Arthritis National Research Foundation

Arthritis National Research FoundationGrant Application Cover Sheet

Principal Investigator Name: RADHASHREE MAITRA

Position/Title: ASSISTANT PROFESSOR

Institution: ALBERT EINSTEIN COLLEGE OF MEDICINE OF YESHIVA UNIVERSITY

Address: 1300 MORRIS PARK AVENUE, JACK AND PEARL RESNICK CAMPUS

City, State, Zip: BRONX, NY-10461

Phone: 718-430-2050 ( OFFICE) 718-801-1359 ( CELL)

FAX: 347-851-5586

E-mail: [email protected]

Name of the Institutional Official responsible for grants: Prof. Edward Burns Executive Dean

Phone: 718-430-4106 email: [email protected]

Animal Subjects: Protocol no: 20060708 approval date: 08.10.07

Human Subjects: Protocol no. 00000140 approval date: CCI exempt 08.01.07

ABSTRACTTitle of Proposed Project: MOLECULAR MECHANISM OF ASEPTIC OSTEOLYSIS

Several inflammatory, autoimmune and infective conditions cause the destruction of the weight bearing surfaces of major joints causing arthritis. Over one million people in the United States each year undergo total joint arthroplasty. A commonly utilized surface material is ultra high molecular weight polyethylene (UHMWPE). Over time different size particles are generated from the UHMWPE implant. These wear debris are responsible for the initiation of an aseptic inflammatory response known as osteolysis. We have identified that “in vivo” UHMWPE breakdown generates short alkane polymers in addition to the previously reported nanometers and micrometers size particles. Majority of these polymers presented oxidized side chain modifications. Alkane side chain oxidation greatly enhanced their ability to activate dendritic cells as compared to the general inertness of the non-oxidized polymers. We have reported that mixture of oxidized alkane polymers triggered a pro-inflammatory response upon binding to TLR2 homodimers and TLR1/2 heterodimers. This is the first report of a synthetic polymer engaging a TLR on immune cells. Understanding the molecular recognition of oxidized alkane polymers by TLR-2 is very important since the oxidative process is greatly facilitated by the prosthetic implant manufacture’s procedure that creates macro-radicals. Furthermore, a model for monocytes recruitment and differentiation at the inflamed site will be developed using GFP-labeled monocytes and the murine model of calvarial osteolysis. All together the study will identify the immunological mechanisms underlying UHMWPE immuno-recognition and the pathological basis of aseptic osteolysis. These findings will also provide the basis to set up biological assays for material testing and biocompatibility, thus, help implementing changes to provide a better therapeutic and biocompatible alternative.

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The major focus of this grant is to determine why and how the immune system becomes aware of UHMWPE and is triggered to initiate an inflammatory response.

Specific Aims:The single most common cause of total joint replacement is a process known as aseptic osteolysis. Billions of dollars are spent each year on joint replacement worldwide and being able to understand and impact the process is a tremendously important undertaking. Over time different size particles and varied length polymers of wear debris are generated from the UHMWPE implant. These alkane wear debris are oxidized in vivo by introduction of side chain modifications such as carbonyl, hydroxyl, ketonic and aldehyde groups. We recently reported that organic synthetic polymers with an alkane subunit backbone and different carbonyl/amide side chain modifications act as potent TLR2 activators (Maitra, 2008). This was the first observation of a synthetic polymer interacting with a cell surface TLR.Understanding the molecular recognition of alkane polymers by TLR-2 is very important. The implant manufacturers selected UHMWPE as the material of choice for prosthetic implants, not only for its strength and durability, but also for its inertness. Our preliminary observations imply that the oxidative process introduces modifications in the side chains of the polymeric wear debris that overturns its inertness (Maitra, 2008). This in vivo oxidation process is greatly facilitated by some of the manufacture’s procedure following the implant molding, namely UV cross-linking that creates macroradicals. Macroradicals are easily converted into hydroperoxyl radicals and then further oxidized. Thus, understanding the contribution of each side chain modification/oxidation for alkane recognition by the immune system is fundamental since it would help implementing changes in the implants manufacture aimed at reducing its ability to oxidize and thus be a better therapeutic material.

The major focus of this grant is to determine why and how the immune system becomes aware of UHMWPE and is triggered to initiate an inflammatory response.

These data lead to the central hypothesis for this application; oxidized UHMWPE wear debris polymers induce activation of “local antigen presenting cells” by mimicking TLR2 receptor ligands and inducing a pro-inflammatory response. The inflammation is further amplified by recruitment of circulating monocytes and immature dendritic cells, which can differentiate into osteoclasts under the influence of the bone microenvironment, thus further advancing bone resorption.

Two aims are proposed to advance this hypothesisA1. Molecular interaction of oxidized alkane polymers with soluble TLR2 molecules. Binding affinity of chemically defined oxidized alkane polymers (with either aldehyde, ketonic, ether or hydroxyl groups) to soluble recombinant TLR2 will be determined by monitoring changes in fluorescence intensity of tyrosine present in the TLR2 (Tyr 326) binding grove. Assays will be performed in the presence or absence of anti TLR-2 mAb known to block the receptors binding groove. Additionally the biological activity of each modified polymer will be tested for dendritic cell activation and release of pro-inflammatory cytokines. Important implication for designing medical implants using alkane polymers with low oxidative potential, thus with a low interaction affinity for TLR, will be drawn by the proposed experiments.A2. Role of circulating dendritic cells in UHMWPE induced osteolysisAn in vivo model for dendritic cells recruitment and differentiation at the site of bone resorption will be developed using GFP-labeled cells and the murine calvarial model of alkane polymer

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induced osteolysis. This model recapitulates the osteolysis observed in patients with alkane implants. Oxidized alkane polymers will be injected in the mouse calvaria, development of osteolysis will be monitored by CT scan and cell recruitment by GFP labeling. Staining of GFP+

cells with cathepsin K and αVβ3 integrin will determine their differentiation into osteoclasts. To fully appreciate the role of dendritic cells in bone resorption the same experiments will be conducted in MCSF-R -/- mice, which lack endogenous osteoclasts and macrophages. Osteolysis observed in those mice will derive from recruited dendritic cells which differentiate into osteoclasts. The results will identify the role played by recruited dendritic cells in “de novo” osteoclastogenesis during bone inflammation.

Experimental DesignSpecific Aim 1:Characterization of the molecular interaction between oxidized alkane polymers and soluble TLR2 molecules. RationaleThe UHMWPE utilized in the manufacturing of prosthetic implant is characterized by long chain

sequence of -CH2- backbone, which is artificially cross-linked between carbon moieties, using UV radiation, to increase the stability and durability of the implant. Surface modifications are also introduced (OH and COOH groups) to facilitate UHMWPE coating with hydroxyapatite (Chiesa et al., 2000). Mass Spectra analysis (acquired on a Varian Fourier transform Mass Spectrometer (FTMS) equipped with a MALDI source) of the polymers generated by UHMWPE breakdown indicated the majority of the compounds presented side chain modification consisting of aldehyde, ketonic and hydroxyl groups (Maitra et al., 2008). Likely, under hyper-oxidative conditions and in

presence of many enzymatic complexes, the free radicals (formed upon breakage of the C-C cross-linking) react with oxygen giving rise to peroxide ROO radicals. Subsequently the peroxide radicals will be further converted to aldehyde, ketonic or hydroxyl groups as we previously determined by FTIR, and FTMS (Maitra et al., 2008) (Abu-Amer et al., 2007; Brach del Prever et al., 2003; Fiorito et al., 2001; Goodman, 2005; Kurth et al., 1988; Kurtz et al., 2005; Kurtz et al., 2007).

Figure 1 UHMWPE Oxidationa) UHMWPE comprises long chain alkanes. Upon molding of the implant electron beam and gamma rays are employed to sterilize the implant. b) The high energy transfer created by the gamma rays is several orders of magnitude higher than that of chemical bonds. Thus the radiation process promotes scission of C-C and C-H bonds giving rise to H● radicals disperse throughout the polymer c) the macroradicals in the presence of oxygen react readily producing hydroperoxyl radicals d) further modification include ketonic, carboxyl and ester groups.

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Even though, UHMWPE oxidation was previously reported, and is widely accepted to be the primary reason for implant failure (Kurth et al., 1988; Kurtz et al., 2005), the mechanisms responsible for the ability of alkane polymer to induce a strong inflammatory response was still unknown.In our previous analysis a mixture of oxidized polymer was utilized for the binding experiments (Maitra et al., 2008). In this Aim, using a series of chemically defined oxidized alkane polymers, we propose to pin point the effect of each side chain modification for alkane binding to TLR2 receptor. Each oxidized polymer will be compared with the non-oxidized counterpart, and the

binding affinity (Kd) to TLR2 will be determined. Furthermore, the biological activity of each modified alkane will be tested in regard to their ability to activate the TLR signal transduction pathway and production of pro-inflammatory cytokines. Identification of alkane side-chain modifications, that trigger TLR2 binding and initiation of an inflammatory cascade, would help designing strategies to prevent such modifications to occur. Altogether this would help retaining the immunological inertness of alkane polymers.

Hypothesis to be tested

Side chains modifications of UHMWPE polymers added by the in vivo oxidative process greatly enhance the binding affinity for the TLR2 receptors. Increased binding affinity results in greater production of pro-inflammatory cytokines and chemokines furthering the osteolysis.

How to evaluate the hypothesisA chemically defined set of oxidized alkane polymers will be tested in binding assay using

soluble TLR2 receptors. A binding affinity (Kd value) will be measured for each compound. The biological activity of each compound will be analyzed using TLR2 transfectant as well as primary cultures of DC. A detailed experimental plan is presented below

Methods to valuate the hypothesisOxidized alkane compounds to be testedThe oxidation of UHMWPE has been extensively studied by several groups including ours ((Abu-Amer et al., 2007; Chiesa et al., 2000; Costa et al.,1998; Fiorito et al., 2001; Goodman, 2005; Maitra et al., 2008). There is consensus in the literature that the major form of oxidation include alkane side chain modification with ketonic, carboxyl and ester groups. Our laboratory was the first one to report that oxidized alkane polymers induce an inflammatory response by binding to TLR2 and TLR1/2 receptors (Maitra, 2008). However, in our initial analysis we utilized a mixture of oxidized compounds. In this Aim we plan to utilize chemically defined alkane polymer to investigate the effect of side chain modification on TLR2 binding (Fig 2). Alkane polymers will be synthesized with a CH2 backbone comprising between 8 and 18 C since it was previously shown that optimal TLR1/2 ligands require at least 10 but

no more then 16 -CH2- groups (Buwitt-Beckmann et al., 2005). Also, polymers of similar length were found in surgical materials from patient undergoing surgery for UHMWPE induced osteolysis (Maitra, 2008). Unmodified alkane polymers will be used as control in all experiments.

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Each compound will be tested for binding affinity to soluble TLR2. Biological experiments to test their efficiency to stimulate a pro-inflammatory response will also be performed on each compound.Figure 2. Basic chemical structure of synthesized alkanes.The figure depicts alkane polymers with one side chain oxidation. Each polymer will be synthesized with 2, 3 or 4 modified groups according to the length of the polymer. Polymers will also be synthesized with either multiple number of the same side chain oxidation or multiple numbers of different side chain oxidation.

TLR2 binding activity of oxidized alkane polymersTo analyze the TLR2 binding activity of modified alkane polymers a soluble form of human recombinant TLR2 (extracellular domain Glu 21-Leu 590) will be utilized in a binding assay that monitored changes in fluorescence intensity (λexcitation= 277 nm and λemission = 335 nm) of a tyrosine present in the TLR2 binding grove (Tyr 326) (Jin et al., 2007). Changes in the binding grove environment, due to ligand occupancy would change the Tyr fluorescence intensity and the wavelength of its fluorescence emission. Soluble TLR-2 will be incubated with increasing concentrations of each polyethylene derivatives and the positive control Pam2CSK4 lipopeptide (known to be a specific ligand for TLR-2)(Jin et al., 2007). The emission scans (between 290 and 420 nm) will be collected for each complex separately and the change in maximum fluorescence signal at 335 nm (due to tyrosinate ion) will be used to generate the binding curves, after subtracting the contribution of the free protein (in the absence of any compounds). Mean and standard deviation of the fluorescence readout will be calculated for each one of the tested polymers in a set of three independent experiments. The normalized fluorescence data will be fitted to a hyperbolic function using the software GraphPad Prism 4. The same binding assays will be performed following TLR2 incubation with a monoclonal antibody (anti human TLR2 clone 383936 R&D Systems) which is known to prevent ligand access to the TLR2 binding groove (Massari et al., 2006). A Kd value will be calculated for each polymer TLR2 binding curve. A similar analysis was previously conducted to establish the TLR 2 binding activity of a mixture of oxidized alkane polymers (Maitra et al., 2008).

NF-κB activation by oxidized alkane polymersNF-κB activation is one of the most prominent features of the TLR activation pathway and is generally used to evaluate activation of the inflammatory cascade following the TLR1/2 receptor activation which culminates in release of pro-inflammatory cytokines. Human 3T3 HEK cell lines stably expressing TLR1/2 or TLR2 are currently available in the laboratory (Invivogen). These lines have also been transfected with a plasmid encoding the luciferase reporter gene under the control of the NF-κB enhancer element. Thus, NF-κB activation following TLR receptor engagement will in turn, up-regulate luciferase production. Each line will be assayed for activation, measured as luciferase activity with every single one of the oxidized alkane polymers. Each assay will also include Pam2CSK4 lipopeptide as specific positive ligand for both TLR2 and TLR1/2 heterodimers. The luciferase readout will be measured at different time points (3-6-9-12-24 hours) using the standard Luciferase reporter assay kit (Promega).Mean and standard deviation of the luciferase readout will be calculated for each one of the tested polymers in a set of three independent experiments.

Dendritic Cells activation Peripheral blood will be obtained from the New York Blood Bank. Mononuclear cells will be separated over a Ficoll gradient and the monocyte population separated using CD14 conjugated MicroBeads (Miltenyi Biotec). Purified CD14+ cells will be differentiated in presence of GM-CSF/IL4 (30 ng/ml plus 10 ng/ml) to obtain DC for 5 to 6 days in RPMI 1640 (GIBCO,

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Grand Island, NY, USA). Immature DC will be cultured in presence of different amount of each one of the oxidized alkane polymers for 24 and 48 hours. To determine which one of the oxidized alkane polymer was capable of inducing DC activation a surface staining will be performed. DC will be washed with cold PBS, and labeled for 30 min on ice with saturating  amounts  of anti human HLA-DR (clone TU36), B7-1 (CD80) or B7-2 (CD86) (BD Biosciences, Pharmingen, San Diego, CA) in staining buffer  (PBS/0.1 % BSA/  0.01% NaN3). Cells will be analyzed using a FACSCalibur flow cytometer and cellquest software program (BD Biosciences Mountain View, CA, USA).Mean and standard deviation of the mean fluorescence index will be calculated for each one of the tested polymers in a set of three independent experiments.

ELISA for cytokines and chemokines. Cell culture supernatants will be collected from dendritic cells previously cultured with different amount of each one of the oxidized alkane polymers for 24 and 48 hours. A panel of 27 cytokines and chemokines (IL-1 beta, IL-1ra, IL-2, IL-4, IL-5, IL-6 IL-7 IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, Basic FGF, Eotaxin, G-CSF, GM-CSF, IFN-gamma, IP-10, MCP-1, MIP-1 alpha, MIP-1-beta, PGDF-BB, Rantes, TNF-alpha, VEGF) will be quantified by ELISA using the 27-Multiplex panel (Bio-Rad). Mean and standard deviation will be calculated for each cytokine value obtained from each polymer tested in triplicate. The data will determine the set of polymer oxidation(s) eliciting the strongest inflammatory response.

Troubleshooting and Data InterpretationResults from experiments proposed in aim 1 will determine the molecular form(s) of the alkane polymers capable of direct activation of TLR-2. Comparison between Kd values obtained for each polymer will indicate which modification(s) have the highest affinity for TLR-2. Data for the biological activity of each polymer will also be collected; up-regulation of surface MHC-class II and co-stimulatory molecules as well as cytokines release by dendritic cells. Altogether a map of the pro-inflammatory activity of each modified polymers will be developed. Several important information will be gained by these experiments:

* Does an increase in the level of polymer oxidation increase TLR2 binding affinity (hydroperoxyl radicals vs ketonic ether and carbonyl modification)?* Do the number, position and type of side chain modification change the TLR2 binding affinity?* Is there a correlation between TLR2 binding affinity and biological activity (DC activation and cytokine release)?

The results will determine unequivocally whether the immunogenecity of alkane polymers is related to their oxidation potential. Also the data will help designing a strategic approach for producing UHMWPE implants that still retain their strength and durability but prevent polymer modifications/oxidation to occur. As a “side result” of this project we will gain information on a new category of TLR2 agonists, non organic in nature, that could prove useful when immune system activation is needed.

A2. Role of circulating dendritic cells in UHMWPE induced osteolysis.Periprosthetic osteolysis is the major cause of premature failure of total joint replacements. The osteolytic process is due to the immune recognition of the oxidized alkane polymers by TLR2 receptors (Maitra et al., 2008) which results in a strong pro-inflammatory response leading to extensive re-absorption of the bone tissue. De novo osteoclastogenesis is a common feature of UHMWPE induced osteolysis and is a main cause of bone re-absorption (Abu-Amer et al., 2007). De novo formed osteoclasts could in part be derived from the differentiation of osteoclast progenitors, which are present in the bone microenvironment. On the other hand DC attracted to

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the site of inflammation could also potentially differentiate into osteoclast. In vivo DC differentiation into osteoclasts has never been previously analyzed.

Hypothesis to be tested

Circulating DC’s are recruited to the site of inflammation and can differentiate into osteoclasts further enhancing the osteolytic process.

How to valuate the hypothesis Under physiological circumstance osteoclast precursors are generated in the bone marrow,

released into the blood stream and recruited back to the bone tissue to form mature osteoclast (Koulouvaris et al., 2007). A common myeloid progenitor gives rise to monocytes macrophages dendritic cells and osteoclast precursors and is characterized by CD11b+/GR1-

low/C-Fms-/+

phenotype. The differentiation of this early stage precursor into the late stage osteoclast progenitors (CD11b+/GR1-

low/C-Fms-)

involves M-CSF as a major growth factor. Another growth factor that is essential for osteoclast formation is RANK L. RANK L, signaling through its

receptor activator NF-kB (RANK), present on osteoblasts supports osteoclastogenesis.

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Receptors for both MCSF and RANKL are present on different myeloid derived cells indicating that there is a potential for their transformation into osteoclast.MCSF-R KO mice have no osteoclast and reduced number of early and late osteoclast progenitors (Dai et al., 2002; Dai et al., 2004)These mice are currently available in the laboratory (kind gift of Richard Stanley, AECOM and constitute a perfect model to address the osteoclastogenic potential of immature DC.

Figure 3. Experimental flow chart to determine DC recruitment at the site of osteolysisDay 1 MCSF-R ko and littermate heterozygous (both in the C57/Bl6 background) undergo surgery (sham or polymer implanted). Day 10 a micro-CT scan of mouse calvaria is performed. A representative frontal section is shown out of a total 162 sections. Images are collected from each mouse and compared before and after surgery to assess the level of bone resorption (Statistical analysis of the following parameters, collected before and after surgery will be performed: cortical volume, trabecular volume, total volume, cortical bone mass density and total mass density). Day 12; all groups will receive iv. Injection of GFP-labeled DC. Day 16; Calvaria will be collected from each sham and polymer implanted mice; (full calvaria 5X is shown). Fluorescence microscopy performed on the calvarial sections will detect recruitment of GFP-labeled dendritic cells (infiltrates are shown as GFP+ cells (fluorescent channel) or overlay between fluorescent and light microscopy both at 63X). Calvaria from each mouse will be process for confocal microscopy or FACS quantification of GFP-DC recruited at the site of osteolysis.

In these mice all endogenous myeloid precursors’ lacks expression of the M-CSF-R, thus cannot differentiate into osteoclast. Injection of exogenous GFP+ immature DC, which do express M-CSF-R and RANK-L will constitute a clean model to address the osteoclastogenic potential of immature DC.

A mouse model of osteolytic necrosis, closely resembling the aseptic necrosis observed in patients with UHMWPE implants, is already available in the laboratory (Figure 3). Oxidized alkane polymers, manufactured to mimic the oxidations observed in UHMWPE failed prosthesis (Abu-Amer et al., 2007; Brach del Prever et al., 2003; Jahan et al., 1995; Maitra et al., 2008), will be implanted under the periostium of the mouse calvaria (Figure 3) Bone resorption will be quantified by CT scan (preliminary data indicate that bone resorption is already evident 10 days following surgery) (Figure 3). GFP-positive DC will be injected intervenous into the mice and their recruitment to the calvaria will be assessed by confocal analysis of calvaria bone sections. In order to be effective participant in bone remodeling recruited DC cells need to undergo several transformations. Firstly, an actin polarization at the plasma membrane is required to form a close contact between the recruited cells and the bone surface. This polarization is mediated by the αVβ3 integrin which is absent on DC but expressed at high levels on osteoclasts. Secondly a polarization of the lysosomal compartments near the plasma membrane has to occur to enable H+-ATPase mediated extracellular transport of protons. The acidification

of the extracellular environment mobilizes the bone mineral, thereby exposing the organic phase of bone that is degraded by cathepsin K. Such enzyme is absent in immature DC but very much up-regulated in osteoclasts. Confocal analysis will be utilized to determine whether GFP-positive DC, upon recruitment, start expressing markers classically associated with osteoclasts and bone resorption (αVβ3 integrin, cathepsin K, and actin ring polarization). Also, confocal analysis will determine whether recruited DC are observed in the Howship’s lacunae which are the site

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were bone resorption occur. Bone resorbing osteoclasts, organized into multinucleated giant cells, attach to the bone matrix and upon polarization of their lysosomes start secreting H+ ions as well as cathepsin K. The organization into giant cells is required to form a cell wall around the bone matrix to ensure that the lytic enzymes are uniquely released and contained within the resorptive microenvironment. These formations are known as bone resorptive pits or Howship’s lacunae and can be easily identified as pits in the bone tissue surrounded by multinuclear giant cells (Figure 12). The recruitment of injected GFP-DC into multinuclear giant cell formation present around the Howship’s lacunae will also be analyzed by confocal microscopy.

Figure 4. Howship’s lacunae. Immunohistochemistry of CD68+ giant cells osteoclast into a resorptive pit.

Methods of evaluate of the hypothesis MCSF-R ko miceMacrophage colony-stimulating factor (MCSF) is indispensable for both proliferation and differentiation of osteoclast progenitors (Tanaka et al., 1993). MCSF-R knock out mice (Csf1r(-/-) mice) were generated and previously characterized in the laboratory of R. Stanley at Albert Einstein College of Medicine (Dai XM et al., 2002 ). These mice confirmed that MCSF-R mediated signaling is critical for osteoclastogenesis since a severe osteoclast and macrophages deficiency is observed in the homozygous littermates. A set of 10 breeding cages has already been set up in our facility. Genotyping of the transgenic mice (to distinguish heterozygous from homozygous knock down) will be performed as previously reported (Dai XM et al., 2002 ).

Murine Calvarial Model. A murine calvarial model of UHMWPE osteolysis has already been set up in the laboratory (Wedemeyer et al., 2007) Briefly both MCSF-R ko (Csf1r (-/-) or littermates control (-/+) will be randomized in two groups. One group will undergo sham surgery the other group will receive chemically defined modified alkane polymers. Mice in each group will be sedated with isoflurane gas and anesthetized with Ketamine (20% Ketamine HCl, 15% Xylazine and 65% Saline at 0.1ml per 20 grams body weight). A 10 mm incision will be made over the calvaria sagittal midline suture. A 1.0X1.0 cm area of the periosteum will be exposed. In the control group the incision will be closed without any further intervention whereas in the experimental set mice will received 10 microgram of a mixture of oxidized alkane polymers. The polymers will be distributed over the periosteum using a sterile sharp surgical spatula. Pre and post (twelve days) operative micro-computed tomography (micro-CT scan) will be performed on each subject to confirm and verify development of particle induced osteolysis. Micro-CT scan will be performed with a La Theta software (Omnipotent CT) running on an Aloka La Theta laboratory CT scan machine (Japan). The x-ray voltage will be set at high. For each skull 162 slices will be scanned with an average section to section distance of 0.03 nm at slow acquisition speed with face down head front position. Statistical analysis of the following collected parameters will be performed among the groups: cortical volume, trabecular volume, total volume, cortical bone mass density and total mass density. Data will quantify the amount of bone resorption between MCSF-R ko (Csf1r (-/-) or heterozygous (-/+) littermates control.

DC recruitment to the calvaria. In a similar set of experiments 14-16 days after surgery mice will be injected intervenous. with 8-10x106 bone marrow derived DC prepared from male C57/Bl6 beta-actin GFP Transgenic mice (Jackson laboratory). DC will be prepared from total bone marrow (cultured in GM-CSF for 7-8 days) and purified using CD11c+ conjugated magnetic beads (Miltenyi Biotech). In some experiments 96 hours after DC injection calvaria will be isolated and directly observed as a whole under fluorescence microscopy. In other experiments 96 hours following GFP-DC

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injection cells will be retrieved from the calvaria and analyzed by FACS to determine the percentage of recruitment of GFP positive DC at the site of osteolysis.

Confocal Microscopy. Calvaria from surgically implanted mice and sham controls will be extracted and fixed in 4% paraformaldehyde in PBS pH 7.4 and placed at 4oC. After 24 hours, samples will be placed in 10% EDTA in PBS pH 7.4 for 48-72 hours at 4oC until decalcified. Calvaria samples will then be placed in O.C.T. compound (Tissue-Tek, Sakura-Fientek, Torrance, CA) and frozen in 2-methylbutane chilled in dry ice. Ten micrometer frozen sections will be placed on glass slides and stored at -20oC for immunohistochemical evaluation. One set of serial sections will be directly visualized under fluorescent microscope to determine GFP-DC recruitment to be bone and giant cells formation. A second set of slides will be stained with Cathepsin K, beta-actin and αVβ3 integrin antibodies. Injected DC are cathepsin K and αVβ3 integrin negative, also the β-actin staining is spread throughout the cytosol (data not shown). Thus confocal analysis will determine whether GFP+-DC, upon recruitment to the bone matrix acquire cathepsin K and αVβ3 as osteoclasts markers and polarize the β-actin, typical of lysosomal secreting cells. Stained slides were examined using a scanning confocal microscope (Leica AOBS system). All images were collected under identical PMT detector settings.

Troubleshooting and Data InterpretationThe results from these experiments will determine whether DC’s can differentiate into osteoclasts upon recruitment to the bone micro-environment. Importantly, since the experiments will be performed in mice genetically lacking endogenous macrophages and osteoclasts we will be able to address the role of DC in bone re-absorption. The data will open future investigation on the importance of DC in different bone-related pathologies such as rheumatoid arthritis.

Preliminary DataThe preliminary data which supports and initiates the current research proposal has been recently published 1. Maitra, R., Clement, C.C., Crisi, G.M., Cobelli, N., Santambrogio L. Immunogenecity of modified alkane polymers is mediated through TLR1/2 activation. PLoS ONE. 2008 Jun 18;3 (6):e2438) and 2. Maitra R, Clement CC, Scharf B, Crisi GM, Chitta S, Paget D, Purdue PE, Cobelli N, Santambrogio L. Endosomal damage and TLR2 mediated inflammasome activation by alkane particles in the generation of aseptic osteolysis. Mol Immunol. 2009 Oct 3. [Epub ahead of print]

TIME LINE FOR THE PROJECTIn the first year emphasis will be given towards the molecular characterization of the

oxidative substitution of the alkane polymers. Experiments like the TLR2 binding assay, determination of NF-κB activation and up-regulation of pro-inflammatory cytokines will be done extensively to pinpoint and characterize the specific side chain oxidation that is most immuno-potent. Identification of the exact nature of oxidation will have a major contribution in future implant design. Altogether the findings will help in achieving better therapies for arthritis patients with total joint destruction.During the second year efforts will be vested on evaluating the in vivo consequences. Although the study will commence from the first year, the generation of sufficient number of homozygous MCSF-R knockout mice is anticipated to take time. The homozygous litters are extremely small in size with stunned growth and 40% of them die within the first month of life. This mouse model is extremely important for the study and subject has to be 10-12 weeks age before it can be

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utilized for calvarial surgery. Analysis of the calvarial osteolysis pre and post surgery by CT scan and immuno-histochemistry will be extensively utilized to follow the fate and contributions of antigen presenting cells during initiation and development of osteolysis. Studies will be directed to follow if any osteoclastogenesis occur as a consequence of the presence of UHMWPE and the exact mechanism of the formation of osteoclasts.

Selected References

BIOGRAPHICAL SKETCH

NAME: RADHASHREE MAITRA POSITION: ASSISTANT PROFESSOR

Summary of Education

Year Institution level Subject Grade1996 Calcutta

UniversityPh.D Biophysics, Molecular Biology & Genetics

1989 Calcutta University

MS Biochemistry I

1987 Calcutta University

BS Chemistry honors Physics & Mathematics I

Professional Experience

2009- Assistant Professor , Department of Surgery and Department of Pathology, Albert Einstein College of Medicine, Bronx, NY2006-2008 NIH training Research Fellow Department of Pathology, Albert Einstein College of Medicine, Bronx, NY2002- 2006 Research Associate, Department of Pathology, Albert Einstein College of Medicine, Bronx NY2001-2002 Research Associate in Department of Biotechnology, Jadavpur University, Calcutta, India1999-2001 Research Scientist in Bioinformatics Calcutta University, Calcutta, India.1996-1999 Research Associate Department of Biology Washington University, St.Louis MO.

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Publications

1.Maitra R, Clement CC, Scharf B, Crisi GM, Chitta S, Paget D, Purdue PE, Cobelli N, Santambrogio L. Endosomal damage and TLR2 mediated inflammasome activation by alkane particles in the generation of aseptic osteolysis. Mol Immunol. 2009 Oct 3. [Epub ahead of print]2. Maitra R, Sadofsky M.J. "A WW-like module in the RAG1 N-terminal domain contributes to previously unidentified protein-protein interactions. "Nucleic Acids Res.2009 Jun; 37(10):3301-9.3. Maitra, R., Clement, C.C., Crisi, G.M., Cobelli, N., Santambrogio L. Immunogenecity of modified alkane polymers is mediated through TLR1/2 activation. PLoS ONE. 2008 Jun 18;3 (6):e24384. Bunbury, A., Potolicchio, I., Maitra, R., Santambrogio L. Functional analysis of monocyte MHC class II compartments. FASEB J. 2009 Jan;23(1):164-715..Zak, E.A.., Norling, B., Maitra, R., Huang F., Anderson, B., and Pakrasi, H.B. (2001) The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes. Proc Natl Acad Sci U S A. 98, 13443-8.6.Inagaki, N.,Maitra, R.,Satoh, K. and Pakrasi, H.B. (2001) Amino acid residues that are critical for in vivo catalytic activity of CtpA, the carboxyl-terminal processing protease for the D1 protein of photosystem II. J Biol Chem, 276, 30099-105.7.Maitra, R. and Thakur, A.R. (1993) Multiple fragment ligation on glass surface: A novel approach. Indian Journal of Biochemistry and Biophysics, 31, 97-99. 8. Maitra, R. and Thakur, A.R . (1993) Hydroxyl radical induced DNA damage: choice of an in vitro model. International Journal of Toxicology, Occupational and Environmental Health. 56, 6-7. 9. Maitra, R. and Thakur, A.R. (1992) Silanisation of glass bound baked DNA permits enhanced polymerization by DNA polymerase. Current Science 62, 586-588.

Honors

•Winner of "SHANTI BHAKTA MEMORIAL AWARD" for the best speaker at the student seminar held between 15th-21st January 1990 in the Department of Biochemistry ,Calcutta University. •Qualified at the "ALL INDIA NATIONAL ELIGIBILITY TEST (NET)"of 1989 in LIFE SCIENCES and was awarded scholarship and eligibility to carry out research for the attainment of doctorate degree from any Indian University or Institute. The scholarship was valid for five years. •Qualified for Senior Research Fellowship at all India level in the examinations conducted by "COUNCIL FOR SCIENTIFIC AND INDUSTRIAL RESEARCH (CSIR) of 1993. The fellowship was valid for three years.

Membership

•.Member of the "SOCIETY OF BIOLOGICAL CHEMISTS (INDIA)". • Member of "INDIAN BIOPHYSICAL SOCIETY". •. Member of “ NEW YORK ACADEMY OF SCIENCE”

Scientific Review

Invited reviewer of Journal of Clinical and developmental Immunology.

Scientific meetings

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•Endosomal damage and TLR2 mediated inflammasome activation by alkane particles in the generation of aseptic osteolysis Radhashree Maitra, Cristina C. Clement, Brian Scharf, Giovanna M, Crisi, Sriram Chitta Daniel Paget, P. Edward Purdue, Neil Cobelli, Laura Santambrogio. Albert Einstein College of Medicine (AECOM), Bronx, NY, USA. Oral Presentation. 4th International Meeting on UHMWPE for arthroplasty from powder to debris, 16th

-18th September 2009 . Turin, Italy.• Self-Peptidomic Repertoire of the human pre-nodal lymph.Clement, Cristina C.; Maitra, Radhashree, Sahu, Ranjit, Santambrogio, Laura. Pathology, Albert Einstein College of Medicine (AECOM), Bronx, NY, USA. Young Investigator Award talk, 21st American Peptide Symposium, Bloomington, Indiana-July 2009.• Binding of modified alkane polymers to human recombinant TLR-2 receptor monitored by intrinsic Tyr fluorescence. Clement, Cristina C.; Maitra, Radhashree Santambrogio, Laura. Pathology, Albert Einstein College of Medicine (AECOM), Bronx, NY, USA. Abstracts of Papers, 236th ACS National Meeting, Philadelphia, PA, United States, August 17-21, 2008 (2008), BIOL-178.

Career Development Plan

I am training myself towards becoming an independent investigator. On annual evaluation of my performance I have been promoted from a Research Fellow to Assistant Professor at the Department of Orthopedic Surgery as well as Department of Pathology, Albert Einstein College of Medicine of Yeshiva University with effect from 01.01.09. This is a major step forward towards becoming an independent scientist. I have been associated with AECOM for past 5 years. During the past three years I have solely concentrated in the study of the molecular mechanism underlying the development of aseptic arthritis caused at first by destruction of natural joints and then subsequently by implant failure. I have done series of studies employing both biophysical as well as biochemical techniques to elucidate the exact mechanism of immuno-activation by UHMWPE. The first part of my findings was published in Plos ONE (2008). This study for the first time reported that a synthetic organic compound is capable of activating the TLR pathway. This was followed by a second publication in Molecular Immunology (2009). Two more manuscripts are under review. I plan to establish a well funded research project on osteo-arthritis in the near future.I have also developed association and collaboration with clinicians and orthopedic surgeons for surgical specimen and guidance to further enhance my possibilities of success in the field. The valuable observation regarding the activation of TLR1/2 pathway have urged me to study the clinical pathology of periprosthetic osteolysis at the deep root molecular level for next few years and utilize the data and publications that will be generated to acquire research funding for development of an independent laboratory focusing specifically on the field of osteoimmunology. Once the exact molecular mechanism is clearly understood I of my primary focus would be to try to develop collaboration with implant manufacturing companies and develop a series of standardized testing to find a less immunopotent but more bio-compatible polyethylene substitute that would have a better therapeutic impact and thus ameliorate the sufferings of the implant failure patients with overall improvement in the quality of life.

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Arthritis National Research Foundation

PROPOSED BUDGET

Principal Investigator: DR. RADHASHREE MAITRA

Title of Proposal: MOLECULAR MECHANISM OF ASEPTIC OSTEOLYSIS Category Amount Requested

Personnel/Salary $ 30000.00

Supplies $ 10000.00

Equipment $ 3000.00

Animals $ 5000.00

Travel to Professional Meetings $ 2000.00

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TOTAL REQUESTED $ 50000.00

Please note that ANRF will not fund indirect or overhead expenses, or post-doctoral researchers ” to be named.”

Justification of the Budget

Personnel Radhashree Maitra (PI) of this project will draw a part of her salary which will amount to $30000.Supplies: $ 10000 will be spent towards buying several chemicals, including antibody labeled magnetic beads, cytokines such as GM-CSF, M-CSF, RANK, RANK-L, antibodies to cathepsins K, S, B, D and L for western blotting. Fees for the services used at the in house facilities: confocal imaging, electron microscopy, histology, FACS and proteomics facility are also included.Equipments: A few small equipments will be purchased with in a budget of $ 3000, one vortex machine, one set of pipetteman, one magnetic separation column, one small refrigerator.Animals: $5,000 will be spend towards purchasing TLR2 knock out mice, GFPlabelled beta actin mice, maintenance of CSF-1 -/- knock out mice as well as C57/BL6 mice necessary for setting up of experiments of the mouse calvarial model of osteolysis. Travel to Professional Meetings: $2000.00 will be utilized for presenting the data generated out of the current investigation at appropriate meetings and conferences.

Current and Pending Support

NONE

Name of the Institutional official responsible for acknowledging approval for submission of the proposal

Prof. Edward BurnsExecutive DeanAlbert Einstein College of medicineTel: (718) 430-4106

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Email: [email protected]

Details of Institutional Review Board Approval for study of Animal and Human subjects

Animal Subjects: Protocol no: 20060708 approval date: 08.10.07

Human Subjects: Protocol no. 00000140 approval date: CCI exempt 08.01.07

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