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1.  Macromolecular prodrug of dexamethasone prevents particle-induced peri-implant osteolysis with reduced systemic side effects 
Aseptic implant loosening related to implant wear particle-induced inflammation is the most common cause of failure after joint replacement. Modulation of the inflammatory reaction to the wear products represents a rational approach for preventing aseptic implant failure. Long-term treatment using anti-inflammatory agents, however, can be associated with significant systemic side effects due to the drugs' lack of tissue specificity. To address this issue, N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-dexamethasone conjugate (P-Dex) was developed and evaluated for prevention of wear particle-induced osteolysis and the loss of fixation in a murine prosthesis failure model. Daily administration of free dexamethasone (Dex) was able to prevent wear particle-induced osteolysis, as assessed by micro-CT and histological analysis. Remarkably, monthly P-Dex administration (dose equivalent to free Dex treatment) was equally effective as free dexamethasone, but was not associated with systemic bone loss (a major adverse side effect of glucocorticoids). The reduced systemic toxicity of P-Dex is related to preferential targeting of the sites of wear particle-induced inflammation and its subcellular sequestration and retention by local inflammatory cell populations, resulting in sustained therapeutic action. These results demonstrate the feasibility of utilizing a macromolecular prodrug with reduced systemic toxicity to prevent wear particle-induced osteolysis.
doi:10.1016/j.jconrel.2013.11.024
PMCID: PMC3946775  PMID: 24326124
HPMA copolymer; prodrug; inflammation targeting; dexamethasone; implant loosening; ELVIS
2.  Comprehensive profiling analysis of actively resorbing osteoclasts identifies critical signaling pathways regulated by bone substrate 
Scientific Reports  2014;4:7595.
As the only cells capable of efficiently resorbing bone, osteoclasts are central mediators of both normal bone remodeling and pathologies associates with excessive bone resorption. However, despite the clear evidence of interplay between osteoclasts and the bone surface in vivo, the role of the bone substrate in regulating osteoclast differentiation and activation at a molecular level has not been fully defined. Here, we present the first comprehensive expression profiles of osteoclasts differentiated on authentic resorbable bone substrates. This analysis has identified numerous critical pathways coordinately regulated by osteoclastogenic cytokines and bone substrate, including the transition from proliferation to differentiation, and sphingosine-1-phosphate signaling. Whilst, as expected, much of this program is dependent upon integrin beta 3, the pre-eminent mediator of osteoclast-bone interaction, a surprisingly significant portion of the bone substrate regulated expression signature is independent of this receptor. Together, these findings identify an important hitherto underappreciated role for bone substrate in osteoclastogenesis.
doi:10.1038/srep07595
PMCID: PMC4274512  PMID: 25534583
3.  Implant based differences in adverse local tissue reaction in failed total hip arthroplasties: a morphological and immunohistochemical study 
Background
Adverse local tissue reaction (ALTR) is characterized by periprosthetic soft tissue inflammation composed of a mixed inflammatory cell infiltrate, extensive soft tissue necrosis, and vascular changes. Multiple hip implant classes have been reported to result in ALTR, and clinical differences may represent variation in the soft tissue response at the cellular and tissue levels. The purpose of this study was to describe similarities and differences in periprosthetic tissue structure, organization, and cellular composition by conventional histology and immunohistochemistry in ALTR resulting from two common total hip arthroplasty (THA) implant classes.
Methods
Consecutive patients presenting with ALTR from two major hip implant classes (N = 54 patients with Dual-Modular Neck implant; N = 14 patients with Metal-on-Metal implant) were identified from our prospective Osteolysis Tissue Database and Repository. Clinical characteristics including age, sex, BMI, length of implantation, and serum metal ion levels were recorded. Retrieved synovial tissue morphology was graded using light microscopy and cellular composition was assessed using immunohistochemistry.
Results
Length of implantation was shorter in the DMN group versus MoM THA group (21.3 [8.4] months versus 43.6 [13.8] months respectively; p < 0.005) suggesting differences in implant performance. Morphologic examination revealed a common spectrum of neo-synovial proliferation and necrosis in both groups. Macrophages were more commonly present in diffuse sheets (Grade 3) in the MoM relative to DMN group (p = 0.016). Perivascular lymphocytes with germinal centers (Grade 4) were more common in the DMN group, which trended towards significance (p = 0.066). Qualitative differences in corrosion product morphology were seen between the two groups. Immunohistochemistry showed features of a CD4 and GATA-3 rich lymphocyte reaction in both implants, with increased ratios of perivascular T-cell relative to B-cell markers in the DMN relative to the MoM group (p = 0.032).
Conclusion
Our results demonstrate that both implant classes display common features of neo-synovial proliferation and necrosis with a CD4 and GATA-3 rich inflammatory infiltrate. Qualitative differences in corrosion product appearance, macrophage morphology, and lymphocyte distributions were seen between the two implant types. Our data suggests that ALTR represents a histological spectrum with implant-based features.
doi:10.1186/1472-6890-14-39
PMCID: PMC4169255  PMID: 25242891
Adverse local tissue reaction; Corrosion products; Revision arthroplasty; Synovial inflammation
4.  The influence of synovial inflammation and hyperplasia on symptomatic outcomes up to two-years post-operatively in patients undergoing partial meniscectomy 
Objective
Synovitis is associated with pain and other symptoms in patients with knee OA, and in patients with meniscal tears even in the absence of radiographic OA. Patients undergoing arthroscopic partial meniscectomy were followed for 2 years to determine whether synovitis predicts post-operative symptoms.
Design
Thirty-three patients scheduled for arthroscopy were recruited for this pilot study. Symptoms were assessed using a knee pain scale, the Lysholm score, and the SF-12® pre-operatively and at 16 weeks, 1 year and 2 years post-operatively. Synovial inflammation and hyperplasia were graded on surgical biopsies. Linear mixed effects models were tested to determine whether inflammation or hyperplasia is associated with outcome scores over time.
Results
Lysholm scores and SF-12® physical component sub-scores were worse pre-operatively in patients with inflammation (Lysholm: 52.42 [95%CI 42.37,62.47] vs. 72.38 [66.03,78.72], p<0.001; SF-12: 36.81 [28.26,45.37] vs 46.23 [40.14,52.32], p<0.05). Up to two-years post- operatively, patients with inflammation achieved mean scores similar to those without inflammation. As a result, the mean improvement in Lysholm scores was 13.01 [1.48–24.53] points higher than patients without inflammation, p = 0.03. 33% (4/12) of patients with inflammation still had fair-to-poor Lysholm scores two years after surgery compared to 7% (1/15, (p=0.14) without inflammation. No association between hyperplasia and symptoms was noted.
Conclusions
In this pilot study of patients undergoing partial meniscectomy, synovial inflammation was associated with worse pre-operative symptoms, but not with poorer outcomes in the first two years post-arthroscopy. Larger cohorts and longer follow-up should be pursued to confirm this relationship, and determine if the initial response is sustained.
doi:10.1016/j.joca.2013.05.011
PMCID: PMC3753697  PMID: 23973154
synovitis; osteoarthritis; meniscal tear; inflammation; arthroscopy
5.  In vivo cyclic compression causes cartilage degeneration and subchondral bone changes in mouse tibiae 
Arthritis and rheumatism  2013;65(6):1569-1578.
Objectives
Alterations in the mechanical loading environment in joints may have both beneficial and detrimental effects on articular cartilage and subchondral bone and subsequently influence the development of osteoarthritis (OA). We used an in vivo tibial loading model to investigate the adaptive responses of cartilage and bone to mechanical loading and to assess the influence of load level and duration.
Methods
We applied cyclic compression of 4.5 and 9.0N peak loads to the left tibia via the knee joint of adult (26-week-old) C57Bl/6 male mice for 1, 2, and 6 weeks. Only 9.0N loading was utilized in young (10-week-old) mice. The changes in articular cartilage and subchondral bone were analyzed by histology and microcomputed tomography.
Results
Loading promoted cartilage damage in both age groups, with increased damage severity dependent upon the duration of loading. Metaphyseal bone mass increased in the young mice, but not in the adult mice, whereas epiphyseal cancellous bone mass decreased with loading in both young and adult mice. Articular cartilage thickness decreased, and subchondral cortical bone thickness increased in the posterior tibial plateau in both age groups. Both age groups developed periarticular osteophytes at the tibial plateau in response to the 9.0N load, but no osteophyte formation occurred in adult mice subjected to 4.5N peak loading.
Conclusion
This non-invasive loading model permits dissection of temporal and topographical changes in cartilage and bone and will enable investigation of the efficacy of treatment interventions targeting joint biomechanics or biological events that promote OA onset and progression.
doi:10.1002/art.37906
PMCID: PMC3672344  PMID: 23436303
Mechanical loading; cartilage; subchondral bone; osteoarthritis; mice
6.  Proteomic analysis of synovial fluid from the osteoarthritic knee: comparison with transcriptome analyses of joint tissues 
Arthritis and rheumatism  2013;65(4):981-992.
Objective
The pathophysiology of the most common joint disease, osteoarthritis (OA), remains poorly understood. Since synovial fluid (SF) bathes joint cartilage and synovium, we reasoned that a comparative analysis of its protein constituents in health and OA could identify pathways involved in joint damage. A proteomic analysis of knee SF from OA patients and control subjects was performed and compared to microarray expression data from cartilage and synovium.
Methods
Age-matched knee SF samples from control subjects, and patients with early- and late-stage OA (n=10 per group) were compared using two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A MS with multiplexed peptide selected reaction monitoring (SRM) assay was used to confirm differential expression of a subset of proteins in an independent OA patient cohort. Proteomic results were analyzed by Ingenuity pathway analysis and compared to published synovial tissue and cartilage mRNA profiles.
Results
66 proteins were differentially present in healthy and OA SF. Three major pathways were identified among these proteins: the acute phase response, and the complement and coagulation pathways. Differential expression of 5 proteins was confirmed by SRM assay. A focused analysis of transcripts corresponding to the differentially present proteins indicates that both synovial and cartilage tissues may contribute to the OA SF proteome.
Conclusion
Proteins involved in the acute phase response, complement and coagulation pathways are differentially regulated in SF of OA patients suggesting they contribute to joint damage. Validation of these pathways and their utility as biomarkers or therapeutic targets in OA is warranted.
doi:10.1002/art.37823
PMCID: PMC3618606  PMID: 23400684
7.  The Effect of Paraformaldehyde Fixation on the Delayed Gadolinium-Enhanced MRI of Cartilage (dGEMRIC) Measurement 
The delayed Gadolinium-Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC) method allows for both qualitative and quantitative measurement of the spatial distribution of glycosaminoglycan [GAG] in excised cartilage. The objective of this study was to determine the effect of paraformaldehyde fixation on dGEMRIC measurements. Five bovine and seven human cartilage pieces were punched into 5-mm plugs, fixed for 18 h in 4% paraformaldehyde solution, and washed. The magnetic resonance imaging (MRI) parameter T1 was measured prior and post fixation in cartilage without (T10) and with (T1Gd), the ionically charged MRI contrast agent Gd(DTPA)2−. Images of tissue before and after fixation were qualitatively very similar. The ratios of T10, T1Gd, and calculated [GAG] after fixation, relative to before fixation, were near or slightly higher than 1 for both bovine cartilage (1.01 ± 0.01, 1.04 ± 0.02, 1.05 ± 0.03, respectively) and for human cartilage (0.96 ± 0.11, 1.03 ± 0.05, 1.09 ± 0.13). Thus, these data suggest that dGEMRIC can be used on previously fixed samples to assess the three dimensional spatial distribution of GAG.
doi:10.1002/jor.20767
PMCID: PMC3947557  PMID: 18973237
cartilage; GAG; fixed charge; fixation; dGEMRIC
8.  Development of Polymeric Nanocarrier System for Early Detection and Targeted Therapeutic Treatment of Peri-Implant Osteolysis 
HSS Journal  2012;9(1):79-85.
Background
Extensive research has implicated inflammation as a necessary and causative factor in the development of peri-implant osteolysis, suggesting that such an inflammatory response is the sentinel event for the process. The potential to impact the clinical course of this condition is hampered by the lack of an effective medical therapy, as well as a limited ability for early detection prior to radiographically evident osteolysis. Advances in nanotechnology have allowed for the production of engineered water-soluble nanocarriers, which exploit changes in the microvascular architecture for selective distribution to inflamed tissues. Evaluation of the uptake of the nanocarriers in sites of inflammation has elucidated a novel mechanism of cellular uptake and retention of these particles.
Purpose
The current review discusses the development of a novel, biocompatible, water-soluble nanocarrier utilizing copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA), conjugated to imaging and therapeutic agents for the detection and targeted treatment of inflammatory conditions.
Methods
We performed Medline searches for the terms “periprosthetic osteolysis,” “murine osteolysis model,” “HPMA osteolysis,” and “HPMA inflammation.” These searches identified 631, 306, 1, and 6 articles, respectively. These were then manually searched for articles relevant to the development of mouse models for inflammatory osteolysis and the use of HPMA copolymer technology in mouse models of inflammation.
Results
Promising results in a small animal model of osteolysis have demonstrated the capability for detection prior to the development of bone loss, and have highlighted the utility of nanocarriers for selective drug delivery to the affected tissues.
Conclusions
Challenges to the clinical translation of HPMA nanocarriers in peri-implant osteolysis remain, and the future research directions necessary for human clinical application are reviewed.
doi:10.1007/s11420-012-9307-7
PMCID: PMC3640727  PMID: 24426848
periprosthetic osteolysis; nanocarrier; HPMA; optical imaging
9.  Negative Regulation of Osteoclast Precursor Differentiation by CD11b and β2 Integrin-BCL6 Signaling 
Negative regulation of osteoclastogenesis is important for bone homeostasis and prevention of excessive bone resorption in inflammatory and other diseases. Mechanisms that directly suppress osteoclastogenesis are not well understood. In this study we investigated regulation of osteoclast differentiation by the β2 integrin CD11b/CD18 that is expressed on myeloid lineage osteoclast precursors. CD11b-deficient mice exhibited decreased bone mass that was associated with increased osteoclast numbers and decreased bone formation. Accordingly, CD11b and β2 integrin signaling suppressed osteoclast differentiation by preventing RANKL-induced induction of the master regulator of osteoclastogenesis NFATc1 and of downstream osteoclast-related NFATc1 target genes. CD11b suppressed induction of NFATc1 by the complementary mechanisms of downregulation of RANK expression and induction of recruitment of the transcriptional repressor BCL6 to the NFATC1 gene. These findings identify CD11b as a negative regulator of the earliest stages of osteoclast differentiation, and provide an inducible mechanism by which environmental cues suppress osteoclastogenesis by activating a transcriptional repressor that makes genes refractory to osteoclastogenic signaling.
doi:10.1002/jbmr.1739
PMCID: PMC3522783  PMID: 22893614
osteoclast; signaling; integrin; CD11b; BCL6
10.  Development of macromolecular prodrug for rheumatoid arthritis☆ 
Advanced drug delivery reviews  2012;64(12):1205-1219.
Rheumatoid arthritis (RA) is a chronic autoimmune disease that is considered to be one of the major public health problems worldwide. The development of therapies that target tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and co-stimulatory pathways that regulate the immune system have revolutionized the care of patients with RA. Despite these advances, many patients continue to experience symptomatic and functional impairment. To address this issue, more recent therapies that have been developed are designed to target intracellular signaling pathways involved in immunoregulation. Though this approach has been encouraging, there have been major challenges with respect to off-target organ side effects and systemic toxicities related to the widespread distribution of these signaling pathways in multiple cell types and tissues. These limitations have led to an increasing interest in the development of strategies for the macromolecularization of anti-rheumatic drugs, which could target them to the inflamed joints. This approach enhances the efficacy of the therapeutic agent with respect to synovial inflammation, while markedly reducing non-target organ adverse side effects. In this manuscript, we provide a comprehensive overview of the rational design and optimization of macromolecular prodrugs for treatment of RA. The superior and the sustained efficacy of the prodrug may be partially attributed to their Extravasation through Leaky Vasculature and subsequent Inflammatory cell-mediated Sequestration (ELVIS) in the arthritic joints. This biologic process provides a plausible mechanism, by which macromolecular prodrugs preferentially target arthritic joints and illustrates the potential benefits of applying this therapeutic strategy to the treatment of other inflammatory diseases.
doi:10.1016/j.addr.2012.03.006
PMCID: PMC3572768  PMID: 22433784
Rheumatoid arthritis; Macromolecular prodrug; ELVIS; Targeting inflammation; Drug delivery
11.  The Role of Synovitis in Osteoarthritis pathogenesis 
Bone  2012;51(2):249-257.
Research into the pathophysiology of osteoarthritis (OA) has focused on cartilage and peri-articular bone, but there is increasing recognition that OA affects all of the joint tissues, including the synovium (SM). Under normal physiological conditions the synovial lining consists of a thin layer of cells with phenotypic features of macrophages and fibroblasts. These cells and the underlying vascularized connective tissue stroma form a complex structure that is an important source of synovial fluid (SF) components that are essential for normal cartilage and joint function. The histological changes observed in the SM in OA generally include features indicative of an inflammatory “synovitis”; specifically they encompass a range of abnormalities, such as synovial lining hyperplasia, infiltration of macrophages and lymphocytes, neoangiogenesis and fibrosis. The pattern of synovial reaction varies with disease duration and associated metabolic and structural changes in other joint tissues. Imaging modalities including Magnetic Resonance (MRI) and ultrasound (US) have proved useful in detecting and quantifying synovial abnormalities, but individual studies have varied in their methods of evaluation. Despite these differences, most studies have concluded that the presence of synovitis in OA is associated with more severe pain and joint dysfunction. In addition, synovitis may be predictive of faster rates of cartilage loss in certain patient populations. Recent studies have provided insights into the pathogenic mechanisms underlying the development of synovitis in OA. Available evidence suggests that the inflammatory process involves engagement of Toll-like receptors and activation of the complement cascade by degradation products of extracellular matrices of cartilage and other joint tissues. The ensuing synovial reaction can lead to synthesis and release of a wide variety of cytokines and chemokines. Some of these inflammatory mediators are detected in joint tissues and SF in OA and have catabolic effects on chondrocytes. These inflammatory mediators represent potential targets for therapeutic interventions designed to reduce both symptoms and structural joint damage in OA.
doi:10.1016/j.bone.2012.02.012
PMCID: PMC3372675  PMID: 22387238
12.  Targeting the Giant Cell Tumor Stromal Cell: Functional Characterization and a Novel Therapeutic Strategy 
PLoS ONE  2013;8(7):e69101.
Giant cell tumor of bone (GCTB) is a benign, locally destructive neoplasm, with tumors comprised of mesenchymal fibroblast-like stromal cells; monocytic, mononuclear cells of myeloid lineage; and the characteristic osteoclast-like, multinucleated giant cells. Hampering the study of the complex interaction of its constituent cell types is the propensity of longstanding, repeatedly passaged cell cultures to undergo phenotypic alteration and loss of osteoclast-inducing capacities. In this study, we employed a novel, single-step technique to purify freshly harvested stromal cells using a CD14-negative selection column. Using 9 freshly harvested GCTB specimens and the purified stromal cell component, we performed analyses for markers of osteoblast lineage and analyzed the capacity of the stromal cells to undergo osteoblastic differentiation and induce osteoclastogenesis in co-cultures with monocytic cells. Successful purification of the CD14-negative stromal cells was confirmed via flow cytometric analysis and immunocytochemistry. Osteogenic media upregulated the expression of osteocalcin, suggesting an osteoblastic lineage of the GCTB stromal cells. The effects of the Wnt pathway agonist, SB415286, and recombinant human bone morphogenetic protein (BMP)-2 on osteoblastogenesis varied among samples. Notably, osteogenic media and SB415286 reversed the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) expression ratio resulting in diminished osteoclastogenic capacity. Recombinant human BMP2 had the opposite effect, resulting in enhanced and sustained support of osteoclastogenesis. Targeting the giant cell tumor stromal cell may be an effective adjunct to existing anti-resorptive strategies.
doi:10.1371/journal.pone.0069101
PMCID: PMC3724882  PMID: 23922683
13.  Osteoarthritis: A Disease of the Joint as an Organ 
Arthritis and Rheumatism  2012;64(6):1697-1707.
doi:10.1002/art.34453
PMCID: PMC3366018  PMID: 22392533
14.  Osteoimmunology and Bone Homeostasis: Relevance to Spondyloarthritis 
The seronegative spondyloarthopathies (SpA) share certain common articular and peri-articular features that differ from rheumatoid arthritis (RA) and other forms of inflammatory arthritis. These include the tendency of the SpAs to involve the axial skeleton in addition to the diarthrodial joints, and the prominent involvement of the extra-articular entheses (sites of ligamentous and tendon insertion), which are not common sites of primary pathology in RA and other inflammatory arthropathies. The differential anatomic sites of bone pathology in the SpAs in comparison to the other forms of arthritis suggest that the underlying pathogenic processes and cellular and molecular mechanisms that account for the peri-articular bone pathology involve different underlying disease mechanisms. This review will highlight the molecular and cellular processes that are involved in the pathogenesis of the skeletal pathology in the SpAs, and provide evidence that many of the factors involved in regulation of bone cell function exhibit potent immune-regulatory activity, providing support for the general concept of osteoimmunology.
doi:10.1007/s11926-013-0342-2
PMCID: PMC3689465  PMID: 23709258
Osteoimmunology; Bone homeostasis; Spondyloarthritis; Ankylosing spondylitis; Psoriatic arthritis; Osteoclast; Osteoblast; Osteocyte; Bone remodeling; Inflammation; Cytokine; Bone morphogenic proteins; Wnt/βcatenin; Sclerostin
15.  Cellular Response to Prosthetic Wear Debris Differs in Rheumatoid Versus Non-Rheumatoid Arthritis Patients 
Arthritis and Rheumatism  2011;64(4):1005-1014.
Objective
This study examined whether patients with rheumatoid arthritis (RA) demonstrate different patterns of prosthetic wear or cellular responses to implant wear debris compared to patients without inflammatory joint disease.
Methods
Thirty-eight patients who had a primary revision of a total elbow arthroplasty (TEA) for aseptic loosening between 1996 and 2008 were identified. Twenty-five had RA and 13 had no inflammatory arthritis. Clinical data, gross wear patterns of the removed prostheses, and histopathological analyses of peri-implant tissue were compared between RA and non-RA patients.
Results
Evaluation of the retrieved prostheses showed that conformational change of the humeral polyethylene bushing was associated with the generation of polyethylene and metal particles. The amount and type of wear debris in peri-prosthetic tissues was similar in RA and non-RA patients. RA patients not on anti-tumor necrosis factor (TNF) therapy exhibited a histologic pattern of interstitial and sheet-like lymphocytic infiltrates associated with a high plasma cell composition, which was different from the predominantly perivascular infiltrates with few plasma cells seen in non-RA patients (p-value = 0.04). RA patients on anti-TNF therapy fell in between these two groups.
Conclusion
RA patients exhibit a distinct cellular response to implant wear debris compared with non-RA patients. This reaction was unrelated to differences in the type or amount of wear debris and was mitigated by anti-TNF therapy. These results suggest an intrinsic alteration in immunoregulation in RA and have implications for potential immunologic treatment of osteolysis in these patients.
doi:10.1002/art.33459
PMCID: PMC3292699  PMID: 22127818
16.  Plasma proteins take their toll on the joint in osteoarthritis 
In their recent study, Sohn and colleagues identify multiple plasma proteins in the synovial fluid of patients with osteoarthritis (OA) and demonstrate the capacity of several of the proteins to activate macrophages via the innate immune receptor Toll-like receptor-4 (TLR-4). The authors speculate that the plasma proteins transit into the synovial compartment at sites of tissue damage where the endothelial barrier may be compromised, thus bringing them into contact with the articular surface and cells within the synovium. These results demonstrate a novel mechanism by which synovial inflammation can be initiated in patients with OA and how this process may contribute to the pathogenesis of OA joint pathology.
doi:10.1186/ar3741
PMCID: PMC3446406  PMID: 22385929
17.  Early detection and treatment of wear particle-induced inflammation and bone loss in a mouse calvarial osteolysis model using HPMA copolymer conjugates 
Molecular pharmaceutics  2011;8(4):1043-1051.
Wear particle-induced inflammation is considered to be the major cause of aseptic implant loosening and clinical failure after total joint replacement. Due to the frequent absence of symptoms, early detection and intervention prior to implant failure presents a significant challenge. To address this issue, a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based optical imaging contrast agent (P-IRDye) was developed and used for the detection of wear particle-induced inflammation employing a murine calvaria osteolysis model. The particle-induced osteolysis of calvaria was evaluated by H&E, tartrate-resistant acid phosphatase (TRAP) staining and μ-CT after necropsy. One-day post particles implantation, P-IRDye was administrated to the mice via tail vein injection. Live imaging of the animals 6 days after implantation revealed the preferential distribution and sustained retention of the macromolecular contrast agent at the site of particle implantation. Immunohistochemical staining and FACS analyses of the calvaria-associated soft tissue revealed extensive uptake of the HPMA copolymer by F4/80, Ly-6G (Gr1) and CD11c positive cells, which accounts for the retention of the macromolecular probes at the inflammatory sites. To test the potential of the system for therapeutic intervention, an acid-labile HPMA copolymer-dexamethasone conjugate (P-Dex) was prepared and shown to prevent the particle-induced inflammation and bone damage in the calvaria osteolysis model.
doi:10.1021/mp2000555
PMCID: PMC3148351  PMID: 21438611
HPMA copolymer; aseptic orthopedic implant loosening; theranostics; orthopedic wear particle; inflammation targeting
18.  Alterations in periarticular bone and cross talk between subchondral bone and articular cartilage in osteoarthritis 
The articular cartilage and the subchondral bone form a biocomposite that is uniquely adapted to the transfer of loads across the diarthrodial joint. During the evolution of the osteoarthritic process biomechanical and biological processes result in alterations in the composition, structure and functional properties of these tissues. Given the intimate contact between the cartilage and bone, alterations of either tissue will modulate the properties and function of the other joint component. The changes in periarticular bone tend to occur very early in the development of OA. Although chondrocytes also have the capacity to modulate their functional state in response to loading, the capacity of these cells to repair and modify their surrounding extracellular matrix is relatively limited in comparison to the adjacent subchondral bone. This differential adaptive capacity likely underlies the more rapid appearance of detectable skeletal changes in OA in comparison to the articular cartilage. The OA changes in periarticular bone include increases in subchondral cortical bone thickness, gradual decreases in subchondral trabeular bone mass, formation of marginal joint osteophytes, development of bone cysts and advancement of the zone of calcified cartilage between the articular cartilage and subchondral bone. The expansion of the zone of calcified cartilage contributes to overall thinning of the articular cartilage. The mechanisms involved in this process include the release of soluble mediators from chondrocytes in the deep zones of the articular cartilage and/or the influences of microcracks that have initiated focal remodeling in the calcified cartilage and subchondral bone in an attempt to repair the microdamage. There is the need for further studies to define the pathophysiological mechanisms involved in the interaction between subchondral bone and articular cartilage and for applying this information to the development of therapeutic interventions to improve the outcomes in patients with OA.
doi:10.1177/1759720X12437353
PMCID: PMC3403248  PMID: 22859924
osteoarthritis; bone remodeling; articular cartilage; biomechanics
19.  Identification of a central role for complement in osteoarthritis 
Nature Medicine  2011;17(12):1674-1679.
Osteoarthritis, characterized by the breakdown of articular cartilage in synovial joints, has long been viewed as the result of “wear and tear”1. Although low-grade inflammation is detected in osteoarthritis, its role is unclear2–4. Here we identify a central role for the inflammatory complement system in the pathogenesis of osteoarthritis. Through proteomic and transcriptomic analyses of synovial fluids and membranes from individuals with osteoarthritis, we find that expression and activation of complement is abnormally high in human osteoarthritic joints. Using mice genetically deficient in C5, C6, or CD59a, we show that complement, and specifically the membrane attack complex (MAC)-mediated arm of complement, is critical to the development of arthritis in three different mouse models of osteoarthritis. Pharmacological modulation of complement in wild-type mice confirmed the results obtained with genetically deficient mice. Expression of inflammatory and degradative molecules was lower in chondrocytes from destabilized joints of C5-deficient mice than C5-sufficient mice, and MAC induced production of these molecules in cultured chondrocytes. Furthermore, MAC co-localized with matrix metalloprotease (MMP)-13 and with activated extracellular signal-regulated kinase (ERK) around chondrocytes in human osteoarthritic cartilage. Our findings indicate that dysregulation of complement in synovial joints plays a critical role in the pathogenesis of osteoarthritis.
doi:10.1038/nm.2543
PMCID: PMC3257059  PMID: 22057346
21.  Synovial inflammation in patients undergoing arthroscopic meniscectomy: molecular characterization and relationship with symptoms 
Arthritis and Rheumatism  2011;63(2):391-400.
Objective
Traumatic and degenerative meniscal tears have different anatomic features and different proposed etiologies, yet both are associated with development or progression of osteoarthritis (OA). In established OA, synovitis is associated with pain and progression, but a relationship between synovitis and symptoms in isolated meniscal disease has not been reported. Accordingly, we sought to characterize synovial pathology in patients with traumatic meniscal injuries and determine the relationships between inflammation, meniscal and cartilage pathology, and symptoms.
Methods
Thirty-three patients without evidence of OA undergoing arthroscopic meniscectomy for meniscal injuries were recruited. Pain and function were assessed preoperatively; meniscal and cartilage abnormalities were documented at the time of surgery. Inflammation in synovial biopsies was scored and associations between inflammation and clinical outcomes determined. Microarray analysis of synovial tissue was performed and gene expression patterns in patients with or without inflammation compared.
Results
Synovial inflammation was present in 43% of patients and was associated with worse pre-operative pain and function scores, independent of age, gender, or cartilage pathology. Microarray analysis and real-time PCR revealed a chemokine signature in synovial biopsies with increased inflammation scores.
Conclusion
In patients with traumatic meniscal injury undergoing arthroscopic meniscectomy without clinical or radiographic evidence of OA, synovial inflammation occurs frequently and is associated with increased pain and dysfunction. Synovia with increased inflammation scores exhibit a unique chemokine signature. Chemokines may contribute to the development of synovial inflammation in patients with meniscal pathology; they also represent potential therapeutic targets for reducing inflammatory symptoms.
doi:10.1002/art.30137
PMCID: PMC3260472  PMID: 21279996
Meniscectomy; meniscal injury; inflammation; synovium; synovitis
22.  The Relative Timing of Exposure to Phagocytosable Particulates and to Osteoclastogenic Cytokines Is Critically Important in the Determination of Myeloid Cell Fate 
During granulomatous inflammatory reactions, myeloid cells can differentiate into activated phagocytic macrophages, wound-healing macrophages, foreign body giant cells, and bone-resorbing osteoclasts. Although it is appreciated that a variety of stimuli, including cytokines, cell–matrix interactions, and challenge with foreign materials can influence myeloid cell fate, little is known of how these signals integrate during this process. In this study, we have investigated the cross talk between receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and particle phagocytosis-induced activation of human monocytes. Understanding interconnected signals is of particular importance to disorders, such as periprosthetic osteolysis, in which granulomatous inflammation is initiated by particle phagocytosis in proximity to bone and leads to inflammatory bone loss. Using cell-based osteoclastogenesis and phagocytosis assays together with expression analysis of key regulators of osteoclastogenesis, we show in this study that phagocytosis of disease-relevant particles inhibits RANKL-mediated osteoclastogenesis of human monocytes. Mechanistically, phagocytosis mediates this effect by downregulation of RANK and c-Fms, the receptors for the essential osteoclastogenic cytokines RANKL and M-CSF. RANKL pretreatment of monocytes generates preosteoclasts that are resistant to RANK downregulation and committed to osteoclast formation, even though they retain phagocytic activity. Thus, the relative timing of exposure to phagocytosable particulates and to osteoclastogenic cytokines is critically important in the determination of myeloid cell fate.
doi:10.4049/jimmunol.0902808
PMCID: PMC3016856  PMID: 20543106
23.  Bone and cartilage in osteoarthritis: is what's best for one good or bad for the other? 
The interest in the relationship between articular cartilage and the structural and functional properties of peri-articular bone relates to the intimate contact that exists between these tissues in joints that are susceptible to the development of osteoarthritis (OA). The demonstration in several animal models that osteoporosis and decreased bone tissue modulus leads to an increased propensity for the development of post-traumatic OA is paradoxical in light of the extensive epidemiological literature indicating that individuals with high systemic bone mass, assessed by bone mineral density, are at increased risk for OA. These observations underscore the need for further studies to define the pathophysiological mechanisms involved in the interaction between subchondral bone and articular cartilage and for applying this information to the development of therapeutic interventions to improve the outcomes in patients with OA.
doi:10.1186/ar3135
PMCID: PMC2991002  PMID: 21044355
24.  Inhibition of RANK Expression and Osteoclastogenesis by TLRs and IFN-γ in Human Osteoclast Precursors1 
TLRs have been implicated in promoting osteoclast-mediated bone resorption associated with inflammatory conditions. TLRs also activate homeostatic mechanisms that suppress osteoclastogenesis and can limit the extent of pathologic bone erosion associated with infection and inflammation. We investigated mechanisms by which TLRs suppress osteoclastogenesis. In human cell culture models, TLR ligands suppressed osteoclastogenesis by inhibiting expression of receptor activator of NF-κB (RANK), thereby making precursor cells refractory to the effects of RANKL. Similar but less robust inhibition of RANK expression was observed in murine cells. LPS suppressed generation of osteoclast precursors in mice in vivo, and adsorption of LPS onto bone surfaces resulted in diminished bone resorption. Mechanisms that inhibited RANK expression were down-regulation of RANK transcription, and inhibition of M-CSF signaling that is required for RANK expression. TLRs inhibited M-CSF signaling by rapidly down-regulating cell surface expression of the M-CSF receptor c-Fms by a matrix metalloprotease- and MAPK-dependent mechanism. Additionally, TLRs cooperated with IFN-γ to inhibit expression of RANK and of the CSF1R gene that encodes c-Fms, and to synergistically inhibit osteoclastogenesis. Our findings identify a new mechanism of homeostatic regulation of osteoclastogenesis that targets RANK expression and limits bone resorption during infection and inflammation.
doi:10.4049/jimmunol.0900072
PMCID: PMC2783334  PMID: 19890054
25.  Development of a macromolecular prodrug for the treatment of inflammatory arthritis: mechanisms involved in arthrotropism and sustained therapeutic efficacy 
Arthritis Research & Therapy  2010;12(5):R170.
Introduction
The purpose of the present manuscript is to test the hypothesis that arthrotropic localization and synovial cell internalization account for the unique capacity of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-dexamethasone conjugate (P-Dex, a macromolecular prodrug of dexamethasone) to induce sustained amelioration of joint inflammation and inhibition of tissue damage in an animal model of inflammatory arthritis.
Methods
Rats with adjuvant-induced arthritis (AA) were treated with P-Dex, free dexamethasone, saline or HPMA homopolymer. To define the biodistribution of P-Dex, conjugates with different imaging labels were given to AA rats and analyzed. Isolated joint tissues were evaluated by fluorescence-activated cell sorting (FACS) and immunohistochemical staining. Cellular uptake of P-Dex and its effects on apoptosis and production of proinflammatory cytokines were examined using human monocyte-macrophages and fibroblasts.
Results
A single systemic administration of P-Dex completely suppressed AA for >20 days. Magnetic resonance imaging demonstrated higher HPMA copolymer influx into the inflamed joints than the normal joints. Immunohistochemistry and FACS analyses of arthritic joints revealed extensive uptake of the polymer conjugate by synovial fibroblasts and myeloid lineage cells. The capacity of P-Dex to suppress inflammation was confirmed in monocyte-macrophage cultures in which P-Dex treatment resulted in suppression of lipopolysaccharide-induced IL-6 and TNFα release. Similarly, TNFα-induced expression of matrix metalloproteinases (MMP1 and MMP3) in synovial fibroblasts from a rheumatoid arthritis patient was suppressed by P-Dex. P-Dex showed no detectable effect on monocyte apoptosis.
Conclusions
P-Dex provides superior and sustained amelioration of AA compared with an equivalent dose of free dexamethasone. The arthrotropism and local retention of P-Dex is attributed to the enhanced vascular permeability in arthritic joints and the internalization of P-Dex by synovial cells. The uptake and processing of P-Dex by macrophages and fibroblasts, and downregulation of proinflammatory mediators, provides an explanation for the sustained anti-inflammatory efficacy of P-Dex in this model of inflammatory arthritis.
doi:10.1186/ar3130
PMCID: PMC2990997  PMID: 20836843

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