osteonecrosis of the jaw (onj) - t-space - university of toronto
TRANSCRIPT
THE ROLE OF THE INNATE IMMUNE SYSTEM
IN BISPHOSPHONATE-INDUCED
OSTEONECROSIS OF THE JAW
By
Carol Forster
A thesis submitted in conformity with the requirement
for the Degree of Master of Science
Graduate Department of Dentistry
University of Toronto
© Copyright by Carol Forster 2011
THE ROLE OF THE INNATE IMMUNE SYSTEM IN
BISPHOSPHONATE-INDUCED OSTEONECROSIS OF THE
JAW
Carol Forster 2011, Degree of Master of Science, Graduate Department of Dentistry,
University of Toronto
ABSTRACT Bisphosphonate-induced osteonecrosis of the jaw (BPONJ) has been identified as a severe
complication of dental treatment in 1-10% of patients previously treated with intravenous
bisphosphonates. The mechanism by which bisphosphonates induce BPONJ is uncertain. It has
been noted that necrotic bone from BPONJ sites display signs of bacterial infection that suggests
that an immune defect may play a role in the pathophysiology of BPONJ. The purpose of this
thesis examined the effect of a potent bisphosphonate, zoledronate, on the innate immune
system, specifically, neutrophil function, differentiation and survival with in vitro and in vivo
murine models. Zoledronate exposure leads to decreased neutrophil migration, neutrophil
NADPH oxidase activity, circulating neutrophil counts, as well as neutrophil survival, however
does not appear to affect neutrophil differentiation. We present evidence that bisphosphonates
have the potential to depress the immune system in mice and a subset of patients, possibly
contributing to the pathogenesis of BPONJ.
ii
Acknowledgements
Special thanks to Dr. Glogauer, who kindly accepted me as a summer research student many
years ago and then as a Masters student when I had little knowledge and experience in molecular
biology research. This study would not have been possible without his guidance, support and
encouragement.
I add my sincere thanks to my supervisory committee members, Drs. Sean Peel and Morris
Manoloson for their time in regular attendance to the committee meetings and for their advice
and critical discussion of my work.
I would also like to thank everyone in the CIHR Group in Matrix Dynamics. Special thanks to
Chunxiang Sun, and Jan Kuiper for their help with the GST-pull down assay and apoptosis
assays and other technical support in the Glogauer lab.
Finally, I would not have come this far without the love and support from my parents, my sibling
and my friends.
iii.
TABLE OF CONTENTS
ABSTRACT……………………………………………………………….……………………..ii
ACKNOWLEDGEMENTS………………………………........................…………………… iii
TABLE OF CONTENTS……………………………………………........................……….…iv
LIST OF TABLES & FIGURES……………………………………..........................…….....vii
ABBREVIATIONS………………………………………………..........................……......…viii
Chapter 1: INTRODUCTION…………...………………...…........................….1
I. INTRODUCTION…………………………………………………………..........................2
II. BISPHOSPHONATES
A. Chemical Structure…………………………………………………………………….4
B. Mechanism of Action……………………………………………………………….…5
C. Targeting of Bisphosphonates to Bone and Cellular Uptake………………………….6
D. Distribution of bisphosphonates in bone………………………………………………7
E. Uptake and retention of bisphosphonates in bone……………………………………..8
F. Speed of onset of anti-fracture effects…………………………………………………9
G. Offset of Action……………………………………………………………………...10
H. Relating properties of individual bisphosphonates to their clinical effects………….11
I. Clinical uses of bisphosphonates……………………………………………………...12
i. Osteoporosis……………………………………………………………….…..12
ii. Paget’s disease…………………………………………………………….….12
iii. Bone oncology……………………………………………………………..…13
J. Side-effects of bisphosphonates…………………………………………………......14
III. BISPHOSPHONATE-INDUCED OSTEONECROSIS OF THE JAW (BPONJ)
A. Definition and Diagnosis…………………………………………………………….15
B. Staging Criteria………………………………………………………………………17
C. Incidence………………………………………………………………….………….17
i. Randomized control trials..................................................................................18
ii. Retrospective chart reviews………………………………………...…………18
D. Prevention …………………………………………………………………...………20
E. Management …………………………………………………………………...…….22
IV. NEUTROPHILS
A. Bone Marrow Origin of the Neutrophil………..………….……………….................24
B. The Fate of Circulating Neutrophils……………………………...……......................25
i. Leukocyte Rolling……………………………………………….……..............25
iv.
ii. Leukocyte Arrest and Activation……………………………………….…......26
iii. Transendothelial Migration…………………………………………….........27
C The Role of the Cytoskeleton………………………………………………................29
D. Cell Morphological Changes………………………………………………................29
E. Chemotaxis………………………………………………………………...................30
i The role of the Rho family GTPases……………………………………...........30
F. Neutrophil Chemoattractants…………………………………………........................32
G. Phagocytosis ………………………………....…….………………….......................32
H. Neutrophil Respiratory Burst Oxidase ………………………………...….................33
I. Oral Neutrophils as a Diagnostic Test ………………………….....……………..….35
Hypothesis and Objectives of Thesis………………………………..........………..........…36
Chapter 2: MATERIALS AND METHODS……………………………….….38
2.1. Antibodies and reagents................................................................................39
2.2. Animals.........................................................................................................39
2.3. In vivo Zoledronate treatment……..............................................................39
2.4. Isolation of murine bone marrow neutrophils…........................................40
2.5. Measurement of NADPH oxidase activity............................................. …40
2.6. Zigmond chamber chemotaxis.................................................................... 41
2.7. Sodium periodate peritonitis....................................................................... 41
2.8. Circulating neutrophil levels....................................................................... 41
2.9. GST-pulldown assay.................................................................................. 42
2.10. Apoptosis assay...........................................................................................42
2.11. In vitro generation of neutrophils……………………….…..…………..42
2.12. BPONJ patient selection…………………………………………….…..43
2.13. Patient blood neutrophil chemotaxis and isolation…………………..….43
2.14. Patient oral rinse samples……………………………………………….44
2.15. Statistics………………………………………………………………....44
Chapter 3: RESULTS……………......................................................................45
3.1. Effect of Zoledronate on in vitro Neutrophil Function............................. .46
3.2. Effect of Zoledronate on in vivo Neutrophil Function…………….…. 46
3.3. RhoGTPase Assay.......................................................................................47
3.4. Effect of Zoledronate on Circulating Neutrophil counts……………… 47
3.5. Effect of Zoledronate on Neutrophil Differentiation and survival…….47
3.6. Blood neutrophil chemotaxis in BPONJ patients........................................49
3.7. Oral neutrophil recruitment in BPONJ patients……………………..........49
v.
Chapter 4: DISCUSSION……………….............................................................55
4.1 Pathogenesis………………………………………………………………………...……….56
4.1-1. Anti-angiogenesis and Matrix Necrosis………………………………….……….56
4.1-2. Bone Turnover and Microcracks………………………………………….………57
4.1-3. Soft Tissue Toxicity ……………………………………………………….……..59
4.1-4. Infection …………………………………………………………………..………61
4.2 Purpose of Thesis ………………………………………………………………………...….63
4.3 Plausible Mechanism through which ZOL affects the innate immune system ………….…..63
4.4 Oral Innate Immunity ……………………………………………………………………..…64
4.5 The Effect of Zoledronate on Neutrophil Function and Survival………………………..…..65
4.5-1 Zoledronate negatively affects neutrophil function ……………………….……....66
4.5-2 Zoledronate affects neutrophil function through the small RhoGTPase RhoA …...68
4.5-3 Zoledronate affects mature neutrophil survival ……………………………….…..69
4.5-4 Zoledronate and G-CSF ………………………………………………..……….…69
4.6. Neutrophil Function in BPONJ patients ……………………………………………….…...71
4.7 Questions Arising from our Work …………………………………………………………..72
4.8 Study Limitations ……………………………………………………………………………74
4.9 Future Work …………………………………………………………………………………75
Thesis Summary………….......………………………..………...………………77
References…………………………………………………………..................…79
vi.
List of Tables & Figures
Table 1. The chemical structure, MOA, potency and trade names of common BPs…….…….11
Table 2. Proposed clinical descriptions of BPONJ ……………………………………………16
Table 3. Staging criteria for BPONJ …………………………………………………………..17
Figure 1-A. Effect of Zoledronate on in vitro neutrophil chemotaxis..........................................50
Figure 1-B. Effect of Zoledronate on in vitro NADPH oxidase activity......................................50
Figure 2-A. Effect of in vivo Zoledronate treatment on in vitro chemotaxis..............................50
Figure 2-B. Effect of in vivo Zoledronate treatment on in vivo peritoneal recruitment............ 50
Figure 3. Possible mechanisms through which ZOL affects neutrophil functions.............. 51
Figure 4. Effect of Zoledronate on circulating neutrophil levels........................................... 51
Figure 5. Effect of Zoledronate on differentiation and apoptosis of mature neutrophils….. 52
Figure 5-A. Effect of Zoledronate on in vitro neutrophil differentiation from bone
marrow stem cells …………...……………………………….………………… . 52
Figure 5-B. Quantification of neutrophil cell death following ZOL exposure……………...... .52
Figure 5-C. ZOL affects mature neutrophil survival during the process of differentiation……52
Figure 5-D. Effect of ZOL on neutrophil apoptosis in the absence of G-CSF………………….52
Figure 5-E. Effect of ZOL on neutrophil apoptosis in the presence of G-CSF…………………52
Figure 6-A. Neutrophil Chemotaxis is perturbed in BPONJ patients….......................................54
Figure 6-B. Oral neutrophil counts in BPONJ patients vs. healthy control patients……...........54
vii.
Abbreviations
AIDS Acquired immune deficiency syndrome
AAOMS American Academy of Oral and Maxillofacial Surgery
ASBMR American Society for Bone Mineral Research
BP Bisphosphonate
BPONJ Bisphosphonate-(associated/induced/related) osteonecrosis of the jaw
CTX Cross-linked C-telopeptide
F-actin Filamentous actin
FPP farnesyl diphosphate
fMLP N-Formyl-methionyl-leucyl-phenylalanine
G-actin Globular actin
GAP GTPase Activating Protein
GDI Guanine nucleotide dissociation inhibitor
GEF Guanine nucleotide exchange factor
GST Glutathione-S-transferase
GGPP geranylgeranyl diphosphate
HOCl hypochlorous acid
IV Intravenous
ICAT Isotope-Coded Affinity Tags
LPS lipopolysaccharide
MMP8 Matrix metalloprotein-8
MPO Myeloperoxidase
NADPH Nicotinamide Adenosine Dinucleotide Phosphate
N-BP Nitrogenous bisphosphonate
NonN-BP Non-nitrogenous bisphosphonate
NTX Cross-linked N-telopeptide
ONJ Osteonecrosis of the jaw
ORN Osteoradionecrosis
PMN Polymorphonuclear leukocyte
RCT Randomized control trial
ZOL Zoledronate
viii
1
CHAPTER 1
INTRODUCTION
2
I. INTRODUCTION
Bisphosphonates have become the primary treatment for the prevention of osteoporosis
and are used to treat hypercalcemia of malignancy and to reduce skeletal-related
symptoms and events in patients with multiple myeloma and metastatic bone lesions of
solid tumor cancers such as breast, prostate and lung cancer [1],[2]. Bisphosphonates are
potent inhibitors of osteoclast-mediated bone resorption. An important characteristic of
these drugs is their highly selective localization and retention in bone compared to other
tissues [3]. This quality makes them ideal candidates for treatment of bone diseases.
Bisphosphonates work by reducing bone turnover, increasing bone mass, and decreasing
the risk of fracture.
In recent years it has been noted that a small subset of patients (1-10%) treated with
intravenous bisphosphonates are at an increased risk of developing osteonecrosis of the
jaw (BPONJ) [4, 5]. The publicity surrounding this complication and the lack of
information about its underlying pathogenesis has led to apprehension amongst patients
and dental clinicians when treating patients with a history of bisphosphonate therapy [6].
Critical information that will help alleviate these concerns and improve oral treatment
approaches in this patient population is required.
The mechanism by which BPs induce BPONJ is uncertain. Several possible theories have
been cited in literature including: reduced bone turnover, ischemia, bone toxicity, soft
tissue toxicity and infection.
3
In light of the more consistent observation that bacterial colonization is a common, if not
universal finding in BPONJ, we sought to explore the possibility that BPs may impart an
irreversible effect on the immune system of the host. There have been reports that in
some patients, bisphosphonates can cause neutropenia[7], inhibit neutrophil enzymes that
impact on wound healing such as MMP8 [8] and decreased generation of reactive oxygen
species formation [9, 10] . This is not surprising in view of the mechanism of action of
BPs which target small Rho GTPases that are signaling proteins integral to neutrophil
differentiation and function. It is important to note that the BP-related neutrophil defects
indicate that this side effect does not have any severe clinical effects such as increased
susceptibility to infection [11]. Clearly other aspects of the immune system are able to
compensate for the neutrophil effects systemically. However it is possible that in a small
subset of these patients, prolonged BP use may effect neutrophil differentiation and
function which could manifest in poor oral innate immunity, poor oral wound healing and
susceptibility to BPONJ
Being able to identify and understand the possible mechanisms through which
bisphosphonates affect normal bone/wound healing in the jaw will help identify patients
at risk of developing BPONJ and improve patient management. The goal of this thesis is
to look into the possible role that the immune system plays in the pathogenesis of this
rare condition.
4
II. Bisphosphonates
A. Chemical Structure
Bisphosphonates (BPs) are pyrophosphate analogues, meaning that the central oxygen
atom in the molecule is replaced by a carbon atom, resulting in a P-C-P bond. The
existence of this bond not only enables the binding of the bisphosphonate to
hydroxyapatite, the naturally occurring mineral in bone, but also renders the molecule
highly resistant to hydrolysis under acidic conditions or by pyrophosphatases[12]. As a
result, BPs are not converted into metabolites in the body and are excreted unaltered. It
has been suggested that the two phosphate groups are required for binding to the bone
mineral, where varying one or both phosphonate groups can dramatically reduce the
affinity of the BP for bone mineral[13]. In the bisphosphonate molecule, two side chains
are attached to the central carbon atom (R1-the short side chain and R2-the long side
chain) which determine the potency of the particular bisphosphonate[12]. R1 substituents
such as hydroxyl or amino enhance chemisorption to mineral[14], while varying the R2
substituent‟s results in differences in anti-resorptive potency of several orders of
magnitude. Risedronate and zoledronate have been shown to be two of the most potent
anti-resorptive BPs in several animal models[15], due to the nitrogen atom within the
heterocyclic ring side group at R2. The increased anti-resorptive potency observed with
the different R2 groups is linked to its ability to affect the biochemical activity e.g.,
inhibition of the farnesyl diphosphate (FPP) enzyme[16]. The presence of nitrogen in the
R2 chain, as is the case in the second-generation bisphosphonates, increases the potency
of the drug by allowing the formation of a tridentate conformation that is able to bind
Ca2+
more effectively[17, 18].
5
B. Mechanisms of Action
(i) Non-nitrogenous Bisphosphonates
Two classes of bisphosphonates exist depending on their chemical structure and
mechanism of function. As the name suggests, the non-nitrogen bisphosphonates (e.g.
clodronate, tiludronate and etidronate) do not have a nitrogen moiety within their side
chains, leading to a relatively lower potency drug. The non-nitrogenous BPs are
metabolized in the cell to generate cytotoxic analogs of ATP[19, 20]. These ATP analogs
form a non-functional molecule that competes with ATP in the cellular energy
metabolism and as a result, interferes with mitochondrial function[21]. In essence, the
induction of osteoclast apoptosis following the accumulation of such metabolites, appears
to be the major mode of action of these BPs in inhibiting bone resorption[3].
(ii) Nitrogenous Bisphosphonates
In contrast, the highly potent nitrogenous bisphosphonates (N-BPs) (e.g., pamidronate,
alendronate, ibandronate, risedronate and zoledronate) are not metabolized by osteoclasts
but instead bind to and inhibit farnesyl diphosphate (FPP) and geranylgeranyl
disphosphate (GGPP) [22] in the HMG-CoA reductase pathway (also known as the
mevalonate pathway). Inhibition of the HMG-CoA-reductase pathway at the level of
FPP and GGPP prevents the formation of two metabolites; farnesol and geranylgernaiol.
This ultimately disrupts the prenylation and membrane targeting of members of the Rho
family of small GTPases such as Ras, Rho, Rac, Rab and Cdc42 [23, 24]. These small
GTPases are essential for numerous immune and osteoclast cell functions [25]. As a
result, N-BPs may interfere with various intracellular processes such as cytoskeleton
6
reorganization, vesicle transport, exocytosis and ultimately, apoptosis in the targeted cells
[26, 27].
C. Targeting of Bisphosphonates to Bone and Cellular Uptake
Nitrogenous BPs
The pronounced selectivity of bisphosphonates for bone rather than other tissues is the
basis for their value in clinical practice. The bone targeting property of the
bisphosphonates comes from the fact that they have a high avidity for Ca2+
ions[18].
Once in the circulation, the bisphosphonates selectively absorb onto exposed bone
surfaces in active areas of bone remodeling and concentrate there[28]. During the bone
resorptive process, the space beneath the osteoclast, known as the ruffled border is
acidified by the action of vacuolar-type proton pumps. The acidic pH of this environment
causes dissolution of the hydroxyapatite bone mineral and as a result leads to the release
of free, non-mineral bound bisphosphonate. Since the osteoclasts are here in the highest
proportion, they are therefore exposed to the highest concentration of drug[28, 29].
Because the osteoclasts are highly endocytic during the bone resorption process,
bisphosphonates located in the resorption lacuna in bone are internalized in the
osteoclast. The N-BPs carry a highly negative charge and are therefore considered
membrane impermeable, and the manner in which bisphosphonates cross the vacuolar
membrane to enter the cytoplasm is not well established[30, 31]. In a previous study,
Felix et al. used radiolabeled bisphosphonates to study uptake by calvarial cells in vitro
and confirmed that the bisphosphonates could enter the cytoplasm as well as
mitochondria and other organelles[32]. Studies with slime mold amoebae, where the
growth is inhibited by bisphosphonates[33], have also demonstrated that cellular uptake
7
occurs by fluid-phase endocytosis. The endocytosis of the BPs by the osteoclasts leads to
a disruption of the cytoskeleton, loss of ruffled border, inhibition of lysosomal enzymes,
and protein tyrosine phosphatases[34]. In addition to their primary action on the
osteoclast activity and life span, bisphosphonates also reduce the number of active
osteoclasts by inhibiting their recruitment and finally they inhibit the osteoclast-
stimulating activity of osteoblasts[34].
D. Distribution of Bisphosphonates in bone
At the microscopic level, BPs initially localize to areas of active bone turnover[28]. This
pattern of localization has the advantage of targeting BPs specifically to disease sites in
Paget‟s disease and metastatic cancer in bone, and possibly even in osteoporosis. A
majority of the BPs that attach to the bone surface become quickly buried in the bone
where they can be retained for long periods of time.
Small amount of BPs can be measured over several weeks or months in the urine
following cessation of the drug due to the release from the skeleton[35, 36]. This suggests
that BPs must be present in the circulation and ready for re-absorption into bone for
prolonged periods, and may be responsible for their ongoing pharmacological actions.
In theory, the release of BPs from bone can occur through at least two mechanisms; either
chemical desorption and/or ostoclastic resorption. The binding of BPs to hydroxyapatite
surfaces is a reversible physiochemical process[16, 37], such that BP absorption will
occur when extracellular concentrations are high and desorption will occur when
extracellular levels are low[16, 37].
8
Bisphosphonates will also be released from bone during osteoclastic resorption. It is
unknown the relative contribution of desorption versus cell-mediated resorption. It is
assumed however that physico-chemical desorption to be more important immediately
after dosing, before the drug becomes buried under bone. However in the longer term,
after the cessation of dosing, release of BPs may occur readily as a result of liberation
during bone resorption[38].
E. Uptake and retention of BPs in bone
The retention of BPs in the skeleton has been studied in both animal and human models.
The use of radiolabeled BPs has been the main method used to measure the uptake and
retention of BPs in different parts of the skeleton. From these studies, approximately one
to two thirds of the administered BP dose becomes incorporated into the skeletons,
whereas the remainder is predominantly excreted into the urine within the first few hours
following administration. One of the difficulties in these retention studies has been the
fact that retention is affected by the disease state of the individual, renal function and
retention is also dependent on the specific BP used[39].
The concept of half-lives of retention can be quite misleading based on the fact that the
half-lives of the drugs are not equivalent to the half-lives of their effects. Much of the
BPs kept for long periods of time within the skeleton is likely to be rapidly “buried”
within the mineral phase rendering it pharmacologically inactive[40]. Thus the release of
BPs from bone is highly dependent on the remodeling and resorption. It is reasonable to
assume that the figures quoted in the literature that have previously referred to half-lives
of 10 years or greater, compared with days or months, do not offer valid comparisons
9
among BPs. These figures also fail to give a true indication of the persistence of their
pharmacological effects[17].
F. Speed of onset of anti-fracture effects
From a clinical standpoint, it is advantageous to know the speed with which various BPs
achieve fracture reduction. There is evidence from individual randomized control trials
(RCTs), post-hoc analysis and observational studies to suggest that these effects occur to
varying degrees with the different BPs. However these results are not directly comparable
between the different BPs since there have been few head to head studies. One of these
studies performed was a 15 month, randomized study that compared intravenous
pamidronate to zoledronate in 90 subjects with active Paget‟s disease of bone [41].
Paget's disease is a disorder that involves abnormal bone destruction and regrowth, which
is typically monitored with blood levels of alkaline phosphatase (ALP), where elevated
levels suggest increased bone descruction. The primary efficacy endpoint used in this
study was therapeutic response at 6 months, which was defined as normalization of
alkaline phosphatase (ALP) or a reduction of at least 75% in total ALP excess. At 6
months, 97% of patients receiving zoledronate had a therapeutic response compared with
45% of patients receiving pamidronate. In the zoledronate group, normalization of ALP
was achieved in 93% of patients and in 35% of patients in the pamidronate group. ALP
normalization was maintained in 79% and 65% of zoledronate-treated patients after 12
and 15 months, respectively; loss of therapeutic response was observed in 2 of 30 (6%) at
12 and 15 months[41].
10
When looking at radiographic vertebral fractures, several RCTs with aledronate[42] and
ibandronate[43] show a significant reduction following 24 months. While risedronate and
zoledronate show a significant reduction following only 12 months[11, 44].
G. Offset of Action
Information about the speed of reversal of effect following discontinuation of the BP is
limiting as no head-to-head studies of the different BPs have be undertaken. Bone
turnover markers, such as type-1 collagen crosslinked N-telopeptide (NTX) and type-1
collagen cross-linked C-telopeptide (CTX), can be used as an indirect measure of bone
turnover. Following 5 years of treatment with aledronate, bone turnover markers
appeared to be at least partially suppressed for up to 5 years after treatment was
discontinued[45, 46]. Similarly, bone turnover markers remained reduced for 1 year after
a single IV dose of zoledronate at 4 or 5-mg and remained significantly reduced at the 2
year follow up [11, 47]. While a variety of BPs have been shown to affect the duration of
suppression of bone turnover, data from animal and human studies suggest that the dose
of the BP also plays a role. In general, the higher the dose or the higher the bone affinity
of the drug, the longer bone turnover remains reduced[47, 48].
11
H. Relating properties of individual BPs to their clinical effects
Due to the diverse structures of the BPs, each one can display unique biochemical
potency on FPP and potential differences in mineral targeting and release (Table 1). The
rank order of the most critical properties is as follows[17]:
Mineral affinity: clodronate < etidronate < risedronate < ibandronate < alendronate <
pamidronate < zoledronate
FPP enzyme inhibition: etidronate < clondronate (extremely weak inhibitors) <<<<<
pamidronate < alendronate < ibandronate < risedronate < zoledronate
Table 1. The chemical structure, mechanism of action, potency and trade
names of common bisphosphonates
12
I. Clinical Uses of Bisphosphonates
Since it was first recognized that BPs are selectively absorbed by the skeleton, oral and
intravenous forms of BPs have become widely used for the treatment of osteoporosis,
Paget‟s disease, and in oncology.
(i) Osteoporosis
Oral and in a fewer cases, intravenous bisphosphonates, are the preferred
pharmacological agents in the treatment of osteoporosis. Bisphosphonates are effective
in increasing bone mineral density and lowering the risk of fractures at the spine by 30-
70%, and also to varying degrees at other skeletal sites in postmenopausal women[42,
49, 50]. Etidronate was the first BP approved for osteoporosis[51, 52], followed by
alendronate, risedronate[44], and ibandronate[43], and most recently zoledronate[11].
(ii) Paget’s Disease
Many different BPs have been prescribed to treat Paget‟s disease. The order of potency
of the different BPs used to suppress the rapid bone turnover observed in Paget‟s disease
appears to be similar to their order of anti-resorptive potency observed in an animal
model: etidronate< clodronate < pamidronate < alendronate < risedronate <
zoledronate[53, 54]. Recently, zoledronate and risendronate, newer and more potent BPs,
have been observed to produce an even greater suppression in disease activity. In the
most recent study[48], a single 5mg infusion of zoledronate was found to produce a
greater and longer lasting suppression of excess bone turnover than oral risedronate
given at 30 mg daily over 2 months.
13
(iii) Bone Oncology
Bisphosphonates are quite effective in the treatment of bone complications associated
with malignancy, including hypercalcemia and/or increased bone destruction and now
are considered the standard of care when treating these patients. The concept behind
using these osteoclastic inhibitors in the therapeutic armamentarium for bone metastases
is explained by the pathophysiology of tumor-induced osteolysis. Once cancer cells
colonize the bone marrow, they are attracted to the surface of the bone by products from
bone resorption. The cancer cells have the ability to increase osteoclast differentiation of
hematopoietic stem cells and over time destroy the bone via direct osteoclast
stimulation[55, 56]. As well, in the case of cancers of the breast, the propensity to
metastasize and proliferate is best explained by the “seed and soil” theory [57]. The
“seed” (the breast cancer cell), secrete osteolytic factors, such as parathyroid hormone-
related peptide (PTHrP), which tend to increase the development of metastases in the
skeleton, which is thought of as the “soil” since it is rich in cytokines and growth factors
that are known to stimulate cancer cell growth. These cytokines are thought to induce
osteoclast differentiation from hematopoietic stem cell as well as activate mature
osteoclasts in the bone. The overall increase in osteoclast number and activity leads to
local foci of osteolysis, and enhanced released of growth factors that further stimulate
cancer cell proliferation. Thus a viscous cycle is created where the bone resorption-
induced release of growth factors from the bone matrix tends to encourage growth of
breast cancer cells and the production of osteolytic factors.
Zoledronate, clodronate, ibandronate, and pamidronate are all approved for clinical use
based on placebo-controlled trails, though not all are available in all countries.
14
J. Side Effects of Bisphosphonates
Adverse effects are similar with oral bisphosphonates and include
upper gastrointestinal irritation, which may be manifested with heartburn,
esophagitis, abdominal pain, or diarrhea. Rarer adverse effects include bone, joint, and
muscle pain that subsides after the drug is discontinued. The i.v. preparation is associated
with osteonecrosis of the jaw, ocular inflammation manifesting as scleritis, uveitis, or
conjunctivitis, fever, leukopenia and nephritic syndrome[58, 59].
15
III. Bisphosphonate-induced osteonecrosis of the Jaw
(BPONJ)
A. Definition and Diagnosis
Bisphosphonate-(induced/associated/related) osteonecrosis of the jaw (BPONJ) was first
described in the dental literature in 2003 [60]. There is no one agreed upon definition of
BPONJ however several professional groups have attempted to develop a more uniform
definition of BPONJ (Table 2). The diagnosis of BPONJ is primarily based on clinical
findings. The clinical findings that appears to be central to each associations definition of
BPONJ is a patient who has had current or previous treatment with a bisphosphonate, and
has exposed, necrotic bone in the maxillofacial region that has persisted for >8weeks; and
has no history of radiation therapy in the jaws[61].
16
Table 2. Proposed clinical descriptions of osteonecrosis of the jaw (ONJ) American Society for
Bone and Mineral
Research (ASBMR)
Task Force (2007)
BPONJ is defined as an area of exposed bone in the maxillofacial region that does not heal
within 8 wk after identification by a healthcare provider, in a patient who is receiving of has
been exposed to a bisphosphonate and has not had radiation therapy to the craniofacial region.
Additional signs and symptoms may include pain, swelling, paresthesia, suppuration, soft
tissue, ulceration, intra-or extraoral sinus tracts, loosening of teeth, and radiographic
variability. However, these are neither individually nor collectively sufficient for a diagnosis
in the absence of exposed bone as described above.
American Academy of
Oral and Maxillofacial
Surgeons (AAOMS)
(2006)
Patients may be considered to have bisphosphonate-related ONJ if all of the following 3
characteristics are present:
1. Current or previous treatment with a bisphosphonate
2. Exposed bone in the maxillofacial region that has persisted for >8wk
3. No history of radiation therapy to the jaws
American College of
Rheumatology (ACR)
(2006)
ONJ typically appears as an intraoral lesion with areas of exposed yellow-white hard bone
with smooth or ragged borders, sometimes associated with extraoral or intraoral sinus tracts.
Painful ulcers may be present in the soft tissues adjacent to the ragged bony margins of the
lesion. Dental x-rays may be unremarkable in the early cases, but advanced cases demonstrate
poorly defined areas of „moth-eaten‟ radiolucencies, with or without radio-opaque bone
sequestra. Pathologic jaw fractures have occurred in some cases. In its most severe form, ONJ
may cause loss of a significant part of the mandible or maxilla.
American Association
of Endodontics (AAE)
(2006)
Patients presenting with bisphosphonate-associated ONJ typically present with at least some
of the following signs and symptoms:
1. An irregular mucosal ulceration with exposed bone in the mandible or maxilla
2. Pain or swelling in the affected jaw
3. Infection, possibly with purulence
4. Altered sensation (e.g., numbness or heavy sensation)
American Dental
Association (ADA)
(2006)
The typical clinical presentation of BON includes pain, soft-tissue swelling and infection,
loosening of teeth, drainage, and exposed bone. Symptoms may occur spontaneously in the
bone or, more commonly, at the site of a previous tooth extraction. In some cases, patients
seek dental care complaining of pain that may mimic a dental problem. Infection may or may
not be present. The “dental” problem does not respond to routine dental therapy, and there is
no clinically visible osteonecrosis. However, BON also may remain asymptomatic for weeks
or months and may become evident only after the finding of exposed bone in the jaw during a
routine examination. In some cases, the symptoms of BON can mimic dental or periodontal
disease. Routine dental and periodontal treatment will not resolve the symptoms. In this case,
if the patient is receiving bisphosphonate therapy, BON must be considered as a possible
diagnosis, even in the absence of exposed bone. If BON is suspected, dentists are encouraged
to contact the FDA‟s MedWatch program.
Multidisciplinary
expert panel
recommendations
(2006)
ONJ may remain asymptomatic for many weeks or months and is usually identified by its
unique clinical presentation of exposed bone in the oral cavity. These lesions typically
become symptomatic when sites become secondarily infected or it there is trauma to adjacent
and/or opposing healthy soft tissue from irregular surfaces of the exposed bone. Signs and
symptoms of ONJ include localized pain, soft-tissue swelling and inflammation, loosening of
previously stable teeth, drainage, and exposed bone. These symptoms most commonly occur
at the site of previous tooth extraction or other dental surgical interventions but may occur
spontaneously. Some patients may present with atypical complaints such as “numbness”, the
feeling of a “heavy jaw”, and various dysesthesias. Objective signs Adapted from American Association of Endodontics[62], J Am Dent Assoc[63], American Academy of Oral and
Maxillofacial Surgeons[64], J Bone Miner Res[65], Aust Fam Phys[66], American College of Rheymatology[67], and J
Oncol Pract[68]
17
B. Staging Criteria
The model for cancer tumor staging has been classically used as the staging criteria for
BPONJ (Table 3.). The American Academy of Oral and Maxillofacial Surgery (AAOMS)
has been the dominant figure to date in the development of a more comprehensive staging
and grading system[69]. In order to better define the severity and assess the treatment
responses, this new system is not solely based on physical examinations and symptoms
but also incorporates the imaging results[61].
Table 3. Staging Criteria of BPONJ
Stage Description
At risk Patients that have been treated with oral or intravenous BPs that show
no apparent exposed/necrotic bone
Stage1 Exposed/necrotic bone in patients who are asymptomatic and have no
evidence of infection
Stage 2 Exposed/necrotic bone associated with infection with pain and
erythema in the region of exposed bone with or without purulence
Stage 3 Exposed/necrotic bone in patients with infection, pain, and ≥ 1 of the
following: pathologic fracture, extraoral fistula, or osteolysis
extending to the inferior border Adapted from American Academy of Oral Maxillofacial Surgeons[64]
C. Incidence Many of the estimates on the prevalence and incidence of BPONJ are weak, arising
primarily from anecdotal reports and single institutions‟ case reports and series. These
data are hindered by inconsistent definitions, as well as incomplete reporting. The risk of
BPONJ in patients receiving high potency BPs has been estimated to be about 1-10 per
100 patients depending on the duration of treatment.
18
(i) Randomized Clinical Trials
In the United States, RCTs of clinical trials of bisphosphonates marketed for the
treatment of osteoporosis reported no cases of BPONJ, out of >17, 000 patient exposed to
aledronate, > 44, 000 patient-years of exposure to risedronate, and >12, 000 patients
exposed to ibandronate[61].
(ii) Retrospective Chart Reviews
A large systemic chart review of 4,019 caner patients receiving IV bisphosphonates
revealed 29 cases of ONJ, giving an overall prevalence of 0.72%. The criteria to be
considered ONJ used in this study was exposed non-healing bone with or without pain
≥ 3months duration. Of the 29 cases, 13 patients had multiple myeloma (13 of 548;
2.4%) and 16 had breast cancer (1.2%)[70].
A similar retrospective study was performed by Murad and colleagues[71] with 1,951
patients who had received oral or IV bisphosphonates or had a listed diagnosis suggestive
of ONJ. Two patients, both with multiple myeloma were identified with ONJ, who had
initially been treated with pamidronate for several years before being treated with
zoledronate. They identified dental extractions as the precipitating event in both cases.
A case control study performed by Bamias and colleagues[72] found that the incidence
ONJ increased with time exposed to intravenous BPs from 1.5% among cancer patients
treated for 4-12 months to 7.7% for treatment for 37-48 months. BPONJ was most
prevalent in the mandible (65.3%) than the maxilla (29.2%) and combination of maxilla
and mandible (4.8%).
King et al[73] reviewed 44 published case reports and case series describing 481 patients
with BPONJ. They concluded that BPONJ occurs more frequently in patients receiving
19
intravenous bisphosphonates (94.2%) than in patients taking oral bisphosphonates
(5.8%). With the intravenous patients with BPONJ, 43% were taking Zoledronic acid,
29.1% were taking Pamidronate, with 26.7% taking a combination of Zoledronic acid and
Pamidronate with the remainder taking other combinations. In those patients who
developed BPONJ following oral BP exposure, 85.7% took Aledronate, while 10.7%
took Risedronate.
Cancer patients had a higher incidence of BPONJ (93.8%) than non-cancer patients.
Among cancer patients, multiple myeloma was the most common (52.3%), followed by
breast cancer (33%), other malignancies (14.6%). Among the non-cancer patients,
osteoporosis was the most common (4.8%), followed by Paget‟s disease (0.8%) then
osteopenia (0.6%)[73]. The most common inciting event of ONJ was investigated and
tooth extraction or other surgical or invasive dental procedure was reported as the most
common (68.8%), followed by spontaneous occurrences (20.7%), and (7.6%) had
periodontal disease, (0.4%) had oral ulcers, with (0.2%) having prior ONJ, maxillofacial
trauma, tooth abscess and new oral prosthesis[73].
Abu-Id et al. [74] recently reviewed 7500 patients from seven case series and found ONJ
to occur in 2-11% of multiple myeloma patients, 1-7% of breast cancer patients and 6-
15% of those with prostate cancer and suggest an overall prevalence of 5%. These results
suggest that these three malignancies do not differ in the risk of ONJ.
Hoff et al.[75] also reviewed charts of 4000 cancer patients treated with bisphosphonates
at MD Anderson Cancer Center and found that half of the patients had either breast
cancer or multiple myeloma, of which 1.2% and 2.8%, respectively developed BPONJ.
20
Of the other half suffering from other malignancies, only two cases of BPONJ were
observed, giving a prevalence of 0.09%.
Recently a systematic review by Khan et al.[76] showed that prospective data evaluating
the incidence of BPONJ are very limited. In cancer patients receiving a high-dose
intravenous BP, BPONJ is dependent on the dose and duration of therapy, where 36
months of exposure has an estimated incidence of 1-12%. BPONJ was found to be quite
rare in osteoporosis patients, with an estimated incidence of <1 case per 100, 000 person-
years of exposure. They found that there was insufficient evidence to confirm a causal
link between low-dose BP therapy in the osteoporosis patient population and BPONJ,
where high-dose BP therapy, primarily in the oncology patient population, is associated
with BPONJ.
Prevention
Due to the absence of any randomized controlled trials in the area of prevention and
treatment of BPONJ, the recommendations regarding the prevention of BPONJ are based
on the available literature. Currently a number of institutions have developed
recommendations for the prevention of bisphosphonate-induced osteonecrosis of the jaw.
Current guidelines recommend that prior to starting BP therapy, a comprehensive dental
exam take place that allows optimization of dental health including tooth cleaning, tooth
extractions and any invasive surgical procedures. If the patient requires invasive surgical
procedures to be completed, it is recommended they wait until the wounds have healed,
generally 2-3 weeks after tooth extraction in the same manner as for patients who receive
radiotherapy to the head and neck region[6, 68, 77, 78].
21
Once bisphosphonate therapy has begun (oral or intravenous), patients should avoid any
invasive dental procedures if possible. If the patient requires oral surgical intervention
while on oral bisphosphonates, it is recommended that they use chlorhexidine 0.12%
mouth rinse for 2 months after surgery[6, 77]. The American academy of Oral and
Maxillofacial Surgeons suggests that discontinuing oral bisphosphonates for 3 months
before and 3 months after invasive procedures (a “drug holiday”) may decrease the risk
of developing ONJ[64]. The idea of a drug holiday is supported by a study which uses a
breakdown product of bone resportion known as C-terminal telopeptide (CTX), as a
surrogate marker of bone turnover[79]. This telopeptide fragment is cleaved from the
main crosslink chains of collagen by the osteoclast during bone resorption. Its level in the
blood is therefore proportional to the amount of osteoclastic resorption occurring at the
time the blood is drawn. The authors suggest that CTX values less than 100 pg/mL
represent a high risk of BPONJ, CTX values between 100 pg/mL and 150 represent a
moderate risk, and CTX values above 150 pg/mL represent a minimal risk. Of clinical
importance, this study showed evidence that a 6-month drug holiday from oral
bisphosphonates had direct and significant improvements in the CTX values in every
patient. This improvement correlated to either spontaneous resolution of the exposed
bone, a significant improvement in the amount of exposed bone, or an uncomplicated
healing response after surgery[79]. The results of this study are controversial to many
who criticize that the lack of a control group render the results questionable, ie. patients
taking BP that had not developed BPONJ were not included.
22
It is important to keep in mind that many of these recommendations are based on
anecdotal evidence, not data from clinical trials, and physicians must consider the
potential risks of discontinuing bisphosphonate therapy.
During intravenous bisphosphonate therapy, the oral cavity must be frequently monitored
for any signs of change. The optimal frequency for dental examinations for a patient
treated with intravenous BPs without signs of osteonecrosis is between two to four times
per year taking into account the dental hygiene of the patient[6, 77]. Migliorati et al
suggest that a maxillofacial CT-scan in the follow-up of the malignant disease may be
beneficial to provide additional information with regard to early development of
subclinical ONJ lesions. However, the number of exams are not well defined[80].
E. Management
In the absence of a clear case definition of BPONJ, the development of guidelines to
manage the condition is a considerable challenge. Several groups have developed
guideline for recommendations. For example, the AAOMS Position Paper states that the
management of BPONJ primarily depends on the staging categories of the disease[77],
where the ASBMR task force recommendations provide general guidelines regarding
management of infection, surgical management, and discontinuation of bisphosphonate
therapy[65]. In a majority of the available guidelines[77],[65, 68, 81], pain control,
treatment of infection and prevention of disease progression are the main goals of therapy
where no one therapeutic modality has proven efficacy.
23
The treatment of BPONJ should initially include the treatment of infection. Once the
infection has been managed, the clinician should then focus on the treatment of necrotic
bone.
Treatment of an isolated infection consists first of antimicrobial testing to identify the
microbes specific to the environment then the appropriate antibiotics (possibly
clindamycin or amoxicillin in combination with metronidazole)[82]. It is also
recommended to combine systemic antibiotics with a local antiseptic treatment such as
chlorhexidine in combination with analgesics if necessary. If the infection has spread to
the adjacent soft tissues, then drainage of the abscess must be completed[83].
As a majority of the patients who have BPONJ were initially being treated for an
underlying malignant disease, one must take into account the life expectancy of patients
when considering how to properly manage the condition. In patients with a short-life
expectancy, supportive cares are recommended where is it suitable to leave the
spontaneously evolved disease. In individuals with longer-life expectancies, a curative
attitude may be more effective with the removal of exposed and surrounding bone[77,
83]. Biological factors such as fibrin or autologous platlet-rich plasma to cover the
osseous surface may provide some benefit in wound healing and bone regeneration[84,
85]. Primary closure of the surgical site with tension-free sutures is also advisable to
isolate the surgical site from the diseased environment[84].
In the posterior maxilla, most cases of BPONJ also present with an underlying sinusitis
due to a communication between the oral environment and the maxillary sinus. Before
considering closure of this communication, it is first advisable to resolve the sinusitis
with antibiotics[83].
24
IV. NEUTROPHILS
1. THE LIFE OF THE NEUTROPHIL
A. Bone Marrow Origin of the Neutrophil
The bone marrow is the site of origin for neutrophils. Neutrophils originate from
committed progenitor cells, which arise from pluripotential hematopoietic stem cells
differentiating in response to specific growth factors. The hematopoietic stem cell upon
replication, generates a new stem cell along with a committed progenitor[86].
Neutrophils delivered to the circulation are mature, and are around 12-15μm in diameter.
They have a characteristic segmented nucleus with abundant cytoplasmic granules
(commonly referred to as a polymorphonuclear neutrophilic granulocyte), while its
immature myelocytic progenitor, known as the „band neutrophil‟, possess a band-shaped
nucleus with few cytoplasmic granules. Much of the machinery and molecules needed for
neutrophil responses are prepackaged into the cells‟ cytoplasmic granules or plasma
membrane, and thereby is available for cellular functions when needed when the cells are
initially isolated from blood[86].
For the purposes of having only functionally capable cells present at the front line of an
infection, only functional mature neutrophils are released from the bone marrow into the
blood stream. In healthy individuals, the levels of blood neutrophils remains fairly
constant at approximately 109cells/L blood[87]. The lifespan of the neutrophil in the
circulation is brief, displaying a half-life of approximately 6 hours. During the onset of
infection, the levels of blood PMNs rise quickly, in response to endotoxins released by
25
gram-negative bacteria, and the neutrophils adhere to the endothelial cells and migrate
into infected tissues where the neutrophils survive for only 1-2 days.
B. The Fate of Circulating Neutrophils
At the sites of inflammation in the systemic circulation, post-capillary and collecting
venules are the principal sites for neutrophil adhesion and emigration. Once neutrophil
have left the blood circulation, they will never return. Neutrophils leave the circulating
blood stream by first tethering to and then rolling on the inflamed endothelium lining the
blood vessel lumen. Adhesion of neutrophils to the venule endothelium occurs normally
in the presence of fluid shear rates ranging from 150 to 1600 per second[86].
i) Leukocyte rolling
The Role of Selectins in neutrophil rolling
All the known members of the selectin family mediate neutrophil rolling on venule
endothelium[88-96]. The selectin family has three members, L-selectin (CD62L), P-
selectin (CD62P), and E-selectin (CD62E). L-selectins are expressed on the surface of
neutrophils, lymphocytes, monocytes and eosinophils[97]. Their localization to the
surface projections (ruffles), allows for close positioning to facilitate interactions with the
inflamed endothelial surface[98, 99]. Shedding of L-selectins occurs rapidly (minutes)
following neutrophil activation. P-selectin are expressed on the surface of activated
platelets and endothelial cells and translocate to the cell surface following stimulation
with thrombin, LTC4, histamine calcium ionophore A23187, complement proteins C5b-9
or phorbol esters. E-selectins on the other hand, are only expressed on the surface of
activated endothelial cells and a wide range of inflammatory mediators induce E-selectin
expression, including IL-1β, TNFα, bacterial endotoxin and substance P [100, 101]. With
26
the development of mice deficient in one or more selectins, it is now apparent that each
of the selectins may mediate leukocyte rolling at different velocities which is also
dependent on the dose, type and timing of inflammatory stimuli [86, 102-104].
Selectins are constitutively active when expressed and bind with rapid rates of association
and dissociation that allow rolling in response to high forces of flowing blood.
ii) Leukocyte Arrest and Activation
The Role of Integrins
The function of integrins is to arrest rolling cells on the inflamed endothelial surface.
Integrins are a family of transmembrane glycoproteins that mediate cell-cell and cell-
substrate interactions[105]. They are intimately involved in the regulation of a variety of
cellular functions including embryonic development, metastasis, programmed cell death,
hemostatsis and leukocyte homing and activation. Each integrin is composed of an α and
β subunit that associate to form a non-covalently bound heterodimer. The β2 family of
integrins is exclusively expressed on leukocytes. The involvement of the leukocyte
integrin family β2 (CD18), in contrast to selectins, exhibit low binding avidity unless
activated[86, 106-109]. Endothelial ICAM-1 (CD54) is the principal ligand for the β2
integrins, LFA-1 (CD11a/CD18) and Mac-1 (cd11b/CD18). While many vessels
constitutively express ICAM-1, its expression is greatly increased following stimulation
with inflammatory mediators (e.g. IL-1β, TNFα, INFγ and endotoxin). β2 integrin
adhesion function can also be strengthened with cross-linking L-selectin [110-112] and
the leukocyte receptor(s) for E-selectin[113].
27
Chemokines on the surface of endothelial cells are thought to trigger the rapid transition
of neutrophil rolling to stationary adhesion[108, 114] . The binding of integrins to
immunoglobulin superfamily members, such as ICAM1 and VCAM1, expressed by
endothelial cells during the rolling process, results in rapid increases in chemokines and
other chemoattractants. Modulation of the affinity of β2 integrin for its ligand is well
documented as a crucial step in chemokine-induced arrest under static conditions[115],
however the evidence documenting the role of surface bound chemokines in the transition
of rolling to stationary adhesion to endothelium under flow conditions in neutrophils is
less convincing[108].
Neutrophils have numerous ligands for selectins on their surface[116, 117] which may
not only be important in the tethering and rolling function but may also allow
transduction of signals for neutrophil activation[118]. For example, transient increases in
Ca2+
flux, the production of reactive oxygen species, IL-8 and TNFα have been observed
following L-selectin crosslinking [119, 120].
iii) Transendothelial Migration
A short time following arrest on the endothelial cells, most neutrophils begin to crawl,
searching for a gateway to cross the endothelium[121]. Transendothelial migration, also
referred to as diapedesis, is the step of leukocyte recruitment in which leukocytes move
across the endothelial cell layer toward a site of inflammation. While the preceding steps
(e.g. tethering, rolling, and firm adhesion) are reversible, diapedesis is the “point of no
return” for a leukocyte. While the events leading up to transendothelial migration are well
characterized in neutrophils, for instance, the selectin-mediated rolling and integrin-
mediated firm adhesion, controversy exists in the mode by which neutrophils migrate
28
across the endothelium[122, 123]. Currently, two models for neutrophil transendothelial
migration have been proposed: (1) Paracellular migration and (2) Transcellular migration
Paracellular Migration: It is widely believed for years and popularized in review articles
that neutrophil migration across the endothelium is primarily paracellular with leukocytes
squeezing through and between adjacent endothelial cells[124, 125]. This involves
penetration (distruption) of the intercellular junctions (zonula occludens or tight
junctions; zona adherens or adherens junctions). Within the cleft, the neutrophil is
thought to interact with endothelial gap junctions and resident adhesion molecules
(PECAM-1 (CD31), CD99, JAMs) [126]. This model is supported by previous electron
microscopic observations of neutrophil transmigration in in vivo models of inflammation.
Transcellular Migration: By this pathway, neutrophils migrate through an individual
endothelial cell. More recently, Feng and colleagues [127] showed that neutrophils
migrate through the endothelium of inflamed venules via a transcytotic pathway in vivo.
In particular, the activation state of endothelial cells, and the ICAM-1 expression level
appears to influence whether migrating leukocytes take the paracellular or the
transcellular pathway. Yang et al.[128] reported that human neutrophils preferentially
utilize the paracellular pathway to cross a monolayers of cultured human endothelial
cells, when endothelial cells are stimulated in vitro for 4 h with tumor necrosis factor-
α[128]. However, 24 h stimulus with this cytokine, which strongly up-regulates ICAM-1
expression, shifted the ratio of transmigrating neutrophils towards the transcellular
pathway, with up to 20% of the cells taking the non-junctional route[128].
29
C. The Role of the Cytoskeleton
The driving machinery of neutrophils is the classical and ubiquitous contractile protein
actin. In the resting spherical cell, the actin exists in two forms, globular monomeric (g-
actin) and polymerized, filamentous actin (F-actin), with the polymerized form primarily
located at the cell periphery[129]. Upon stimulation, nearly all of the g-actin rapidly
polymerizes, and as the cell migrates forward, a seemingly constant amount of
polymerized actin (F-actin) remains towards the front leading edge[129]. The filamentous
actin is then crosslinked into bundles and networks by actin binding proteins. This allows
the cell to tailor the dimensions and mechanical properties of the bundles to suit specific
biological functions[130, 131]. Examples of actins binding proteins include filamins, α-
actinin, fibrins, and spectrins.
D. Cell Morphology changes
Neutrophils are spherical in shape when in suspension, but when they are allowed to
adhere to a surface, the cytosol spreads from the contact site in all directions with no
obvious polarity. The presence of a chemical stimuli results in the neutrophil rapidly
organizing itself into a clearly defined polarized structure which is essential for cell
motility. The polarized structure consists of a leading edge “lamellipodium”, and a
trailing end also known as the uropod. The lamellipodium present at the leading edge of
the mobile cell is made up of an actin mesh which is believed to be the actual motor that
pulls the cell forward during migration[132]. Within the lamellipodium are small, finger-
like projections of actin filaments which, when spread beyond the lamellipodium frontier
are called filopodia[132]. Filopodia are thought to be responsible for probing the
extracellular milieu and detecting the chemical gradient[133]. On the other end of a
30
polarized neutrophil, myosin-based contraction and retraction of the uropod facilitates
substrate de-adhesion, resulting in forward propulsion.
E. Chemotaxis
Neutrophil chemotaxis involves a complex array of individual components, leading to an
organized development and dissolution of actin filaments and focal adhesions along a
highly structured cytoskeletal network, as well as contractile processes that operate in a
coordinated manner along the length of the cell.
i) The Rho Family of Small GTPases:
The Rho family of small GTPases have been shown to play pivotal roles in regulating the
biochemical pathways involved in cell migration [134]. They constitute a family of
intracellular messengers that are regulated both by their location and state of
activation[135].
Rho-related small GTPases, including Rho, Rac, and Cdc42, play key roles in regulating
cell shape, motility, and adhesion through the reorganization of the actin
cytoskeleton[136]. Small GTPases are monomeric guanine nucleotide-binding proteins
(molecular masses=20-25 kDa) that serve as molecular switches to regulate growth,
morphogenesis, and cell motility. The Rho family of small GTPases is of special interest
because they transduce extracellular stimuli into intracellular signals. Small GTPases
cycle between inactive (GDP-bound) and active (GTP-bound) states. Following
stimulation, GTPases release GDP and bind to GTP, a reaction mediated by guanine
nucleotide exchange factors (GEFs). In their active GTP-bound state, Rho GTPases
interact with a variety of effector proteins to promote cellular responses. The active state
of GTPases is transient because of their intrinsic GTPase activity, which is stimulated
31
further by GTPase activating proteins (GAPs).Guanine nucleotide dissociation inhibitors
(GDIs) are thought to block the GTPase cycle by sequestering and solubilizing the GDP-
bound form [137],[138] . Upon activation, the RhoGTPases become prenylated allowing
them to translocate and anchore to the plasma membrane where they can then interact
with cellular target proteins (effectors) to generate a downstream response[139]. The
three main RhoGTPases are Rac (Rac1 and Rac2), Cdc42, and RhoA. In general, Rac
localizes to the leading edge of a migrating cell and its primary role is to generate a
protrusive force through the localized polymerization of actin to generate lamellipodia.
Specifically, it has been shown that the two Rac isofoms, Rac1 and Rac2, play distinct
roles in neutrophils during immune cell function, including cell migration and bacterial
killing[140, 141]. While Rac1 has been shown to be essential for neutrophil
chemoattractant gradient detection and orientation toward the chemoattractant source,
Rac2 is the primary regulator of actin assembly providing the molecular motor for
neutrophil translocation during chemotaxis[141].
Cdc42 also induces actin polymerization to generate filopodia observed at the leading
edge, but also plays a crucial role at the front of cells in controlling the direction of
migration. RhoA on the other hand, localizes to the rear of the migrating cell and is
associated with regulating the assembly of contractile, actin:myosin filaments necessary
for cell body contraction and rear end retraction [26, 133, 136, 142]. Similar to other
motile cells, dynamic reorganization of the neutrophils actin cytoskeletion allows for
changes in cell shape and also generates the forces necessary for cell locomotion.
32
F. Neutrophil chemoattractants
Neutrophils respond to a variety of soluble stimuli including those that act on seven
transmembrane-spanning domain receptors (f-met-leu-phe, C5a, IL-8, Substance P, PAF)
and those that are receptor independent (the phobol esters, PMA)[143]. These agents
increase random neutrophil motility when in the absence of a chemoattractant gradient
[144](chemokinesis) and also direct the migration of the neutrophil towards the source in
the presence of a chemoattractant gradient (chemotaxis). The majority of neutrophil
chemotaxis studies use fMLP as the stimulus. Unlike mammalian protein synthesis in
which methionine is the “starter” amino acid, in bacteria the methionine is
formylated[145]. This difference allows neutrophils to detect bacteria at a distance[146].
This mechanism may be the most important in the body, since it does not rely on the
cooperation of other cells or systems. All of the agonists stimulate, as an early event, the
hydrolysis of PIP2 with the consequent generation of IP3 leading to Ca2+
mobilization[147]. Another response to a chemotactic agonist is activation of tyrosine
kinases, resulting in the tyrosine phosphorylation of multiple intracellular substrates[148-
150].
G. Phagocytosis
Following extravasation from the circulation and chemotaxis to the site of infection, the
neutrophil arrives at the location and encounters the invading microbe. The process of
phagocytosis may be divided into recognition and attachment of a particle to the surface
of the phagocyte and internalization of the bound particle[151-153]. In contrast to
attachment, engulfment is a process that requires energy from glycolysis. Physiologically,
the particle to be internalized will be opsonized, meaning that it will be coated with a
33
molecule for which the neutrophil has receptors[151, 152]. There are essentially two
types of opsonization, either with the complement fragment C3bi or with antibodies
specific for antigens on the particle surface, thus displaying the Fc region of the antibody
to the neutrophil. Interaction between the opsonic ligands and their receptors on the
neutrophil surface induces a circumferential flow of phagocyte membrane which
gradually surrounds the microorganism to be ingested. The elaborated pseudopod meet in
a single point, and fusion of opposing membranes at this point pinches off the resulting
phagosome into the cytoplasm [153, 154].
H. Neutrophil Respiratory Burst Oxidase
The normal function of neutrophils is to protect the individual from infection by seeking
out and destroying invading microbes. These cells possess a membrane-bound enzyme
system which can be activated to produce toxic oxygen radicals in response to a wide
variety of stimuli[153].
The enzyme responsible for the respiratory burst in neutrophils is the membrane bound
multi-component NADPH-oxidase, which consists of a membrane-spanning electron
transport chain, with cytosolic NADPH as the electron donor and oxygen as the electron
acceptor[155]. In unstimulated resting neutrophils, this NADPH-oxidase system is
normally dormant. The activation of this system can be triggered by phagocytosis or by a
number of other soluble physiological or experimental agents such as the complement
fragment, C5a, fMLP, platelet activating factor and PMA[156]. Once this system is
triggered, the enzyme system is rapidly activated in the walls of the phagocytic vacuole,
where electrons are shuttled from cytosolic NADPH to oxygen present in the
34
extracellular fluid. Under normal circumstances, one single electron is donated to
molecule of oxygen. This results in the generation of one molecule of superoxide (O2.-).
The overall reaction catalyzed by the oxidase is:
2O2 + NADPH 2O2. -
+ NADP+ + H
+
The 2 molecules of superoxide (O2. -
) then interact either spontaneously (dismutase
reaction) or enzymatically to form one molecule of hydrogen peroxide (H2O2):
2O2. -
+ 2H+ H2O2 + O2
Further reactions of O2. –
and H2O2 are possible and in the presence of transition metals
such as iron and copper salts a complex series of redox reactions is seen to occur with the
generation of other more powerful reactive oxidant species such as the hydroxyl radical
(OH.)[157, 158].
O2. -
+ H2O2 OH. + OH
- + O2
The bulk of the O2. –
generated by triggering the NADPH-oxidase system dismutates to
H2O2.
The heme protein myeloperoxidase is found in the azurophilic granules of neutrophils.
This enzyme can use Cl-, Br , or I as a reducing substrate to greatly potentiate H2O2
toxicity by converting it into hypocholorous acid (HOCl)[159]. HOCl is the major
hypohalous acid produced during neutrophil oxidase activation because the plasma
concentration of Cl- is more than a thousand times that of other halides[143]. HOCl is an
extremely potent cytotoxin for bacteria, viruses, fungi, mycoplasmas and cultured
mammalian cells[152].
35
I. Oral Neutrophils as a Diagnostic Test
Recent research from our lab and others suggest that the mouth provides an opportunity to
monitor the innate immune system non-invasively through the use of an oral rinse that
measures oral neutrophil levels[160-162] . The underlying principle of this test is that for
normal innate immunity, neutrophils must be able to enter the bacteria-rich oral cavity in
sufficient numbers to maintain health. Any defects in this normal recruitment can be
monitored using a standardized rinse test to quantify oral neutrophil levels much like a
standard complete blood count. During infections or following surgery neutrophil levels
spike indicating an active immune and healing response. In addition to using an oral rinse
to monitor periodontal disease [163] we have also used an oral rinse assay to monitor
susceptibility to infection and acute neutropenia (low blood neutrophil counts) during
hematopoietic stem cell transplantation. We have confirmed that oral neutrophil levels can
be used to monitor and report on the state of the innate immune system in pediatric and
adult patients with successful bone marrow transplants [161, 162, 164] .
36
Hypothesis and Objectives of Thesis
Bisphosphonate-associateed osteonecrosis of the jaw (BPONJ) has been identified as a
severe complication of dental treatment and oral trauma in 1-10% of patients previously
treated with intravenous bisphosphonates[165]. It is believed that in the subset of
susceptible patients, bisphosphonates perturb normal osseous wound healing through
inhibition of osteoclast formation and function. However, it has been noted that necrotic
bone from ONJ sites also display signs of bacterial infection that suggest to us that an
immune defect may play a role in BPONJ pathophysiology in affected patients [166]. This
thesis focuses on the possible role of neutrophils which are critical elements of normal
wound healing, particularly in the oral cavity where bacterial biofilms have such a strong
impact on oral health and disease. This thesis determined in a novel fashion whether
bisphosphonates alter neutrophil function and if this defect could explain the development
of BPONJ, and the physiological mechanisms through which bisphosphonates mediate
BPONJ in susceptible patients. This thesis tested the novel hypothesis that bisphosphonates
dampen neutrophil functions in an animal and human model and may result in poor innate
immunity and an increased risk of BPONJ following oral surgery or trauma. The specific
objectives of this thesis were:
Objective 1). Determine if bisphosphonates cause functional defects in neutrophils
i) Analyze neutrophil functions in mice treated with the bisphosphonate zoledronate.
Neutrophils will be isolated from mice treated one month previously with a single
dose of zoledronate. Using standard neutrophil function assays for chemotaxis
37
[141, 167] and NADPH oxidase function, we will characterize the effect of
previous BP therapy on neutrophil functions in vitro. The neutrophil function
results from the in vitro assays will be confirmed in vivo using standard murine
acute models that measure neutrophil influx into inflamed/infected sites and
neutrophil mediated bacterial killing efficiency in those sites.
ii) Analysis of neutrophil functions from patients being treated with
bisphosphonates. In order to determine if neutrophil functions are impaired by BP
therapy, blood neutrophils will be isolated from patients diagnosed with BPONJ.
In vitro functional tests (as in i, above) will be used to monitor and compare
neutrophil functionality during the high dose BP therapy.
iii) Analysis of neutrophil delivery to in the oral cavity in patients diagnosed with
BPONJ
In order to determine if neutrophil delivery in the mouth is impaired in patients
diagnosed with BPONJ, oral rinses will be collected from these patients and
compared to healthy controls.
38
CHAPTER 2
MATERIALS AND METHODS
39
2.1 Antibodies and reagents
A mouse monoclonal antibody against Rho A (sc-418) and a mouse monoclonal
antibody against Cdc42 (B-8) were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). A mouse monoclonal antibody against Rac was purchased from Upstate Cell
Signaling Solutions (Charlottesville, VA). The QIAamp DNA Blood Extraction mini
Kit(Cat.no.51104) was purchased from QIAGEN (Santa Clarita, CA). N-formyl-
methyonyl-leucyl-phenylalanine (fMLP) and Percoll were purchased from Sigma (St
Louis, MO). Glutathinone-Sepharose beads, horseradish peroxidase-coupled anti-mouse
antibody and horseradish peroxidase-coupled anti-Rabbit antibody were purchased from
Amersham Pharmacia Biotech UK (Buckinghamshire, England). 50X protease inhibitor
cocktail was purchased from BD PharMingen.
2.2 Animals
All experiments were performed with 3-month old mice bred at the University of Toronto
Animal Care Facility. All procedures were carried out in accordance with the Guide for
the Humane Use and Care of Laboratory Animals, and were approved by the University
of Toronto Animal Care Committee.
2.3 in vivo zoledronate treatment
Using a modification of a previously described rat protocol [168, 169], 3 month old mice
were treated with 2 or 20µg of Zoledronate or vehicle alone. The dosing was based on the
recommended human therapeutic dose of 4mg every 4 weeks. Thus the dose on a µg
ZOL per gram BW basis (assuming an average weight of 60Kg) is 4,000µg ZOL divided
40
by 60,000g BW = 0.06667µg/g. For a 30g mouse then the equivalent dose would be
2µg/mouse. The higher dose was used to model longer durations of treatment.
2.4 Isolation of murine bone marrow neutropihls
As described previously[170], mouse neutrophils were isolated from the bone marrow of
mice which is the primary neutrophil storage site in the mouse. Briefly, Zoledronate
treated or the vehicle control treated mice were euthanized by cervical dislocation. The
femur and tibia were dissected, cut proximally and distally, and flushed with ice-cold 1x
HBSS (supplemented with 10 mM HEPES, pH 7.5), and the eluant was centrifuged at
2,600 rpm for 30 min at 4 °C, and layered over a three-layer Percoll gradient (52, 65, and
85%). The neutrophil-rich fraction was then collected at the interface of the 65 and 85%
layers, resuspended in 1x HBSS, and centrifuged at 1,500 rpm for 5 min at 4 °C. Pelleted
cells were resuspended in 1x HBSS containing 2.5% heat-inactivated fetal bovine serum
and used immediately. The murine neutrophil isolation protocol routinely yields cell
suspensions that are >90% neutrophils with >98% viability as judged by Wright stain and
trypan blue exclusion, respectively. All of the neutrophil studies are carried out at 37 °C.
2.5 Measurement of NADPH oxidase activity
Neutrophil associated oxidant content was measured using dihydrorhodamine 123 (DHR;
Molecular Probes, Eugene, OR). Isolated bone marrow neutrophils were incubated in the
presence of 1 mM DHR for 15 min, followed by incubation with 10-5
M PMA and 10-6
M
fMLP. Cell-associated fluorescence was then measured 10 min later using a flowcytometer
[171].
41
2.6 Zigmond chamber chemotaxis
To assess activation and determine whether there was a chemotactic defect,
isolated bone marrow neutrophils were suspended in HBSS and 1% gelatin. A neutrophil
suspension (1 x 106/mL) was allowed to attach to
bovine serum albumin (BSA)–coated
glass coverslips (22 x 40 mm) at 37°C for 20 minutes. The coverslip was inverted
onto a
Zigmond chamber, and 100 µL HBSS media was added to the left chamber with 100 µL
HBSS media containing fMLP[10
-6] added to the right chamber. Time-lapse video
microscopy was used to examine neutrophil movements in the Zigmond chamber.
The
images were captured at 20-second intervals with Nikon Eclipse E1000 Microscope.
Cell-
tracking software (Retrac version 2.1.01 Freeware) was used to characterize cellular
chemotaxis from the captured images.
2.7 Sodium periodate peritonitis
To induce an experimental peritonitis, 1 mL of 5 mM sodium periodate (Sigma,
Oakville, ON, Canada) in phosphate-buffered saline (PBS) was injected intraperitoneally.
The mice are then euthanized 3 hours later and the peritoneal exudate is collected by lavage
with chilled PBS (5 mL/mouse). Neutrophils were counted by a hemocytometer
and an
electronic cell counter (Becton Dickinson). The neutrophil numbers between the BP treated
mice and the vehicle controls are compared to their untreated (no peritonitis) controls.
2.8 Circulating neutrophil levels
Prior to euthanasia, 200 l of blood was taken from the great saphenous leg vein of the
mouse. A complete blood count was measured using a hemavet (Hemavet®950, Drew
Scientific, Oxford, CT).
42
2.9 GST-Pull down Assay
The Pak-binding domain (PBD) and Rhotekin-binding domain (RBD) assays
were carried out as described previously[172]. Briefly, isolated bone marrow neutrophils
were exposed to Zoledronate (20 M) for 15 minutes, followed by a one minute
exposure to fMLP(10-6
M) at 37°C. They were immediately lysed with and glutathione-S-
transferase (GST)–PBD or GST-RBD
beads (Amersham Pharmacia Biotech
Buckinghamshire, England) were added to the lysates. The GST-PBD or GST-RBD were
recovered with Sepharose beads and immunoblotting was completed using either a Cdc42
antibody
(B-8; Santa Cruz Biotechnology), Rac antibody (Upstate Cell Signaling
Solutions (Charlottesville, VA), or a RhoA antibody (sc-418; Santa Cruz Biotechnology).
Image J software version 1.31v
(National Institutes of Health) was used for blot
densitometry. Data were normalized to resting control neutrophils and are
expressed as
the mean value from 3 separate experiments.
2.10 Apoptosis Assay
The percentage of apoptotic neutrophils was determined by Annexin-V staining
using the Annexin V-FITC Apoptosis Detection Kit from BioVision according to the
manufacturer's protocol. Stained cells were analyzed by FACS and cells positive for both
annexin-V and propidium iodide (PI) or annexin-V alone were considered apoptotic.
2.11 In vitro generation of neutrophils
Hematopoetic progenitors were isolated from bone marrow using the EasySep™
Hematopoietic Progenitor Enrichment kit from Stemcell technologies (Vancouver,
43
Canada). Stem cells were subsequently differentiated into neutrophils as described
previously [173]. In brief, progenitors were cultured for 3 days in IMDM (Gibco), 10%
fetal calf serum (FCS), 50 uM beta-mercaptoethanol, 10 u/ml pencillin/streptomycin, 2 mM
glutamine, SCF, 50 ng/ml), FLT-3 (50ng/ml), GM-CSF (0.1 nM), IL-3 (0.1 nM) and G-
CSF (30 ng/ml). After this initial period the medium was replaced every 3 days with
medium containing only G-CSF (30ng/ml). Cells were harvested between day 9 and 12 (as
mentioned in the results section).
2.12 BPONJ patient selection
Five patients diagnosed with bisphosphonate-induced osteonecrosis of the jaw
and three patients diagnosed with osteoradionecrosis were recruited from the dental clinic
at Sunnybrook Health Sciences Centre in Toronto, Canada by dental staff practitioners.
The patients diagnosed with BPONJ met the criteria for diagnosis set by the
AAOMS[77]. This study was run in accordance with the institutions Research Ethics
Board-approved protocols and after informed consent had been obtained.
2.13 Patient Blood Neutrophil Isolation and chemotaxis
A 10-ml blood sample was drawn into an EDTA-containing vacutainer. Neutrophils
were isolated using a one-step neutrophil isolation solution. Briefly, 4 ml of the blood was
layered carefully over 4 ml of Optiprep (Sigma, Oakville CA), and the tube was centrifuged
at 1,600 revolutions per minute (rpm) for 30 minutes at 4C. The neutrophils were harvested
from the lower of two bands and washed by centrifugation with Hanks balanced salt
solution at 2,500 rpm for 5 minutes. Any remaining red blood cells were lysed with 1 ml
distilled water, and a neutrophil-rich pellet is obtained by centrifugation at 2,500 rpm for 5
44
minutes. The cells were resuspended in 1 ml Hanks balanced salt solution. A cell count was
obtained with a hemocytometer. This method yields neutrophils with >95% cell viability as
determined by trypan blue staining.
To assess activation and determine whether there was a chemotactic defect, isolated
neutrophils were placed into the upper well of a transwell chamber which is separated from
the bottom chamber, containing vehicle alone or 10-6
M fMLP (Sigma) and a glass cover
slip, by a membrane with 3 micron pores (COSTAR). The transwell plates were placed at
37C for 1 hour and cells migrating through the membrane and onto the bottom cover slip
were washed once and quantified. Chemotaxis was assayed by counting the mean number
of cell per field over 6 fields [174].
2.14 Patient Oral Rinse Samples
The neutrophils from the oral cavity were collected following a one-minute oral
rinse with 5-ml of a sterile bicarbonate solution. One rinse was collected from each patient
in a sealable falcon tube and immediately transported to the Glogauer laboratory at the
University of Toronto for analysis. Neutrophils in each oral rinse sample was quantified
using the acridine orange staining technique [162]
. This technique gives a very characteristic
fluorescent bi-lobed nuclear staining pattern in neutrophils facilitating easy identification
and counting on a hemocytometer chamber.
2.15 Statistics
Normally distributed data were analyzed by unpaired Student‟s t tests. Analysis of
variance (ANOVA) with Bonferroni correction was used for multiple comparisons.
A P value of <0.05 was considered to be significant. Data are expressed as mean ± SD
unless indicated otherwise.
45
CHAPTER 3
RESULTS
46
RESULTS
Although bacterial colonization is a common finding in patients with BPONJ, it remains
unclear whether infection plays a primary causative or is secondary to the appearance of
the lesion. We chose to test the hypothesis that zoledronate exposure leads to impaired
neutrophil function in an in vitro and in vivo murine model.
3.1 Effect of Zoledronate on in vitro Neutrophil Function
Isolated murine neutrophils exposed to increasing concentrations of zoledronate for 15
minutes showed a dose dependent decrease in neutrophil function (p<0.05). Both in vitro
chemotaxis speed (Figure 1A) as well as in vitro NADPH oxidase activity (Figure 1B)
were diminished with increasing concentrations of zoledronate (p<0.01), suggesting that a
brief direct exposure to the drug may not only detrimentally effect the neutrophils ability to
migrate to the site of infection but also diminish its ability to destroy the invader at the site.
3.2 Effect of Zoledronate on in vivo Neutrophil Function
To determine if in vivo zoledronate treatment has any residual effects of
neutrophil function, mice were treated 4 weeks prior to experimentation with 0, 2 or 20g
of Zoledronate. While a dose dependent relation was not observed with systemic
treatment of zoledronate, an obvious decrease is chemotaxis speed was observed in mice
treated previously with zoledronate at both 2 and 20µg (p<0.05). Both sodium periodate-
induced in vivo neutrophil recruitment (Figure 2A) as well as fMLP-mediated in vitro
chemotaxis (Figure 2B) show decreases in neutrophil chemotaxis speed (p<0.05).
47
3.3 RhoGTPase Assay
As bisphosphonates have been well documented to inhibit a crucial step in
RhoGTPases prenylation, we hypothesized that zoledronate treatment may affect the
function of these key signaling proteins involved in neutrophil function. As indicated in
Figure 3, while fMLP activation of Rac1, and Cdc42 was normal in cells pretreated with 20
micromolar zoledronate for 15 minutes in vitro, RhoA activity downstream of fMLP was
depressed by almost 50% at the same 20 micromolar zoledronate dose (P<0.05). This data
confirms that this bisphosphonate can affect a key regulator of neutrophil functions.
3.4 Effect of Zoledronate on Circulating Neutrophil Counts
While abnormalities in bacterial colonization and impaired wound healing
observed in patients with BPONJ may be related to neutrophil functional defects, one
must also consider a possible defect in circulating neutrophil numbers. Neutropenia was
observed in mice 2-weeks following a 2 micromolar pretreatment with zoledronate
(P<0.05) (Figure 4). This finding led us to consider the possibility that zoledronate may
decrease neutrophil survival and/or neutrophil differentiation in the bone marrow.
3.5 Effect of Zoledronate on Neutrophil Differentiation and Survival
In order to verify that zoledronate could impact neutrophil differentiation in the
bone marrow we used a standard in vitro neutrophil differentiation assay to monitor
neutrophil differentiation in the presence of this commonly used bisphosphonate. Control
stem cells were differentiated into neutrophils for 12-14 days in the presence of G-CSF.
48
During this differentiation period, cells were treated with varying concentrations of
zoledronate. After 12 and 14 days of differentiation, cells were analyzed by Diff-Quick
staining. Figures 5A illustrates a dose dependent decrease in viable mature neutrophils
(dark blue nucleus) with increasing zoledronate concentration, suggesting that zoledronate
exposure leads to increased cell death during neutrophil differentiation in vitro (p<0.05).
We were also interested in determining if ZOL‟s effect on neutrophil survival during
differentiation is irreversible in nature. Stem cells from mice pre-treated 4 weeks prior with
ZOL were isolated in vitro and differentiated into neutrophils for 10-12 days in the absence
of ZOL. Figure 5C illustrates that stem cells from zoledronate-pretreated mice („Z-M‟) are
able to differentiate normally in vitro with similar survival rates as control cells (“C-M‟) if
zoledronate is absent during differentiation. However, the addition of zoledronate to control
stem cells during in vitro differentiation increases the number of apoptotic events (Figure
5C; “C-M+ZOL”) (p<0.05). These results suggest that ZOL must be present during
neutrophil differentiation to affect mature neutrophil survival.
We also determined that a key growth factor, G-CSF must be present in the immediate
environment in order for us to observe the negative affects ZOL has on mature neutrophil
survival. When mature neutrophils were isolated from ZOL pre-treated mice and left to
undergo physiologic apoptosis, there was no difference in levels of apoptosis compared
with control cells (Figure 5D). However, when G-CSF was added to the media, increased
levels of neutrophils apoptosis was observed (Figure 5E).
49
3.6 Blood neutrophil chemotaxis in BPONJ patients
In order to determine if a similar neutrophil chemotaxis defect is observed in a human
disease model, blood samples were taken from five patients diagnosed with BPONJ, three
patients with diagnosed ORN and three control patients. Neutrophils were isolated and their
ability to migrate through a porous membrane was measured using fMLP as a
chemoattractant in a Boyden chamber set-up. Significantly fewer numbers of neutrophil
from BPONJ patients were observed to migrate toward the fMLP stimulus compared with
control neutrophils and ORN neutrophils (p<0.05) (Figure 6A).
3.7 Oral neutrophil recruitment in BPONJ patients
To determine if the in vitro defects manifest as reduced neutrophil function in the
oral cavity in these patients, we used an oral rinse protocol to quantify neutrophil delivery
to the oral cavity. Figure 6B illustrates a trend toward increased neutrophil delivery to the
oral cavity in patients with BPONJ as well as ORN, however significance was not
achieved for either group (p>0.05).
50
FIGURES
Figure 1-Effect of Zoledronate on in vitro neutrophil chemotaxis (A) and NADPH oxidase activity (B):
In order to determine if Zoledronate had a direct effect on neutrophil functions, mature neutrophils were
isolated from mouse bone marrow as described previously [141] . Neutrophils were treated for 15 minutes at
the indicated concentration of Zoledronate followed by the assessment of either A) fMLP mediated
chemotaxis in a Zigmond chamber or B) fMLP mediated NADPH oxidase activity. As indicated in A and B,
zoledronate inhibits both fMLP mediated chemotaxis in a dose dependent manner and NADPH oxidase
activity. [All experiments were repeated 3 times with each experiment using 3 mice per group. Errors are
standard deviation and all points are significantly different from control p<0.01].
Figure 2-Effect of in vivo Zoledronate treatment on A) in vitro chemotaxis and B) in vivo peritoneal
recruitment: A) In order to determine if in vivo Zoledronate treatment had a residual effect on in vitro
neutrophil functions, mature neutrophils were isolated from bone marrow of 4 month old mice treated 30 days
previously with 0, 2 or 20 micrograms of Zoledronate [168, 169]. fMLP mediated neutrophil chemotaxis was
assessed in a Zigmond chamber [167]. As indicated in A) zoledronate given 30 days prior has a residual
inhibitory effect on neutrophil chemotaxis. B) In order to verify these results, the same mouse treatment and
Zoledronate dosage protocol was used to test in vivo neutrophil recruitment to sites of inflammation. Briefly a
peritonitis model was used in which an irritant (sodium periodate) is injected into the peritoneum and 3 hours
later the cellular infiltrate is collected and quantified [141, 175]. In this model greater than 95% of cells
collected are neutrophils. As indicated in B, mice previously treated with Zoledronate display a clear
reduction in neutrophil recruitment to the inflamed peritoneum. [All experiments were repeated 3 times with
each experiment using 3 mice per group. Errors are standard deviation and all points are significantly
different from control p<0.01].
Effect of Zoledronate on in vitro chemotaxis Effect of Zoledronate on in vitro NADPH activity
*
*
*
* * * *
51
Figure 3- Possible mechanisms through which ZOL affects neutrophil functions: In order to verify that
Zoledronate could affect key signaling proteins involved in neutrophil functions we measured
chemoattractant mediated Rho small GTPase activity in neutrophils treated with Zoledronate in vitro. As
indicated in this figure, while fMLP activation of Rac1, and Cdc42 was normal in cells pretreated with 20
micromolar Zoledronate for 15 minutes, Rho activity downstream of fMLP was depressed by almost 50% at
the same 20 micromolar Zoledronate dose. This data confirms that this bisphosphonate can affect a key
regulator of neutrophil functions. Errors are standard deviation and * is different from control p<0.01]
Figure 4-Effect of zoledronate on circulating neutrophil levels: In order to determine if Zoledronate had
a direct effect on neutrophil numbers, circulating blood was collected from 4 months old mice that had
been pre-treated with a 2mg dose of zoledronate 2 weeks prior and compared with controls. A significant
decrease in circulating neutrophils is observed following zoledronate treatment, suggesting that zoledronate
may be either causing early apoptosis of cells or is negatively affecting the production of neutrophils.
[Experiments were repeated 2 times with each experiment using 5 mice per group. Errors are standard
deviation and ( *) is significance at p<0.05].
*
52
Figure 5: Effect of Zoledronate on differentiation and apoptosis of mature neutrophils -In order to
determine if zoledronate treatment has an effect on neutrophil differentiation and apoptosis, we quantified
the numbers of mature cells that had been derived from either (A-C) in vitro neutrophil differentiated
precurors cells or (D, E) mature neutrophils extracted from bone marrow . (A) In order to determine if
Zoledronate could impact neutrophil differentiation in the bone marrow we used a standard in vitro
neutrophil differentiation assay to monitor neutrophil differentiation in the presence of this commonly used
bisphosphonate. Bone marrow cells were harvested from 3 control mice and hematopoetic progenitors were
isolated using a negative selection kit from StemCell Technologies. Progenitors were cultured for 3 days in
IMDM medium containing 10% FSC, 50 μM β-mercaptoethanol, SCF (50 ng/ml), FLT-3 (50 ng/ml), IL-3
(10 ng/ml), GM-CSF (1 ng/ml) G-CSF and (30 ng/ml). The medium was replaced every three days with
IMDM medium containing 10% FSC, 50 μM β-mercaptoethanol, G-CSF (30 ng/ml) and Zoledronate (1,
10, 50 and 200 μM). After 12 and 14 days the differentiation of neutrophils was assessed by Diff-quick
staining. The panels in A, shows representative images of Diff-quick stained cells after 12 days in vitro.
Live cells stain with a dark blue nucleus, while dead cells stain with a pale blue nucleus. Cells were
counted in 4 random fields and the percentage of neutrophils was calculated for 12 days. As demonstrated
in this figure zoledronate reduces mature neutrophil survival in a dose dependent manner, but does not
appear to affect neutrophil differentiation since similar numbers of total cells are present (live and dead)
compared with control. (B) Quantification of the percentage of cells that have undergone mature
C-M Z-M C-M +ZOL
53
neutrophil apoptosis in response to increasing concentrations of zoledronate during the process of
differentiation. A significant increase in apoptosis compared with control is observed when neutrophils are
differentiated in the presence of zoledronate concentrations greater that 10uM. (C) Progenitors from 3 mice
pretreated with 2ug of zoledronate 2 months prior („Z-M‟) and 3 control mice („C-M‟) were differentiated
similar to that in (A) but without the addition of zoledroante to the media. No significant difference is noted
in neutrophil survival between (Z-M and C-M). As a positive control, 3 control mice were differentiated in
an exact manner as was performed in (A) in the presence of 20 uM zoledronate (C-M + Zol). The presence
of ZOL to the media during differentiation leads to increase neutrophil apoptosis. (D) Mature neutrophils
were extracted from the bone marrow of mice that had been pre-treated 2 weeks prior with 2ug of
zoledronate and subjected to a spontaneous 20H apoptosis assay in the absence of G-CSF. The levels of
apoptosis were not significantly different („NS‟) between mature neutrophils from control and zoledronate
pre-treated mice. (E) Mature neutrophils were extracted from the bone marrow of mice that had been pre-
treated 2 weeks prior with 2ug of zoledronate and subjected to a spontaneous 20H apoptosis assay in the
presence of G-CSF (30 ng/ml). Zoledronate pre-treated mice in the presence of G-CSF show significantly
higher levels of apoptosis of mature neutrophils compared with control mice. These levels of apoptosis are
similar those observed during the in vitro differentiation of precursors cells in (A). Annexin-positive but PI-
negative cells are defined as early apoptotic cells, whereas annexin -positive and PI-positive cells are
defined as late apoptotic cells. [Experiment was preformed with n=4 per group. Errors are standard
deviation and * is significance of p<0.05].
54
Figure 6 (A) Neutrophil Chemotaxis is perturbed in BPONJ patients. The final data that confirms that
there is value in testing the novel hypothesis proposed comes from comparing neutrophil chemotaxis in
control neutrophils, patients with osteoradionecrosis (ORN) and from patient with bisphosphonate induced
osteonecrosis of the jaw (BPONJ). We measured the neutrophils ability to migrate through a porous
membrane using fMLP (10-6M) as a chemoattractant in a Boyden chamber set-up. Neutrophils from 5
patients with BPONJ were analyzed and compared with neutrophils from 3 patients with ORN and 3 healthy
control patients for migratory impairments. Cells that migrated through the 3μm pores were DAPI-stained,
and 5 random fields of view (FOV) were counted using a fluorescent microscope under 40X magnification.
The level of migration was expressed as the average number of neutrophils per FOV. Control, ORN and
BPONJ neutrophils show significantly increased migration toward fMLP. However, significantly fewer
numbers of BPONJ neutrophils were observed to migrate toward the fMLP stimulus compared with control
neutrophils as well as ORN neutrophils, suggesting a neutrophil migratory defect in the BPONJ patients.
*p<0.05, error bars are S.D. (B) Oral neutrophil counts in BPONJ patients vs. healthy control patients-
Oral Neutrophils in the rinse samples were easily identified using the acridine orange staining technique
described previously[162]. The neutrophils were visualized with fluorescence microscopy and counted using
a hemocytometer. Although there was a trend toward increased neutrophil recruitment in the BPONJ and
ORN patients, the levels did not reach statistical significance with p>0.05, error bars are S.E.M.
55
CHAPTER 4
DISCUSSION
56
In recent years it has been noted that a small subset of patients (1-10%) treated with
intravenous bisphosphonates are at an increased risk of developing osteonecrosis of the
jaw (BPONJ) [4, 5]. The publicity surrounding this complication and the lack of
information about its underlying pathogenesis has led to apprehension amongst patients
and dental clinicians when treating patients with a history of bisphosphonate therapy [6].
4.1 Pathogensis
Several mechanisms by which bisphosphonates (BP) are thought to cause
bisphosphonate-induced osteonecrosis of the jaw (BPONJ) have been discussed in the
literature. Apart from the earliest idea that BPs lead to a decrease in bone remodeling due
to an increase in oseoclastic cell death, several other theories have emerged.
4.1-1 Anti-angiogenesis and Matrix Necrosis
One of such theories is the idea that the anti-angiogenic properties of the
bisphosphonates which compromise the blood supply in the bone, is the initiating event
of BPONJ[176]. Angiogensesis is the physiological process involving the growth of new
blood vessels from pre-existing vessels. There has been evidence that bisphosphonates
can interfere with the proliferation, adhesion, migration, and vessel sprouting of
endothelial cells thereby reducing the formation of new blood vessels[177],[178].
However the effects observed in the aforementioned studies were found at greater-than-
human equivalent dose, and have not been demonstrated in humans. The most recent
human study to support this theory looked at 31 patients with BPONJ who underwent jaw
bone biopsy and tissue sampling. Light microscopy and confocal scanning laser
microscopy examination demonstrated prominent and dense woven bone formation with
57
fewer and distant blood vessels along with reduced osteoclastic activity. This study also
supported the notion that anti-angiogenic effects by BPONJ may be caused from an
ischemic necrosis due to the osteogenic and nonosteogenic expansion of the bone without
an increase in blood supply. The authors propose that this new dense bone with less blood
supply would eventually lead to necrosis and secondary infection [179].
With this being said, it is believed by some that these drugs have the potential to
cause avascular necrosis. This etiology has been reported to be unsubstantiated by many
who argue that if the bone was in fact avascular, the ONJ tissue should not bleed at the
time of surgery which has been reported [180]. As well, Hansen et al[181] noted patent
vessels in seven out of eight BPONJ cases studied histologically. The vascular supply of
the mandible is inherently restricted, and interferences with this blood supply. Moreover,
other more potent anti-angiogenic medications such as thalidomide, used for
chemotherapy, are not associated with BPONJ in humans[176].
In a beagle dog model of BPONJ, Burr and Allen [182] showed that bone matrix
necrosis occurs in bones with higher remodeling rates such as the jaw bone that has been
treated with with clinical doses of aledronate and zoledronate, while none of the control
animals in the study acquired osteonecrosis.
4.1-2. Bone Turnover and Microcracks
Another theory arises from the well documented mechanisms of action of the
bisphosphonates in reducing bone turnover. It is believed by some that an excessive
reduction in bone turnover exists in some individuals taking bisphosphonates leading to
an accumulation of microcracks. In essence, the microcracks may make the bone more
susceptible to necrosis when there is increased demand for osseous repair such as in cases
58
of trauma to the jaw[176, 183, 184]. This theory is supported by a recent human study
using scanning electron microscopy[185]. Microcracks were present in 54% of patients
with BPONJ compared with 29% in patients without BPONJ but taking BPs, and 17% in
osteoporosis patients not taking BP, whereas no microcracks were observed in patients
with osteoradionecrosis of the jaw or osteomyelitis of the jaw without BP therapy.
The results of this study have been questioned by some who criticize the use of scanning
electron microscopy to evaluate microcrack damage. Allen [186] proposes that the gold
standard method for evaluating microdamage in bone is basic tuchsin staining, which
stains the bone prior to processing. Allen argues that the importance of staining
specimens prior to processing is that it allows separation of microcracks that existed prior
to processing and those that are due to specimen preparation. Without such staining, it is
impossible to know whether any damage is due to processing or not. In his own study,
Allen et al. [187] showed that three years of daily oral alendronate treatment in beagle
dogs increases microdamage in vertebral bone but does not significantly increase it
beyond levels of microdamage found after 1 yr of treatment. He suggests microdamage
accumulation peaks during the early period of bisphosphonate treatment and does not
continue to accumulate with longer periods of treatment.
Refuting the notion that BPs lead to increase in microcracks in bone is Chapurlat et
al[188] who examined transiliac bone biopsies from postmenopausal women and found
no significant differences in microcracks between these women and control groups
following at least 3 years‟ treatment with a bisphosphonate (3 subjects on I.V.
pamidronate, 37 on oral alendronate, and 10 on oral risedronate) despite a marked
reduction in bone turnover. A similar study was conducted by Stepan et al[189] where
59
they compared transiliac bone biopsies from 38 postmenopausal women that were being
treated with daily or weekly doses of aledronate for at least 5 years. They found no
differences in crack density and crack surface density between the control and study
groups. A major drawback of these sudies however, is that the majority of the women in
the study population were on oral doses of bisphosphate rather than intravenous
administration of the drug. It is well documented that BPONJ is associated with
intravenous administration rather than oral administration [65, 184]. As well, the bone
examined in these studies were not taken from the jaw bone where BPONJ occurs.
One point to keep in mind is the idea that if reduced bone turnover was in fact the
etiology of BPONJ, it seems odd that other conditions associated with chronically
reduced bone turnover, such as hypoparathyroidism, do not show BPONJ-like
lesions[61].
Other compelling evidence suggests that bone turnover is not even reduced within
BPONJ lesions. Several groups[181, 190, 191] have observed deep osteoclastic pits in
BPONJ lesions, as well, Hansen et al.[181] showed up to a four-fold increase in
osteoclast number in BPONJ patients compared with those from osteoradionecrosis and
control patients suggesting that bone turnover is not suppressed at sites of BPONJ.
Enhanced bone turnover has also been demonstrated with bone scintigraphy, where Abu-
Id et al. found that areas of necrotic bone had increased tracer uptake in 15 of the 16
BPONJ subjects examined[74].
4.1-3. Soft Tissue Toxicity
Soft tissue toxicity to the oral mucosa may lead to compromised healing of
traumatic lesions that arise in the soft tissue, with extension of the lesion to the
60
underlying bone[192]. Much of the focus of BPONJ has centered upon bisphosphonates
and bone, an „inside-out‟ theory. However the toxic effects of bisphosphonates on
traumatized oral epithelium may play a critical role in the development of BPONJ, a so-
called “outside-in” hypothesis. Several studies have confirmed that idea that BPs can
negatively affect the function of several cell types including macrophages, periodontal
ligament fibroblasts [193], endothelial cell[177, 194-196], a variety of tumor cells,
osteoblasts and epithelial cells[197-199]. Bisphosphonate toxicity to epithelial cells is a
well documented side effect with the use of oral nitrogen-bisphosphonates with the
formation of gastric erosions and ulcers. Coxon et al[200] explored the idea that
bisphosphonates on bone surfaces are toxic to adjacent cells. Interestingly, they observed
that when the bisphosphonates were not bound to bone but present free in solution, they
appeared to have toxic affects on cells. However, when the bisphosphonates were in the
presence of bone, the toxicity was greatly reduced, suggesting that the when the
bisphosphonates are bound to bone, they are unable to concentrate and toxify the
surrounding cells. On the other hand, when osteoclasts were added to the cultures in an
attempt to simulate a normal physiologic environment, the bisphosphonates were
mobilized from the bone and began to concentrate in the surrounding solution leading to
toxicity in the adjacent cells[201].
With respect to bone, the amount of drug that is deposited in the maxillofacial bones is
unknown. Likewise, the amount of active bisphosphonate present in the gingival
crevicular fluid is unknown, however the presence of active periodontal disease would be
expected to increase the level of bisphosphonates in the area[61].This is supported by
61
Marx [176], who suggests that approximately 85% of affected patients have had
peridontitis during the diagnosis of BPONJ.
Similarly, areas of high bone turnover observed in the alveolar bone following a
traumatic extraction would be expected to present with higher concentrations of
bisphosphonates. Studies using 99m
Tc-labeled bishosphonates have confirmed the
hypothesis that there is preferential uptake of tracer at these sites[202], however the exact
concentration of the bisphosphonates is unknown. Given this potential mechanism, it is
not surprising how one may believe a scenario in which trauma to the oral mucosa can
represent an initiating event of BPONJ and release of bisphosphonates from the
underlying bone can inhibit mucosal wound healing with spread to the adjacent bone[61].
4.1-4. Infection
One of the final common theories discussed in literature as a possible initiating factor in
the pathogenesis of BPONJ is presence of an infection. This idea came about from the
consistent, if not universal histological finding of bacterial colonizationin biopsies taken
from BPONJ patients[74, 181, 203, 204].
Recently, a study looking at biopsies of four BPONJ patients using scanning electron
microscopy, made the observation that all four patients had the presence of microbial
biofilms[190]. A biofilm is a complex aggregation of microorganisms growing on a solid
substrate. Biofilms are characterized by structural heterogeneity, genetic diversity,
complex community interactions, and an extracellular matrix of polymeric
substances[205]. They have a characteristic feature of having resistance to both host
defenses (antibodies and phagocytes) and to antibacterial agents, making them difficult to
62
penetrate. Physical removal of the biofilm is usually required to effect a cure. The
presence of infection may be significant in producing one of the unpredicted but constant
characteristics of this condition; increased bone resorption despite the presence of
bisphosphonate in bone[201]. Several bacteria that reside in the biofilm, such as gram
negative bacteria, have the ability to stimulate bone resorption, and some even have the
capability to inhibit bone formation. Many species of Gram-negative bacteria are
pathogenic, meaning that they can cause disease in a host organism. This pathogenic
capability is usually associated with certain components of Gram-negative cell walls, in
particular the lipopolysaccharide (also known as LPS or endotoxin) layer. In humans, the
LPS mediates osteolysis through stimulation of local cytokine production and immune
system activation[206]. However, there are several other factors produced by bacteria
that have similar negative effects on the host that act through different mechanisms. For
example, studies have shown that several proteins from Porphyromonas gingivalis can
directly regulate the production of RANKL and osteoprotegerin in human periodontal
ligament cells and gingival fibroblasts, increasing the stimulation of
osteoclastogenesis[207].
The theory that infection may play a role in the pathogenesis of BPONJ may explain the
exclusive predisposition of BPONJ to the oral cavity. The oral cavity, unlike other parts
of the body, represents an open environment where bone has the potential to be easily
colonized by an abundant flora of bacteria. Some authors have even suggested that
BPONJ can be subclinical, where no mucosal breakdown and bone exposure occurs in
the oral cavity. In cases such as these, it not unreasonable to suggest that an infection can
63
ensue from bacterial colonization through odontogenic or periodontal infection extending
to the bone through fistulas in the absence of mucosal breakdown. The jaws are typically
resistant to microbial colonization, in part, by the contant influx of neutrophils which
migrate into the mouth from the periodontal tissues that surround and support the teeth
[192, 208]. However in a situation where the immune system in comprimised, the
presence of oral infection is not uncommon[209, 210].
4.2 Purpose of Thesis
The goal of this thesis tested the novel hypothesis that bisphosphonates dampen
neutrophil functions and differentiation resulting in poor oral innate immunity. The
compromised oral immunity increases the risk of infection following surgery or trauma
which can subsequently develop into BPONJ.
This theory is based on the fact that there have been reports that some bisphosphonates
can cause neutropenia[7], inhibit neutrophil enzymes that impact on wound healing such
as MMP8 [8] and decreased generation of reactive oxygen species formation [9, 10] .
This is not surprising in view of the mechanism of action of bisphosphonates which target
small RhoGTPases that are signaling proteins integral to neutrophil differentiation and
function. Despite this fact, the idea that BPs may negatively affect the innate immune
system has not received much focus in the literature.
4.3 Plausible Mechanism through which ZOL Affects the Innate Immune System
The plausible mechanism for this hypothesis is based on the biology of bisphosphonates
and small GTPases where 1) it is known that BP target the Rho family of small GTPases
which are signaling proteins [211] not only in osteoclasts but also in neutrophils and 2)
64
neutrophils with defects in small GTPases display perturbed differentiation and defective
antibacterial functions [170, 212].
The mechanism of action of the nitrogenous bisphosphonates involves the perturbation of
several metabolic reactions, most notably the mevalonate biosynthetic pathway. This
affects cell activity and cell survival by interfering with protein prenylation and therefore,
the signaling function of key regulatory proteins. The mevalonate pathway is a
biosynthetic route responsible for the synthesis of cholesterol, other sterols, and
isoprenoid lipids such as isopentyl diphosphate, farnesyl diphosphate (FPP) and
geranylgeranyl diphosphate (GGPP)[213, 214]. FPP and GGPP are involved in post-
translational modification (prenylation) of small GTPases such as Ras, Rab, Rho, and
Rac[215]. Prenylation is necessary for the correct function of these proteins because the
lipid prenyl group serves as an anchor for the proteins in the cell membranes[23]. The
small GTPases are important signaling proteins that regulate many aspects of intracellular
actin dynamics, and are found in all eukaryotic organisms including yeasts and some
plants. Three members of the family have been studied a great deal: Cdc42, Rac1, and
RhoA. Rho proteins have been described as "molecular switches" and play a role in cell
proliferation, apoptosis, gene expression, and multiple other common cellular
functions[216]. In osteoclasts, the small RhoGTPases are essential for cell morphology,
cytoskeletal arrangement, membrane ruffling, trafficking of vesicles, and apoptosis[40].
Neutrophil function is also dependent on the rhoGTPases, where they control among
other things, filodopida and lamellopodia formation and neutrophil chemotaxis[216].
While many of the theories regarding the pathogenesis of BPONJ have focused on how
bisphosphonates negatively affect osteoclast function, very little has been written on the
65
effect that the bisphosphonates may impart on other cell types in the myeloid lineage
such as the neutrophil.
4.4 Oral Innate Immunity
The mouth contains a constant supply of bacteria in the form of surface coating biofilms on
teeth, tongue and other shedding surfaces [217]. This bacterial presence is kept under
control, in part, by the constant influx of neutrophils which migrate into the mouth from the
periodontal tissues that surround and support the teeth. In order to maintain a state of oral
health, an individual‟s immune system must be kept in tight control. A shift to either side
of this equilibrium may result in a state of immune under-activity, observed in cases of
AIDS, where a reduced number of neutrophils in the oral cavity can result in an
overgrowth of unwanted fungi termed “thrush or candidiasis” or lead to a rapid
destruction of the soft and hard tissue of the periodontium known as „necrotizing
ulcerative periodontitis‟ (NUP) [218]. Interestingly, Marx has shown that approximately
85% of affected patients have had periodontitis during the diagnosis of BPONJ [176].
Conversely, excessive neutrophil levels or cell hyperactivity may result in a state of
immune over-activity and can lead to diseases of the periodontium known as „refractory
periodontitis‟[219, 220].
The observation that a majority of cases of BPONJ, present with an active infection lead
us to suspect that the immune system may be perturbed in these individuals.
4.5 The Effect of Zoledronate on Neutrophil Function and Survival
A state of immune under-activity can result from either (1) a decrease in the functional
capability of the immune cells, or (2) a decrease in the number of immune cells that are
present to fight off the infection, or a combination of the two.
66
We chose to test this hypothesis by first performing functional test on neutrophils
following both in vitro and in vivo zoledronate exposure.
4.5-1. Zoledronate negatively affects Neutrophil Function
Our results show that both in vitro and in vivo zoledronate exposure leads to diminished
neutrophil chemotaxis in vitro, diminished neutrophil recruitment to the peritoneum in
vivo, as well as diminished NAPDH oxidase activity in vitro.
Although both in vivo and in vitro models confirm that zoledronate exposure leads to
diminished neutrophil chemotaxis, only the in vitro model resulted in a dose dependent
response; where incremental increases in concentration of zoledronate, resulted in near
proportional decreases in neutrophil speed. The differences in response with increasing
BP dose observed between the in vitro and in vivo models may be the fact that the level
of exposure in vivo may have been different to what the neutrophils were exposed to in
vitro where the in vivo dose may have already been maxed out. For the zoledronate pre-
treated mice, we chose to use a dose of 2ug/mouse. This dose has been previously
calculated for mice to correspond to the dose that is typical for a human patient (detail
explanation found in „Materials and Methods‟).
Since there are no studies that have determined the concentration of zoledronate in the
bone marrow, we chose to use data from a previous study in rats to determine the
appropriate concentration of zoledronate to use for our in vitro mouse model. Using
radiolabled zoledronate in rats, Green et al. [221] quantifed the concentration of
zoledronate in bone, soft tissue and blood from 1 to 280 days after a single 2ug infusion.
Since the bone marrow is apposed to the bone, we estimated that the concentration of
ZOL in the bone marrow is somewhere in between the bone value and the soft tissue
67
value. Taking this into account, we used values from 20-100 uM in vitro to try and
reproduce the 2ug dose in vivo. The differences in dose response between the in vitro and
in vivo models, is possibly due to the fact that although we attempted to expose the
neutrophils to similar ZOL concentrations in both models, we were unable to do so
precisely. It is possible that a 2ug dose of zoledronate elicits the maximum effect on
neutrophils that can be achieved, where any further increase in dose does not have any
further negative effect. Whereas in vitro, the dose from 20uM-100uM may be still low
enough that incremental increases lead to further decreases in cell speed.
All of our initial experiments were performed after 30 days post-zoledronate injection to
recapitulate the dosing cycle in humans. Typically human patients are given a regime of
4mg/30days of a bisphosphonate (usually zoledroante) for a variable number of months
depending on the severity of their condition. Interestingly the results that were obtained
following 30 days post-zoledronate infusion were the same as those obtained after only
14 days of exposure. The lack of difference between 14 and 30 days post injection is
consistent with previous work in a rat model, where the levels of zoledronate in bone and
soft tissue remained relatively constant from the day of infusion to approximately 60 days
post-infusion. These long term effects observed with ZOL can be attributed to the idea
that there is continual recycling of BP off and on back onto the bone mineral surface [28,
29]. This notion is supported by the observation the BPs can be found in plasma and
urine many months after dosing [54] . The observation that neutrophil function is also
perturbed at 14 days post-ZOL exposure implies that; because of the intrinsically short
life of the neutrophils, one will see the deficits in the neutrophils within a few days and
68
this will continue until the levels of circulating BP drop below some critical
concentration, possibly months or years after the last injection.
4.5-2. Zoledronate Affects Neutrophil Function through the small RhoGTPase RhoA
Neutrophil chemotaxis and NADPH oxidase activity is dependent upon the appropriate
localization and activation of the small RhoGTPases; Rac, Rho and Cdc42, within the
neutrophil. Unlike Cdc42 and Rac which localize to and are responsible for the
formation and protrusion of the leading edge of the neutrophil, RhoA localizes to and is
responsible for regulating the myosin II-based contraction and retraction of the trailing
uropod during neutrophil chemotaxis[26, 133, 136, 222-224]. Our results confirm that
zoledronate perturbs the activation of RhoA but does not appear to disrupt the activation
of either Cdc42 or Rac1 following fMLP stimulation. The diminished RhoA activation
may explain the reduced migratory ability of the zoledronate-exposed neutrophils
compared with controls, where these neutrophils may have an inhibility to fully retracting
their tails during migration. These results are in accordance with several other groups
who have also observed neutrophil migration defects when RhoA activation is impaired
[225, 226].
To address the second possible scenario that a decrease in the number of immune cells
leads to diminished immune function, we quantified the number of circulating neutrophils
in zoledronate-treated mice at 14 and 30 days following a 2ug dose of ZOL. We
demonstrate that both time points result in a similar significant reduction in circulating
neutrophil counts, leading us to propose that ZOL may decrease either mature neutrophil
survival and/or neutrophil differentiation in the bone marrow.
69
4.5-3. Zoledronate Decreases Mature Neutrophil Survival
Neutrophil progenitors isolated from control mice and differentiated in the presence of
ZOL in vitro demonstrated an increase in apoptosis in a concentration dependent manner
(Figure 5A). Since the total number of mature neutrophils derived from the progenitors
(live + dead) were unaffected by ZOL treatment we conclude that local zoledronate
exposure does not affect the process of neutrophil differentiation, but instead increases
mature neutrophil apoptosis. While our results show that; (i) ZOL elicits its effect
during neutrophil differentiation (Figure 5A), and (ii) the effects that ZOL imparts on
neutrophils are long lasting, where mature neutrophils from ZOL pre-treated mice exhibit
increased apoptosis even when in a ZOL-free environment (Figure 5E); we are still
unable to determine at what phase of neutrophil differentitaiton that ZOL is eliciting its
affects on neutrophils.
4.5-4. Zoledronate and G-CSF
Interestingly, when we attempted to recapitulate the above findings by subjecting mature
neutrophils isolated from ZOL-treated mice to a 20 hour spontaneous apoptosis assay, no
increase in mature neutrophil apoptosis was observed (Figure 5D). In an attempt to
understand why ZOL exposure affects the survival of mature neutropils when
differentiated in vitro (Figure 5A), while no difference in survival was observed when the
neutrophils were directly isolated from ZOL treated mice (Figure 5D), we considered that
the major difference between these two experiments was the presence of a growth factor
G-CSF. Granulocyte colony stimulating factor (G-CSF) is produced by a number of
different tissues to stimulate the bone marrow to produce granulocytes and stem cells and
70
to promote survival, proliferation, differentiation, and function of neutrophil precursors
and mature neutrophils[227]. These complex functions necessitate G-CSF‟s presence
during in vivo and in vitro neutrophil differentiation from precursor cells. For this reason,
G-CSF was only added to the initial experiment (Figure 5A) to encourage neutrophil
differentiation from the progenitors and not to the later assay (Figure 5D) in which only
mature neutrophils were tested. To adjust for this, G-CSF was added to the media
containing mature neutrophils and apoptosis was re-analyzed. Figure 5E shows that the
addition of G-CSF leads to a similar increase in mature neutrophil apoptosis, as was
observed in Figure 5A. G-CSF is known to dramatically prolong the half-life of mature
neutrophils, having a so-called „anti-apoptotic effect‟, which supports the observation that
even control cells show lower levels of apoptosis when in the presence of G-CSF (Figure
5D and E). The observation that G-CSF must be present for zoledronate to have a
negative effect on neutrophil survival suggests that zoledronate inhibits the anti-apoptotic
effects of G-CSF. Previous groups have shown that G-CSF signals via the G-CSF
receptor requiring various GTPases, specifically Ras and Rap1[228, 229]. Our results
suggest that ZOL may alter G-CSF receptor signaling, impeding the anti-apoptotic effect
of G-CSF on neutrophils and ultimately resulting in higher levels of neutrophil apoptosis.
Our theory that zoledronate negatively effects the function and lifespan of
neutrophils is consistent with previous work by Coxon et al. who has shown that
bisphosphonates have the potential to leave the bone surface in the presence of
osteoclasts and concentrate in solution to disrupt the function of several other cell types
[200].
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4.6 Neutrophil Function in BPONJ patients
We present convincing evidence that suggests that neutrophil function and differentiation
in the presence of bisphosphonates are severely impaired. However some may argue that
the effects that are observed in mice may not be recapitulated in human BPONJ patients.
To address this issue, a study was set up to observe neutrophil function in diagnosed
BPONJ patients. As expected, the preliminary data from these trials suggest that
neutrophil chemotaxis is also perturbed in BPONJ patients, where neutrophils isolated
from the blood of BPONJ patients did not migrate as well as control blood neutrophils
(Figure 6A). As a positive control, we selected three patients that had been diagnosed
with osteoradionecrosis (ORN), a condition which has a similar clinical presentation to
BPONJ, however the pathogenesis is known to be the result from radiation therapy that
negatively affects the vasculature of jaws. The use of these patients as a control is
beneficial since we may observe what true neutrophil recruitment to a site with increased
inflammation is in the absence of bisphosphonate therapy. No difference in chemotaxis
was observed between control and ORN patients, however BPONJ patients had poorer
chemotaxis in comparison to ORN neutrophils (p<0.05), confirming that the
bisphosphonates do in fact perturb the migration of neutrophils, which may result in an
overall reduced immune response. Although we intially expected to see a decrease in oral
neutrophil recruitment in BPONJ patients, this was not the case. Both ORN and BPONJ
patients exhibited a trend toward an increase in oral neutrophil delivery (p>0.05) however
both did not reach statistical significance. The trend toward an increase in neutrophil
delivery in this patient population is not surprising since an open area with active
inflammation is present in the oral cavity in both cases. It may have been more
72
informative to test the specific bacterial killing and NADPH oxidase activity of
neutrophils in the BPONJ group and compare it to the neutrophils present in the ORN
group to identify if the in vitro neutrophil defects can be recapitulated in the human oral
cavity, which may contribute to the ongoing infection in BPONJ patients.
4.7 Questions Arising from our Study
The results presented in this thesis may lead one to then ask the question, if
zoledronate does cause affects on neutrophils, then why are there limited reports of
increased susceptibility to infection in BP treated patients? As well, if bisphosphonates
do lead to neutrophil defects, then why is this condition only observed in 1-10% of BP-
treated patients?
These questions can be addressed by the idea that not all patients with neutropenia for
example, display equal susceptibility to infection, ie. While the BP may be imparting a
defect in neutrophils, possibly only a subset of these patients (1-10%) actually show a
phenotype for increased infection susceptibility.
We believe that the neutrophil defect observed in the 1-10% of BP users is quite mild and
in normal circumstances is mild enough that it does not predispose the patient to
increased risk of systemic infections.
However, in a case where the jaw of a BP-treated patient is now subjected to trauma, the
immune cells which are typically recruited are now being brought to a site with impaired
vascularity and accordingly, a reduced number of these cells are able to penetrate the site.
Consequently, the neutrophils that are able to penetrate the wound are not functioning to
100% capacity, and their ability to combat the abundant oral bacteria which normally
resides in the mouth is impaired and an infection ensues. It is also worth mentioning that
73
along with ZOL imparting mild defects in neutrophils, a majority of patients who develop
BPONJ are cancer patients that are also treated concurrently with corticosteroids.
Corticosteroids act on the immune system by blocking the production of substances that
trigger allergic and inflammatory actions, such as prostaglandins. As well, they also
impede the function of white blood cells impeding the immune system from functioning
properly. Hence it may be combination of both medications (ZOL and corticosteroids)
that is imparting a deleterious effect on the innate immune response which predisposes
certain individuals to BPONJ more than others.
Furthermore, it is likely that the individual defects that have been noted in BP-treated
patients such as, impaired bone and soft tissue remodeling, impaired angiogenesis, and
the impaired immune response, are mild enough that on their own do not cause BPONJ,
but when all of these mild defects are coupled together, a vicious cycle is established
where the bone and soft tissue lesion caused by a tooth extraction or even as small as oral
ulceration is unable to heal because of persisting infection, resulting in sustained bone
resorption and the release of cytotoxic bisphosphonates. The proposed multifactorial
etiology of BPONJ may explain the rarity of this condition in BP- treated patients.
Despite infection being present in most BPONJ patients, difficulty arises when trying to
determine if infection is a primary or secondary event in the pathophysiology of this
condition. Although our data does not difinitively confirm causality, we believe that
infection is a primary etiology to BPONJ for two reasons. Firstly, our data clearly
demonstrates that ZOL disrupts neutrophil functions as well as limiting neutrophil
surivial. Extrapolating these results suggest that if a BP- treated pt is now subjected to
74
trauma to the oral cavity, such as a tooth extraction, then the neutrophils which are not
functioning or surviving to full capacity, will have difficulty initiating the wound healing
response which potentiates the development of a non-healing wound. Secondly, it is also
important to compare and contrast features of BPONJ with ORN. The pathogenesis of
ORN is defined by a sequence of radiation, hypovascular-hypocellular-hypoxic tissue
formation and trauma induced or spontaneous mucosa breakdown leading to a non-
healing wound [230]. While both BPONJ and ORN show rather identical clinical
presentations, one of the major differences histologically is the incidence of infection and
bacterial colonization in biopsies taken from BPONJ patients. While BPONJ patients
almost exclusively present with an active infection of the nectrotic bone, ORN does not
necessarily present in such a way. This is not to say that infection in ORN patients does
not occur, however most recent studies show that the incidence of Actinomyces bacteria
for example, is almost double in BPONJ patients than in ORN patients[181]. A second
group showed in a 5- year retrospective study of 50 cases of ORN of the jaw, that only
six cases (12%) of Actinomyces infection were found[231]. This is in contrast to several
studies which have shown that almost 100% of the BPONJ specimens are colonized with
multispecies microbial biofilms [190, 232]
This suggests that an additional mechanism, such as an alteration in the immune
response, may be predisposing certain patients to infection which may subsequently
develop into BPONJ.
4.8 Study Limitations
It is important to recognize that a specific limitation of this and many studies looking into
the pathogenesis of BPONJ is the fact that a majority of patients diagnosed with BPONJ
75
are concurrently being treated for malignancy. Thus it may be the fact that not only is this
patient population subjected to BPs with a higher dose and potency, but cancer therapy
usually necessitates the use of several other medications, such as corticosteroids and
cytotoxic drugs, that may either exacerbate the effects of the bisphosphonates or they
themselves may be contributing to the pathogenesis of BPONJ.
A cancer treatment that may also affect this condition is the use of G-CSF. G-CSF is the
new standard of care in certain cancer patients being treated with chemotherapy drugs. In
oncology and hematology, a recombinant form of G-CSF is used to accelerate recovery
from neutropenia and prevent infection following chemotherapy[233, 234]. Although we
are unable to come to any firm conclusion about this, it is still worthwhile pointing out
that it is quite possible that a majority of the patients that have developed BPONJ are also
treated with G-CSF . The results of our study suggest that the assumed benefits from the
exogenous G-CSF treatments in these patients may be diminished in those concurrently
treated with zoledronate.
4.9 Future Work
It would be beneficial in the future to analyze neutrophil delivery to and function in the
oral cavity of patients being treated with bisphosphonates. Oral neutrophil levels and oral
neutrophil functions will be monitored prospectively in these patients where oral rinse
samples will be collected from patients prior to initiation, and during high dose BP
therapy to determine if BPs affect neutrophil oral delivery and function. As well, we
suspect that the bisphosphonates may be disrupting the function of various other proteins
involved in cellular activity apart from RhoA that was identified in this study. We hope
76
the use of Isotope-Coded Affinity Tags (ICAT) technology may provide additional
information on possible protein targets of bisphosphonates by using quantitative
comparison of the two proteomes.
Our study is the first to present clear evidence that the bisphosphonates negatively affect
neutrophil function and survival, possibly compromising immune function.
Several rational theories have been proposed in the literature and it likely that this
condition is multifactorial in origin where bisphosphonates are released at high
concentrations into the immediate environment, and are taken up by various cells types
including epithelial cells, vascular, mesenchymal and immune cells which lead to a state
of impaired wound healing.
77
Thesis Summary
Bisphosphonates are anti-bone resorptive drugs that are primarily used for the prevention of
osteoporosis and more recently as the standard of care to prevent skeletal-related symptoms
and events in patients with multiple myeloma and metastatic bone lesions of solid tumor
cancers such as breast, prostate and lung cancer [1] [2]. A small subset of patients (1-10%)
treated with intravenous bisphosphonates are at an increased risk of developing a severe
bone disease that affects the jaws, known as bisphosphonate-induced osteonecrosis of the
jaw (BPONJ) [4, 5].
In recent years, an increased incidence of BPONJ has been associated with the use of high
intravenous dosages of bisphosphonates, required by some cancer treatment regimens,
especially when the patient undergoes subsequent dental procedures.
The publicity surrounding this complication and the lack of information about its
underlying pathogenesis has led to apprehension amongst patients and dental clinicians
when treating patients with a history of bisphosphonate therapy [6]. Critical information
that will help alleviate these concerns and improve oral treatment approaches in this patient
population is required. Being able to identify and understand the possible mechanisms
through which bisphosphonates affect normal bone and soft tissue wound healing in the
jaw will help identify patients at risk of developing BPONJ and improve patient
management.
In this thesis, we show evidence that bisphosphonate therapy negatively affects the function
and survival of cells that are key players in the immune system (neutrophils), and that
patients with BPONJ, appear to have diminished neutrophil function in comparison to their
healthy counterparts. These results suggest that a once overlooked area, the immune
78
system, may play a role as a contributing risk factor in the development of BPONJ. Future
work will concentrate on determining if worsening neutrophil levels or function
corresponds with a worsening of the disease or increased risk of the disease.
If oral neutrophil function is confirmed to be a contributing risk factor for the
development of BPONJ, we may be able to identify those patients at risk of BPONJ prior
to invasive dental treatment. This would be the first risk assessment screening tool for
this condition. Additionally, in patients on BP therapy who require oral surgery or in
those who develop BPONJ, neutrophil function assays may guide therapy in terms of
discontinuing or modifying BP therapy during the peri-operative and healing or
resolution periods.
79
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