the whole concept of

inhibiting cell proliferation rnay not be relevant to the in vivo situation at ail. since macrophages

rn vivo are fully differentiated cells and they do not divide at the bone-implant interface.

Nevertheless, the fact h t cell prolifemtion was affected in a cell line may suggest that either the

cells 'slow dom' while king occupied with digesting the enplfed material. or the cells secrete

an inhibitor of proliferation as a response to particle phagocytosis. The later hypothesis can be

supported by studies performed with hurnan fibroblasts, where the cells were exposed to

conditioned media from macrophage cultures that had been exposed to particulate material. ' O In

the latter study it was observed that exposure of macrophages to titania particles inhibited

fibroblast proliferation, while the exposure to polystyrene particles had an inhibitory effect at

low concentrations and a stimulating e ffect at high concentrations. '' These results demonstrate

that the cclls wcrete diffèrent "WS" o f cytokines in response to dinèrent types o f prt iclcs

Therefore. it appears that in response to polyethylene phagocytosis, macrophages could

potentially secrete an inhibitor of ce11 proliferation.

Even though image analysis was utilized in this study to asses cell number and to

normalize the cytokine and lysosomal enzyme data. this method w u found not to be very

sensitive for detennining slight différences in cell number. between the groups. The

conventional methods of detennining the rates of cell proliferation, such as tritiated thymidine

incorporation to measure DNA synthrsis4'*h7. and protein rneasurement~.'~ are more sensitive

assays and could be used in future expetirnents. Nevertheless, image analysis is a simple methd

that does not require any additional procedures to be performed on existing histological slides

and can provide a quick estimate of cell number or ceIl area when conventional methods are not

available.

It was noted in the Introduction, that differences exist between the resting and activated

macrophage. The main features exhibited by activated macrophages include increase in size,

ruf'tîing of the plasma membrane, increase in the number of pinocytic vesicles, increase or

decrease of different swfàce recepors and the release of infiammatory rnediator~.~~ Of al1 of

these events the latter has been believed to be of greater Cytokines,

prostagiandins. and lysosomal enzymes are considered to be markers of macrophage

activation.''" Cytokines a d PGE2 have nurnerous activities, including the ability to enhsnce

phagocytosis, to activate other cells and to inhibit or promote cellular proliferation.25~2' On the

other hand, the lysosomal enzymes cany out a completely different set of activities. lt has been

described that the release of lysosomal enzymes by macrophages can occur by undamaged cells

by exocytosis. This is particularly noted with older macrophages and in locations near toxic or

imtating substance^.'^ Saithouse et al." ?as also observed that geometry, surface, and other

characteristics of the implant can affkct the levels of lysosomal enzyme activity. Therefore, in

order to confimi increctsed cell activation during phapcytosis of the particles, the media from

ceIl cultures were collected and cytokine and lysosomal enzyme profiles detemined.

42.1. Cytokines and prostaglandin E2

As shown in Figures 3.12, 3.14, a significant increase of IL-la and IL-IP for the cells

exposai to the particles, at 24 hours of incubation, was demonstrated. The releasc of IL-6, TNF-

a, and PGE2 was not significant at 24 hours of incubation (Figures 3.12, 3.13, 3.15, 3.16). 5.7

These findings correlate in pan with the analyses of the human retrieval specimens . as well as rp.43-15

with cell culture results obtained from the study of different particle types. Al Saffar et al.'

uti lized immunohistochemistry on tissue sections and demonstrated the presence of IL- I P

containing cells (80% of these cdls were macrophages) in the retrieved tissues. Jiranek et al.'

employed hybridization of the tissue sections and observed that IL- 1 P mRNA was predominantl y

associated with macrophages in response to polyethylrne, PMMA, and metal debris present in

al1 specimens. 44.4 5

The results presented in this thesis differ somewhat from Shanbhag et al. who

demonstrated a significant increase in L l P , IL-6. and PGEl production in mponse to the

expswe of fabricated submicron polyethylene particles to hwnan periphenl b l d monocytes

and an increase in PGE2 telease in Rsponse to retrieved polyethylene particles. The mean size

of the retrieved polyethylene particles was 0.47 I 0.2 pm, and the mean sizc of the fabricated

polyethylene particles was 0.66 f 0.6 pm. Howcver, when their data were compared to controls

the only statisticdly significant increase in cytokine production was obsewed at a psrcicle/cell

surface area ratio of 10. This corresponded to appoximately 1 130 particles per cell for

fabricated prticles and 3520 particles per cell for retrieved particles. Not surpnsingly. the cell

viebility at these concentrations for both particle types was only 50%. It is hard to imagine the

relevance of such concentrations to the in vivo situation. Moreover, Shangbag's study showed

no histological evidence to conf in intemalidon of the polyethylene prticulate. Hence. the

cytokine release may have well been due to cell death and not to panicle phagocytosis. To the

contrary, the data presented in this thesis show that an increase in cytokine production (IL-la,

IL-1b) was observed at a ratio of i particle per 10 cells, with a cell viability of 98%.

It must also be considered that the variance in cytokine release data between the thesis

study and Shanbhag's work;' could be explained by the differences in cell types (monocytes'"

versus fully diffrrentiated mouse macrophages) and in particle types (retrieved or fabricateâ

submicron UHMWPE paniculate" versus 4-10 Pm HDPE particles). As well, the remeved

particles in Shanbhag's work could have residual protein adsorbed to the surface o f the particles.

The presence of denaturated proteins on the surface can cause unpredictable responses from the

c e l l s . ' ~ s P i t e the variance in cytokine response. the development of this model will allow

detailed investigation of these di fferences.

The presence of collagen in the systern may also have played an important role in the

macrophage response to the particles. At the same time, the data presented in this thesis are 48

confmned by the results obtained from Shirata et al. This group used IC-2 1 cells and an

inverted tissue culture system that enablcd hem to introduce HDPE prticles into the

macrophages. They demonstrated a statistically significant increase in IL-1P release, however,

the latter was the only cytokine measund in their study. Even though collagen was not

employed in Shirata's model, their results comlate to the results presented in this thesis. Since

both studies were carried by different protocols and different research groups. it is difticult to

determine if collapn's role in the particle phagocytosis was specific. Future studies could

simultaneously examine both models in order to assess if the presence of collagen increases the

rate and levels of particle phigocytosis as well as the production and release of the cytokines.

The rationale for why the levels of M u , one of the major pro-infiammatory mediators,

were not statistically sipikani for particles versus no puticles and why this finding âiffers

43.46 from that of other in vitro studies could possibly lx cxplaineâ by the fact that a mouse cell

line was employed in this work. There may be specific differences between mouse and human

macrophages in relation to interactions with polyethylene paticulate. For this reason, future

studies with the model will be peflormed with hwnan derived macrophages. However, cell type

may not be the only factor contributing to the differences in the ce11 response. As demonstrated

by XPS (Table 3.1) and FTIR (Figure 3.2) data, there can be distinctions in swface chemistry

and bulk properties between UHMWPE and HDPE. The combination of prticle type and

surface chemistry (Le., metal venus PMMA versus PE), differences frorn the specific Wear

particles found in vrvo, along with mouse cells could explain the differenccs in the TNF-a 6.45

response. It hm k e n reported in the ment literature , that particle size, shape, and surface

chemistry are critical factors determining cell response. The particles retricved from human

specimens range from submicron values up to 100 pm and contain not only polyethylene, but

other sibmificant materials, such as metal and polymethyl methacrylate. The particle surface can

be altered by repeated exposure to high concentrations of lysosomal enzymes and other oxiâative

agents produced by the inflammatory cells. Metal ions embedded into polyethylene particles7'

could act as catalysts, increastng the rates of oxidation and changing particle surface chemistry.

One could hypothesize, that oxidized prticles may have the ability to cause enhanced cellular

activation in cornpanson to non-oxidized particles. The significance of the material chemistry

and structure rmphasizes the importance of having a well charactetized tn vitro mode1 available

to study the different variables affecting cellular activation. Particle size is another parameter to

be considered. 12.19-22 It has been reported in literature that the majority of the retricved

polyethylene particles are submicron in sire. 12.19-22 Since the prticles employed in this study

were 4-10 pm in size instead of submicron, this could in pui explain some of the differences in

cytokine response. The cellular reaction could vaiy with the particle size simply due to the

increase in reactive surface a m . Nevertheless, the goal of introâucing polyethylene particles

into macrophages was achieved and an increase in cytokine production demonstrated cellular

activation.

It should be considered that there may be another explanation as to why Mir levels

were consistently not siynificant when companng particlc groups to collagen controls. It has

been observed . that collagen has numemus effects on monocyte/macrophage responses.

including enhancement of phag~c~tosis ,~ increase in surface recepton e ~ ~ r e s s i o n , ? ~ - ~ and

modulation of cytokine expression.M62 Eiennan et al." observed that monocytes adhered to

collagen produced significantly higher levels of TNFa than monocytes cultured on tissue

cultiirc: plastic This phenamenon was npeated in the exprirnents presented in this thesis the

cells cultured on collapn secreted more cytokine than the cells in control groups (glass,

DMSOXsee Figures 3.12 and 3.14). The collagen effect on the cells can bring about new

considerations into the investigation of the rnechanism of awptic loosening. It is generally

blieved, that in aseptic loosening, the pro-intlammatory cytokine TNF-a is secreted by

macrophages in response to wcar debris phagoc ytosis. Jqh,3h,J3.J6 However. the only significant

increases in W-u production that have been reported were in experiments with PMMA

pnicies of a non-specified ~ize,".'~ retrieved polyethylene particles,"h and polyethylene particles

embedded in agarose." In experiments with PMMA."%VO goups of particles (1) < 300 pm

and (2) 300-1000 Pm were utilizcd and the significant increase in T N F a release was

demonstrated. However, this phenomenon can be due to cell death rather than to particle

phagocytosis. In the case of retrieved polyethylene prtic~es,'~ the cells could be stimulated by

proteins still attached to the jmrticles, while Harada et al.'hbserved that agarose caused

monocyte activation even in the absence of particles. That may bring one to hypothesize that

TNF-a could be secreteû not in mponse to the particulate phagocytosis, but in response io

exposure to extracellular matrix proteins, especially collagen, which is abundant in the

inflammatory membrane." Nevertheless, the role of this cytokine still remains the m e :

augmentation of the inflammatory response, enhancement of cytokine production, and

promotion of bone damage, however, the mechanism of the cytokine induction could be

different. This hypothesis is confirmed by the mode1 described in this thesis, since al1 collagen

groups (collagen, collagen with DMSO, and collag«i with DMSO and puticles) secnteô similar

levels of TNF-a and polyethylene particulate phogocytosis did not have any messurable efféct on

the production o f this cytokine. Thus, the mode1 mimics the in vivo environment o f the

inflammatory membrane by providing extracellular matrix to the cells and could be used in

detennining the roles and the mechanisms of induction o f various cytokines in response to

different types o f stimuli.

4.2.2. Lysosamal enzymes

.4s show in Figures 3.13, 3.16 a sigiificant increax of BGAL and HEX levels in cells

rxposed to the particles for 24 hours was demonstrated. Whilr: there were sornr exprnments

(see Tables 5 . 6 in Appendix) which did not show the levels of lysosomal enzymes to be

sibmificûnt, it is important to emphasize that only the levels of released enzymes were measured

in this srudy. Since, the primary function of lysosornal enzymes is to digest foreign material

inside of the lysosomeiphagolysosome27~2' and not to serve as an inflammatory mediator, the

levels of the released enzymes may not have the same significaiit effect as the levels of released

cytokines. At the same time, a knowledge of the amount and types of enzymes relewed by the

cells cm provide valuable information about the condition of the cells. For example, one could

presume that in the case of hstrated phagocytosis the levels of rele~sed enzymes would be

dramatical l y increased due to the processes involved, whereas a nondamaged cell would

mrintain nlatively low levels o f the released enzymes. As well, the levels of the lysosomal

enzymes can also provide information about cell numben. For example, Landegred7

demonstrated that the levels of total hexosaminidase increased with an increase in ceil number

and the results comesponded to the data obtained by the thymidine incorporation assay.

However, since the levels of intracellular HEX were not measured in the current study it was not

possible to apply this technique to confinn the cell count values. Nevertheless, the presence o f

non-specific lysosomal enzymes, such as P-GAI. and HEX, was demonstrated and a significant

increase in enzyme release in puticle groups comporcd to conbds was observed

The preliminary characterization of this novel in vitro mode1 has opened the possibility

of studying the response of cells to wear debris in culture. The mode1 has now been expanded

and human macrophages are used in the systern.'' Histological evaluation of the ceIl response

may allow one to detect minute differences in ceIl rnorphology in reaction to différent types of

wc les . including polvethvlerie. PMMA. and metal. with biochemical confirmation of these

differences. The versatility of the model allows one to utilize co-culture techniques and,

therefore, provides a tool to study the mechanism of aseptic loosening and bone lysis due to

macrophage activation by polyethylene particulate.

S. CONCLUSIONS

1. A novel rn vrtro model that allows one to consistently introduce polyethylene particulate

into cells has been developed. It diffen from other attempts to model polyethylene phagocytosis

in that it utilizes the extracellular matrix protein - collagen - to provide the entmpment for the

panicles as well as a matrix for the cells. This mode1 i s simple and versatile making it an easy

task to perfonn histological as well as biochemical analyses with the cells.

2. The mode1 had been characterized both histologically and biochemically.

3. Histologicd evaluation of the mode1 confinned the intemalization of the polyethylene

panicles and provided unique information about morphological changes in the cells in response

to polyethylene particulate phagocytosis.

4. Biochemical evaluation of the model demonstreted the cytokine and lysosomal enzyme

release in response to pmculate phagocytosis, verifjing cellular activation observed

histologically, and established a peak cytokine response st 24 houn of incuôation.

5 . The presence of type 1 collapn played an important role by providing extracellular

matrix for the cells, entrapment for the particles, and may have facilitated macrophage

phagocytosis of the particles.

6. Image analysis was employed to assess the ceIl number in order to normalize the

cytokine and lysosomal enzyme release data. Even though it is a quick and easy method of

analysis, it may not be sensitive enough to detect minute changes in cell proliferation in response

to particle phapcytosis.

7. While only the levels of the released enzymes have been measured in this study, the data

show significant levels of enzymes for particles venus controls in several experiments.

However, it was concluded that the levels of intracellular enzymes could provide more

in format ion about cellular response to particle phagocytosis.

RECOMMENDATIONS

1 . The model could be used to investigate the response of macrophages to different particle

types (metal, PMMA, polyethy lene), different particle concentrations, and different particle sizes

by utilizing histological as well as biochemical techniques.

2. T L model could be employed to study the response to prticulate phagocytosis by

different cell types (mouse vs. rat vs. human, etc.) in order to provide information about

différences between the species.

3. The model could be utilued to determine the effect of particdate phagocytosis on bone

rrsorption using cotulture systems or macrophage conditioned media.

4. The model could allow for the investigation and identification of pioteins/lysosomal

enzymes adhered to the surface of the particles inside of the phagolysosome by

immunohistochemistry. If particles with a chcmially altered sutface are useâ, the differcnces in

protein adsorption to the particle surface could be detennined and m e r assessed for specific

ce1 l ular responses.

5 . The model could be used in conjunction with in vivo expiments in order to determine

the similarities and differences in onecell type system versus multiple-cell type system.

6. The employment of immunohistochemical techniques could be used to study the effect of

particdate intemal ization on cell structure and morphology .

7. It is highly recommended to utilize conventional methods of determining the rates of cell

proliferetion, such as protein and DNA analysis, for nomalization of cytokine and lysosomal

enzyme release data.

8. A further study on the potential role that collagen plays with respect to the uptake of

particles must by investigated. A cornparison study between collagen mode1 venus the inverted

system would probably be valuable in providing this information.

9. The model could be utilized to study bone resorption by particle-activated macrophages.

since it is believed ihat macrophages are capable of direct bone resorption when stimulated by

pnicle phagocytosis and, therefore, contribute to bone Iysis in aseptic loosening.

REFERENCES

H.C. Amstutz, S.M. Ma, R.H. Jinnah, L. Mai, "Revision of aseptic loose total hip

arthroplasties," CORR, 1 70:2 1-33 ( 1982)

E. Boynton, M. Henry, J . Morton, J.P. Waddell, "The inflammatory response to particulate

Wear debns in total hip arthroplasty", Can. J. Surg., 38(6):507-5 15 ( 1995)

E. Boynton, J.P. Waddell, J . Morton, G.W Gardiner, "Asepiic lmsening in total hip

implants: the role of polyethylene Wear debris", ( 'un. J. Surg., 34(6):599405 ( 1 99 1 )

S.M. Horowi(s S.B. Doty, J.M. Lane, A.H. Burstein, "Studies of the mechanism by which

the mechanical failurr: of polymethylmethacrylate leads to bone resorption." J. Hone cilid

Joint S t q . , 75-A(6):802-8 1 3 ( 1 993)

W.A. Jiranek. M. Machado, M. Justy, D. Jevsevar, H.J. Wolfe. S.R. Goldnng, M.J. Goldberg,

W.H. Harris, "Production of cytokines around loosened cemented acetabular components," J.

Hone and Joint Surg., W A ( 6 ): 863-879 ( 1 993)

W.J. Maloney, R.L. Smith, "Periprosthetic osteolysis in total hip arthroplasty: the role of

paniculate Wear debris," J. Bonr und Joint Surg.. 77-A(9): 1448- 146 1 ( 1995)

N. Al Samar, P.A. Revell, "Interleukin-l production by activated macrophages sunowiding

loosened orthopaedic implants: a potential role in osteolysis," Br. J. Rhcumutol., 33:309-3 16

( 1994)

D.W. Howie, D.R. Haynes, S.D. Rogers, M.A. McGee, M.J. Pearcy, "The response to

particulate debris," Orthopt'drc Clrnrcs cg North Amerrcu 24(4):571-58 1 ( 1993)

S.M. Horowitz, B.P. Rapuano, J.M. Lane, A.H. Burstein, "The interaction of the macrophage

and the osteoblast in the pathophysiology of aseptic loosening of joint replacements", Culc$

Tissue Int., 54:320-324 ( 1994)

IO. A.S. Shanbhag, J.J. Jacobs, J. Black, J.O. Galante, T.T. Glant, "Macrophagdpnrcicle

interactions: effect of size, composition and surface area," J. Bionred Murer. Hes., 28, 81-90

( 1994)

1 1. T.T. Glant, J.J. Jacobs, G. Molnar, A.S. Shanbhag, M. Valyon, J.O. Galante, "Bone

resorption activity of particulate-stimulated macrophages," J. Bone und Minerai Hes.,

8(9): 1 O7 1 - 1079 ( 1993)

12. P. Campbell. S. Ma, B. Yeom, H. McKellop. T.P. Schmal~eâ, H.C. Arstutz, "isolation of

predominantly submicron-sized UHMWPE Wear particles from periprosthetic tissues," J.

Biomed. Muter. Re.s..29, 127-131 (1995)

13. T.P. Schrnalzried. M. Jasty. W.H. Harris. "Periprosthetic bone loss in total hip arthroplasty.

Polyethylene Wear debris and the concept of the effective joint spece," J. Hone und Joint

Surg., 74-A@), 849-863 ( 1992)

14. Willert HG, Buchhom GH, Eyerer P (eds.): Ultra-High Molecular Weight Polyethylene as

Biomaterirl in Orthoprdic S wgery. Hogefe & Huber Publishers, 199 1

15. Friedlaender GE, Goldberg V M (eds.): Bone and cartilage alIograh: biology and clinical

applications. Amencan Academy of Orthopaedic Surgeons, 199 1

16. Pilliar RM: "Manufacturing processes of metals: the processîng and properties of metal

implants". Metal and Ceramic Biomaterials, vol. 1. CRC Press Inc., Boca Raton, USA, 1984

17. K.R. St. John (ed.), "Particulate debris from medical implants:mechanisms of formation and

biological consequences." Anrrrrcun Suorty fvr Ïéstrng a d Mutcr~uis, 199 1

18. D. Pienkowski, R. Jacob, P. Hoglin, K. Sam. H. Kaufer, P.J. Nicholls, "Low-voltage

scanning electron microscopie imaging of UHMWPE," .J. of'Bioned Mat. Hrs., 29: 1 167-74.

(1995)

19. A.S. Shanbhag, J.J. Jacobs, T.T. Glant, J. L. Gilbert, J. Black, J.O. Galante, "Composition and

morphology of Wear debris in faiied uncemented total hip replacement," J. Bone and Jomt

Surg., 76-8( 1 ):60-67 ( 1994)

20. K.J. Morgevicius, T.W. Bauer. J.T. McMalon, S.A. Brown, K. Memtt, "Isolation and

characterization of debris in membranes around total joint prostheses," J. Bone and Jornt

Surg., 76-A( 1 1 ): 1664-1 675 ( 1994)

2 1. J.M. Lee, E.A. Salvati, F. Betts, E.F. DiCarlo, S.B. Doty, P.G. Bullough, "Size of metallic

and polyethylene debris particles in failed cementcd total hip replacements," J. Bone und

Jotni Surg., 74-B(3):380-384 ( 1992)

22. P. Campbell, S. Ma, G. Belcher, "A methoù for metal and particle isolation from

periprosthet ic tissues," Tram Orthop. Res. Soc., 39:494 ( 1993)

33. K.J. Kim, J . Chiba, H.E. Rubash, "in vivo and in vitro analysis of membranes from hip

prostheses inserted without cernent." J. &ne und Jorni Surg., 76-A(2): 1 72- 1 80 ( 1 994)

24. C.E. Shannon, A.F. von Recum, R. Thull, "Collegen types found at the material-tissue

interface," ïiunsuctions ï f W t Wodd H~ornuterruls ( bngrrss, (1 ):266 ( 1996)

25. J.M. Austyn, K.J. Wood, "Principles of cellular and molecular immunology," New York,

Oxford University Press ( 1993 )

26. R. van Fwth (ed.), "Hemopietic growth factors and mononuclear phagocytes," Basel,

Karger er( 1993)

27. C. F. Nathan, "Secretory products of macrophages," J. C 'lin. Inves~. ,79:3 19-326 ( 1987)

28. M. Moonis, 1. Ahmad, B.K. Bachhawat, "Macrophages in host defence - an oveiview,"

Indion Juumul oj'Biuchem~stry und Aiophy.srcs, 29: 1 1 5- 1 2 2 ( 1 992)

29. R.B. Johnson Jr., "Monocytes and macrophages," The New Englund Journul of Mcdrcrne,

38( 1 2):747-752 ( 1988)

30. E. J. Brown, "Phagocytosis," Bro~ssu~v.s, l7(2), 109- 1 17 (1 995)

3 1. R.T. Dean, W. Jessup (eds. ), "Mononuclear phagocytes: physioloby and pathology," Elsevier

Science Publishen B. V. ( 1985)

32. T.N. Salthouse, "Cellular enzyme activity at the polyrner-tissue intefice: a review," J.

B~owted. Muter. R a . , 1 O, 1 97-229 ( 1 976)

33. F.R. Balkwi Il, F. Burke, "The cytokine network," I m u n d o g y T d u y , 10(9):299-304 ( 1989)

34. S. Keshav, L.-P. Chung, S. Gordon, "Macrophage products in inflammation," Diogn.

Microbiul. Infect. Dis.. 1 3 :439-447 ( 1 990)

35. D. Male (cd.), "Cytokines and cytokine recepton" h m "In Focus," 2" edition, iRL Press at

Oxford University Press, 1993

36. S.M. Horowitz, M.A. Purdon, "Mechanisms of cellular recniitment in aseptic lmsening of

prosthetic joint implants," CulciJ Tissue Int., 57:30 1-5 ( 1995)

37. G.D. Roodman, "Role of cytokines in the regulation of bone resorption," Culcif: Tissue Inil,

53(S~ppl 1 ):S94-S98 ( 1993)

38. D. Voet, J.G. Voet, "Biochemistry," John Wiley & Sons, Toronto (1990)

39. A.L. Lehninger, "Biochernisûy", 2nd edition, Worth Publishen Inc., New York, ( 198 1 )

40 R.P Phipps. S.H. Stein, Pl. Roper, ".4 new view of prostpglandin E regulmion o f the

immune response," immund. Ï'duy, 12( 10):349-5 1 ( 199 1 )

4 1 . D. W. Howir, B. Vernon-Roberts, R. Oadeshott, B. Manthey. "A rat model of resorption of

bone at the cememt-bone interface in the presence of polyethylene Wear particles," J. Rune

und Joint Surg., 70A(?): 257-263 ( 1988)

42. M. Spector, S. Shortkoff, H.P. Hsu, S Taylor-Zaptka, N. Lane, C.B. Sledhe, T.S. Thomhill,

"Synovium-like tissue from loose joint replacement prostheses: cornparison of human

material with a canine model," Seminars in Arthritis and Rheumatism. 2 l(5):335-344 (1992)

43 Y. Harada, V.A. Doppaiapudl, A.A. Willis, M. Jasty, W.H. Hams. S.R. Goldnng, "Human

macrophage response to polyethylene particles in vitro. A new experimental Model," 7ium.

Orihopuedzc Xrs. Suc., 19(2), 842 ( 1 994)

44. A.S. Shanbhag, J. Black, J.J. Jacobs, J.O. Galante, T.T. Glant, "Human monocyte response to

submicron fabricated and retneved polyethylene, Ti-6Al-4V and Ti panicles,," 7'run.v.

Orthopuedic Hes. Soc., 1 9(2), û49 ( 1 994)

45. A.S. Shanbhag, J.J. Jacobs, J. Black, J.O. Galante, T.T. Glant, "Hurnan monocyte response to

particulate biomaterials generated in vivo and in vitro," J. Orthop. Res., 13:792-8OI (1995)

46. J. Chiba, W. Meloney, S. Sugawara, K. houe, H.E. Rubash. "Biochemical and

morphological analyses of activated human macrophages and fibroblasts by human

pl yethy lene particles," Combtned Orthopued. Hes. Soc. Meetrng, Novemôer 6-8, 1 995, San

Diego. California, 23

47. C.G. Frondoza, K.T. Tanner, L.C. Jones, D.S. Hungrford, "Polymethylmethacrylate particles

enhance DNA and protein synthesis o f human fibroblasts in vitro," J. Biumed Mut. Res., 27,

61 1-617 (1993)

48. K. Shirata, T. Ushida, T. Tateishi, "Effect o f phqpcytosis o f polyethylene particles on

macrophage line IC-2 1 cells," ïimsuctiom of 5" Workl Biomutvriois Congrrss, Toronto.

Canada, May 28-June2, 1996, vol 2 of 2, page 858

4Q. A.F von Recum (ed.), "Handbook of Riomaterials evaluation. Scientifk, technical, and

clinical testing of implant materials," Macmillan Publishing Company, New York (1 9)

50. C.M. Serre, M. Papillard, P. Chavassieux, G. Boivin, "ln vitro induction of a calcifying

matrix by biomaterials constituted of collagen and/or hydroxyapatite: and ultrastructuml

companson of three types of biomaterials," Riomutcrrul.~, 1 d(2 ), 97- 1 O6 ( 1993)

5 1 . DG. Wallace, J. Rosenblatt, G. A. Ksander, "Tissue compatibility ut' collagen-silicone

composites in a rat subcutaneous model," J. Biomed Mur. /tes., 26( 1 1 ), 15 17-34 ( 1992)

52. P. Ries, "Collagnfleece as a bioimplant for orthopaedic surgery," Archives oj'(lrlhopecliic

K. 7iuurnu Surgery. I 1 1 (2), 66-9 ( 1992)

53. G. Zambonin. M. Grano, "Biomaterials in orthopaedic surgery: effects of different

hydroxyapatites and demineralized bone matrix on pro1 i feration rate and bone matrix

synthesis by human osteoblasts," Biomuteriul.s, 1 6(5), 397402 ( 1995)

54. Y. Kinoshita, T. Kuuhara, M. Kirigakubo, M. Kobayashi, K. Shimuka, Y. Ikada, "Reduction

in tumour formation on porous polyethylene by collagen imrnobil ization." Biomrtwiuls,

14(7), 546-50 ( 1993)

55. J. Damell, H. Lodish, D. Baltimore, "Molecular cell biology," Scientific American Books,

New York (1986)

56. K. Tryggvason, "Molecular proprties and diseases o f collagens," Kidney IntL,

47(Supp1.49):S24-S28 ( 1995)

57. L.J. Kleinsmith, V.M. Kish, "Principles of cell biology," Hnrper & Row, Publishers, New

York (1988)

58. J.D. Andrade, ed. "Surface and interfacial aspects of biomedical polymers." vol. 1, Plenum

Press, New York (1985)

59. G. Kaplan, G. Gaudemack, "ln vitro differentiation of human monocytes. Differences in

monocyte phenotypes induced by cultivation on glass or on collagen," J. Exp. hfed., 156,

!loi-il 14 (1982)

60. S.L. Newman, M.A. Tucci, "Regulation of human monocyte/macrophage function by

extracellular matrix. Adherence of monocytes to collagen matrices enhances phagcrvtosis of

opsonized bactena by activation of complement recepton and enhancement of Fc receptor

function," J. ( 'lin Invesr., 86(3):703- 14 ( 1990)

61. D.F. Eieman, C.E. Johnson, J.S. Haskill, "Human monocyte infiammatory mediator gene

expression is selectively regulated by adherence substrates," .J. Inimunol., 142(6), 1970-1976

( 1989)

62. Z. Xing, M. Jordnna, J. Gauldie, "IL-1P and L-6 gene expression in alveolar macrophages:

modulation by extracellular matrices." ..lm. J. P hysiol., 262(6), L600-L605 (1992)

63. B. W. Callen, M.L. Ridge, S. Lahooli, A. W. Neumann, R.N.S. Sodhi, "Remote plasma and

ultravioletszone modification of polystyrene." J. Vuc. SCI. 72chnol. A., 13(4): 20234029

(1995)

64. P.M.T. van Attekum, J.M. Trooster," ", J. Efectron Spectro.~. M u t . Phenom., 1 1 :363 ( 1977)

65. J.B. Lambert, H.F. Shurvell, D.A. Lightner, R.G. Cooks, "lntroduction to organic

spectroxopy," Macmillan Publishing Company, New York ( 1987)

66. J. Levin, F.B. Bang, "Clottable protein in Limulus: its localization and kinetics of its

coagulation by endotoxin," Thromb. Diath Huemmorrlr , 1 9: 1 86 ( 1968)

67. U. Landegren, "Measurement of cell numben by means of the endogenous enzyme

hexosaminidese. Applications to detection of lymphokines and cell surface antigens", J. of

lmnunul. Methds, 67379-88 ( 1984)

68. A.B. Nathens, O.D, Rodnein, A.P.B. Dack, J.C. Marshall, "Intestinal epithelial cells

downregulote macrophage TNF expression," Surgery, 1 18:343-35 1 (1 995)

69. R.I. Freshney, "Culture of animal cells," 1987, 2nd edition, Alan R. Liss, Inc., New York,

p. 5 8

70. D. H. Cormack, "Introduction to histology," J. B. Lippincott Company, New York (1 984)

71. M. Hubbes, R.M. D'Agarosa, J.W. Callahan, "Human placental beta-galactosidase.

Characterization of the dimer and complex foms of the enzyme," Biochem J. ,285, 827-83 1

(1992)

72. R.D. Meldrum, R.D. Rlwhiirn, L.D. Don, "Metal ion concentrations in retrieved

polyethylene total hip inserts and implications for artifactually high readings in tissue," .J.

H iomed. Muter. Hes., 27, 1 349- 1 35 5 ( 1993)

73. C.M. Rimnac, A.H. Burstein, J.M. Can, R.W. Klein, T.M. Wright, F. Betts, "Chernical and

mechanical degradation of UHMWPE: report of the development of an in vitro test." J. Appl.

Biomur., 5, 1 7-2 1 ( 1 994)

74. E.L. Boynton, J. Sandhu, unpublisheû data

75. Unpublished work from MRC gant d. J.P. Santene. E.L. Boynton, J. W. Callahan,

"Bidegradation of polyethylene Wear particles and its related effect on

monocytesimacrophages." (1 9%)

76. R.B. Johnson, "Monocytes and macrophages, " The New fh&nrl Journui of Medicine,

3 18( 11). 747-752 ( 1988)

77. W.J. Maloney, R.L. Smith, F. Castro, D.J. Schuman, "Fibroblast response to metallic debris

in vitro," J. Rune und Jurnt Surg., 75-A(6). 835-844, 1993

78. J.L. Brash, "Role of plasma protein adsorption in the response of b l d to foreign surfaces"

in C.P. Shanna, M. Szycher, eds., "Blood compatible materials and devices. Perspectives

t owâs the 2 1 st century,"

Partkit type Incu br tion

ptriodr

20 UHMWPE

HDPE

HDPE

HDPE

HDPE

HDPE

HDPE

image uidysis w u not performcd on Expetitnent O

Table 2. Summuy of ce11 and uer % countr

G L U S

COLL+DMSO

HDPE I

Experiment 2

GLASS 1

COLL+DMSO

HDPE l

GLASS b

Are8 96 count

f rtd. mot

DMSO

PP

cd1 count

20

20

20

..

2

2

2

6

COLL+DMSO

HOPE

1 GLASS

Cdl couat

, f atd. cmr

Croup type

1 6

DMSO

lncubitki

tint (b)

2005 f 42.5

2164 f 54.3

1957 f 39.3

1522 f 75.7

1832f 67.4

1817 f 54.5

2022 f 28.1

6

6

24

COLL+DMSO

HDPE r

Eiperiinent 3

G L U S

DMSO

COLLAGEN

COLL+DMSO

HDPE

GLASS

DMSO

1876 f 28.3

r 24

+

Lp=0.0038

' @p=0.8657

6.139 f 0.100

1834 f 57.9

. 1865 f 61.3

1944 f 61.0

24

24

,

24

24

24

24

24

48

48

6.422 f 0.142

7.853 f 0.227

6.566 10.160

, 5.008 f 0.258

5.381 f 0.206

,, 4.944 f 0.141

- 5.299 f 0.103

1995 î 39.1

.p=0.7119

8.722 f 0.138

2288 I43.2

2219 f 74.0

1244 f 35.7

1395 f 41.6

1632 f 40.2

1628 f 25.1

1438 f 45.9

2275 k 44.7

2060 f 59.3

6.643 f 0.25 1

6.114 f 0.258

8.1 12 k 0.282

' ~ 4 . 4 183

, -

*p4I.O8 .

8.873 f 0.218

8.585 f 0.368

5.382 f 0.152

5.417 f 0.162

6.953 f 0.239

6.525 f 0.149

5.653 f 0.185

1 1.91 7 f 0.242

10.347 f 0.327

COLLAGEN 48 2338 f 59.1

COLL+DMSO 48 2410 f 32.8

HDPE 48 1839f 121 1

GLASS 72 2744 f 86.6 r

DMSO 72 2905 f 84.9

COLLAGEN 72 3715 î 65.8

, COLL+DMSO 72 3813 f 81.9

HDPE 72 3336 I 101

1 GLASS 1

5.206 I O . 149 1

DMSO

COLLAGEN

COLL+DMSO

HDPE

Experiment 4

GLASS 1 48 1 2265 f 60.7

COLL+DMSO 48 2184 f 52.9

HDPE 48 2026 f 91.4 I

ExWment 5

GLASS 24 2066 f 84.6 1

COLL+DMSO 24 2086 f 82.4

GLASS 24 . 1277 f 3 1.6 b

, COLL+DMSO 24 , 1169f 22.2

HDPE 24 1089 f 36.1

96

96

96

96

1 HDPE 1 24 1 1859I 81.8

5354 f 42.5

5973 f 55.6

5152f 81.0

3672 f 289

* the mplcs were compared using student's paired t-test

Espcriment 6

G L U S

COLL+DMSO

HDPE 1

G L U S I

, COLL+DMSO

HDPE

24

24

24

48

48

48

TiMe 3. Summuy of TNFu

Croup type

, Experiaent O

GLASS 1

, COUAGEN

UHMWPE

Exgerimen t 1

GLASS

COLL+DMSO

HDPE

Experiment 2

GLASS

DMSO

COLL+DMSO

HDPE

GLASS

DMSO

COLL+DMSO

HDPE

G L U S

DMSO

COLt+DMSO

Incubation

time (b)

n-é

20

20

20

n=3

20

20

20

na3

2

2

2

2

6

6

6

6

24

24

24

GLASS

DMSO

DMSO

COLLAGEN

COU+DMSO

HDPE

G L U S

COLLAGEN

24

24

. , 24

24

48

A 1.337 L O. 144

HDPE

GLASS

DMSO

COLLAGEN

COLL+DMSO

HDPE

GLASS

DMSO

COLL+DMSO

HDPE

246.82 f 27.59

4.402 f 0.34 1

4.7 16 f 0.674

4.437 f 0.5 18

0.508 10.332

COLLAGEN

GLASS

633.11 f 53.66

722.76 f 104.61

784.89 f 95.16

49.34 I 27.88

48

72

72

., 72

72

72

%

96

COLL+DMSO

HDPE

96

GLASS

1.480 f 0.602

0.081 f 0.056

O. 198 f 0,067

0.388 f 0.02 1

0.262 f 0.161

0,633 f 0.04 1

0.000 f 0.000

0.150 k 0.096

COLL+DMSO

HDPE

0.498 f 0.032

165.38 i 68.50

7.12 f 4.92

14.28 f 4.85

22.82 I 1.38

14.39 f 8.85

40.50 f 2.84

0.00 f 0.00

7.87 f 5.04

20.47 f 1.39

*p=û. 1364

8

*p=û.0308

COLL+DMSO

HDPE

GCASS

COLL+DMSO ,

HDPE

Exptrimert 6

GLASS

a the sunples were compared using studentts paircd t-test

24

24

48

48

48

HDPE

G L U S

COLL+DMSO

HDPE

a 4

24

2.590 f 0.531

3.623 f 0.562

0.094 f 0.086

0.315 f 0.063

0.940 f 0.365

24

48

48

48

332.05 f 69.59

559.98 f 92.83

9.03 î 8.33

25.19 f 5.03

88.21 f 34.33

0.400 f 0.163 56.79 f 23.65

1,592 k 0.215

0.384 f 0.024

0.575 I 0.03 1

1.155 f 0.389

252.82 f 42.94

3 1.78 f 3.56

41.10f 3.53

128.49 f 47.21

HDPE 1 24 1 19.462f 1.244 1 2.267f0.174

Croup type

Erperiment 2

GLASS

DMSO

COLL+DMSO

Espcriment 3 ..

GLASS

HDPE 1 48 1 4.210f0.580 1 0.470 f 0.074

I L I @ / a m 96,

pJmVim%

mean f ntd. Enor

0.036 k 0.0 17

0.1 12 f 0.095

0.172 f 0.048

Incubitioi

time (h)

n=3

24

24

24

COLLAGEN

COLL+DMSO

HDPE

GLASS

DMSO

COLLAGEN

L m ~ d m L

mean f std. tmr

0.288 f 0.138

0,978 î 0.828

1 .530 f 0.421

n = 4

24

24

24

24

48

48

48

GLASS

2.020 f 0.720

GLASS

COLL+DMSO

HDPE

0,375 î O. 134

3.290 f 0.460

3.960 f 0.610

48.810 f 4.440

2.1 10 f 0.320

2.190 f 0.710

4.640 f 0.680

24

0.473 * 0.068 0.607 f 0.095

8.634 f 0.835

0.177 i 0.027

0.2 12 f 0.069

0.395 I0 ,059

48

48

48

1.185 f 0.306 0.228 f 0.059

0.243 f 0.092

1 A38 k 0.092

t .622 f 0.460

0.026 f 0.010

O. 177 f 0.013

0.198 f 0.057

HDPE

GLASS

COLL+DMSO

HDPE

Experiment 6 ,,

GLASS

COLL+DMSO

HDPE

the m p l e s were c o m p d using student's paird t-test

24

48

48

48

n 4

24

HDPE

24

24

5.024 f 0.512

0.270 f 0.173

0.332 f 0.232

0.842 f 0.45 1

Of0

48

0.777 f 0.091

0.026 f 0.0 17

0.027 f 0.0 19

0.079 i 0.042

O î O

0.155 f 0.146

7.439 f 0.560

0.020 f 0.019

1.182 î 0.151

0.717 f 0.226 0.080 f 0.028

T h l t 5. Summ~y of a t r ~ ~ U u l i r 8-GAL dam

Croup type Iscubrtior B-c& WAU irea O!!,

mean f rtd. e m r mean * std. e m r

Experiment O n-6

GLASS 20 26.167 I 2.120

COLLAGEN 20 8.667 f 0.2 1 1

UHMWPE 20 9.500 f 0.342

Experiment 1 .. nt3

GLASS 20 29.333 f 2.333 4.568 * 0,377

HDPE

Experiment 2 n-3

DMSO 1 2 1 9.650 f 0.600 1

HDPE

GLASS 6 14.610f 1.150 , 2.757 f 0.224

DMSO 6 _ 13.310f0.080 2.168 f 0.038

COLL+DMSO 6 12.280 f 1,460 1.849 f 0.23 1

HDPE 6 13.070 f 0 . 3 0 2.138 f 0.103

GLASS 24 35,090 f 0,510 4.326 f: O. 163

DMSO 24 35.560 f 0.270 4.077 f 0.072

COU+DMSO 24 31.420 f 1,810 3.541 f 0.222

HDPE 24 34.250 f 0,080 3.990 f 0.171

Esrmiaeat 3 n=û rt 24 1-4 rt U r 4 rt 72

G L U S 24 17.730 * 0.250 2.761 f 0.072

GLASS 1 72

l

I DMSO

, COLLAGEN

COLL+DMSO

HDPE 1

GLUS

DMSO

COLLAGEN

COLL+DMSO

HDPE

DMSO 72

17.980 f 0.150

17.130 f 0.210

24

24

24

24

48

48

48

48

48

2.371 f 0.074

2.609 f 0.100

COLLAGEN

COLL+DMSO

HDPE

GLASS

DMSO

COLLAGEN

COLL+DMSO

HDPE

-

17.090 f 0.260

17.370 f 0.270

25.350 * 1.520

27.770 f 2.360

25.170 f 2.240

23.880 f 1.440

72

72

72

%

96

%

%

%

Esperimcn t 4

GLASS

COLL+DMSO

HDPE

G L U S

COLL+DMSO

HDPE

2.619 f 0.072

3 .O72 f O. 1 1 1

2.127 f 0.135

2.684 f 0.243

2.141 f 0.205

1.935 10.125

n 4

24

24

24

48

48

48

23.970 f 1.630 2.679 f 0.277

G L U S L

COU+DMSO

HDPE 1

GLASS l

, COLL+DMSO

HDPE l

, Espcriment 6

G L U S 1

COLLtDMSO

HDPE

the sampla were cornparcd using studmt's pair4 t-test

GLASS

COLL+DMSO

HDPE

24

. - 24

24

48

48

48

.. 0 4

24

24

24

16.750 f 1.436

14.250 f 0.250

16.250 î 0.947

26.500 f 0.646

23.500 f 0.289

27.750 f 0.479

14.750 f 0.750

15.000 f 0.707

16.250 i 0.947

48

48

48

28.000 f 0.707

24.250 f 0.947

27.500 f 1.323

Table 6. Summuy of extr~ccUulu HEX data

Croup îype

L

Experiment O

GLASS 20 891 f 15 . COLL+DMSO 20 719 f 15

HDPE

Exwriment 2

G L U S

DMSO I

COLL+DMSO

GLASS 1 DMSO

GLASS 20 1295 f 51

Incobrtioi

time (b)

n-é

, COLL+DMSO

HDPE

BE&

nmAûmL

mtrn f ritd. error

20

n=3

2

2

2

6

6

GLASS

DMSO

COLL+DMSO

HDPE

EsPCCJmwt 3

GLASS

781 f 18

4 % f 1

494 f 3

490 f 5

601 f 9

613 f 12

6

6

542 f 54

622 f 6

24

24

24

24

m 4 r t 24

24

1356 f 8

1465 f 25

1465 f 106

1421 f 58

m 4 rt 48

1045 f 27

DMSO

COLLAGEN

COLL+DMSO

HDPE

G L U S

DMSO

COLLAGEN

COLL+DMSO

HDPE

GLASS

DMSO

COLLAGEN

COLL+DMSO

HDPE

GLASS

DMSO

COLLAGEN

COLL+DMSO

HDPE

Expdment 4

G L U S

COLL+DMSO

HDPE

G L A S

COU+DMSO

HOPE

Expdment S

140 f 5.77

152 f 7.91

150 f 4.70

172 f 7.28

215 f 6.99

257f 10.14

i8 i f 11.46

144 f 4.42

219 I 18.96

238 f 13.18

208 f 11.20

158f 11.36

145 f 4.02

172 f 7.41

156 f 9.81

O

*p=0.0222

b

*p=0.0034

b

*p=û.O192

24

24

24

24

48

48

48

96

96

96

96

n 4

24

24

24

48

48

48

r 4

1065 f 30

1001 f 37

980 f 21

975 f 26

2567 f 65

2656 f 63

2126 f 112

2615 I474

2991 f 90

2913 f 51

2924 f 40

96ûf 23

851 f 12

878f 18

1740 f 44

1578 f 31

1586 f 29

48

48

72

72

72

72

72

96

1782 f 36

1957 f 74

2706 f 122

2889 f 125

2693 f 179

2646 f 21

2689 f 9û

2862 f 172

137 f 25.04

123 f 4.61

144 f 3.63

199 * 16.58

184 f 6.88

203 f 6.68

223 f 9.53

189 * 7.40

195 f 7.03

194 f 9.59

b

ap4.0853

I

*p"o. 1280

'

'fl.944 1

G U S

HDPE

HDPE

G L U S

COLL+DMSO

HDPE

Experiment 6

G L U S

COLL+DMSO

GLASS 1 48

24

24

48

48

48

n-4

24

24

the sunples were compued using studcnt's paircd t-test

179 f 10.49 1337 f 27

HDPE

--

1250 f 24

2009f 41

1724 f 43

1732 f 33

965 f 22

911 f 23

48

193 f 11.88

194 f 5.80

138 f 4.63

162 f 5.86

137 f 12.80

116 f 14.37

'p4.0286

'p4.0161

O

1512 f 6 168 f 24.65 .p=0.062 1

Table 7. Summ

G-Q rrpc

y of POE2 d m

Incubrtion

time (b)

na3

DMSO

HDPE 24 14.07 f 0.87 1.639 f 0.123

COLL+DMSO

HDPE

GLASS

DMSO

Experiment 3

GLASS

DMSO

COLLAGEN

COLL+DMSO

mE2~

pgD.1 IL

mean f atd. errot

6

HDPE

GLASS

DMSO

COLLAGEN

COLL+DMSO

HDPE

GLASS

DMSO

COUAGEN

?GE2/arci*!,

p f l l iUarc r%

mmn f rtd, e m r

COLL+DMSO 24 12.8 î 0.4 1.443 f 0.057

1 21.2 f 0.4

3.045 f 0.117

3.500 î O. 162

1.294 f 0.532

1.445 f 0.072

6 1 20.2 0.2

3.453 f 0.086

6 21.4 î 0.4

24

24

r

10.5 f 4.3

12.6 f 0.6

t h runples wete compued using student'r paired t-test

COLL+DMSO

HDPE

GLASS

DMSO

COLLAGEN

COLL+DMSO

HDPE

72

72

%

%

%

96

%

4.2 f 0.53

5.43 f 0.1

17.6f 1.6

19.6 f 0.4

16.4 f 0.4

15.3 f 0.7

16.4 f 0.4

0.23 1 f 0.030

0.347 f 0.01 1

0.962 f: 0.089

1 .O28 f 0.03 1

0.674 f 0.022

0.758 f 0,037

1.1 15 î 0.096

*p=O.OIOS

,

1

*p-1).0740

Erwriment 3

GLASS

n 4

DMSO

a the m p l a werc compared using studcnt's piired t-test

mean f rtd. emr

COLLAGEN

COLL+DMSO

HDPE

GLASS

DMSO

COLLAGEN

COLL+DMSO -

mtin f rtd. error

24

24

24

24

48

48

48

48

7.67 f 0.47 1.420 f 0.097

14.40 f 0.18

19.22 f 0.63

189.83 f 8.58

11.10 f 2.33

10.92 î 2.86

27.71 f 3.91

25.21 f 3.91

2.071 f 0.076

2.946 f 0.118

33.580 f 1.874

0.93 1 f 0.196

1.055 f 0,278

2.357 f 0.343

2.043 k 0.320

Lp<O.OOO1

8

Table 9. Sumrmvy of IL-6 dit

Grorp type Incubation

time (b)

Esperimeat 3 n 4

GLASS 24 1

DMSO 24

COLLAGEN 24

COLL+DMSO ,. 24

HDPE 24

COLLAGEN rn the runpler were compued using studcnt's pwcd t-test