CCL2, CCL5, and IGF-1 participate in the immunomodulation of osteogenesis during M1/M2 transition in vitro.

The modulation of macrophage phenotype from pro-inflammatory (M1) to tissue healing (M2) via exogenous addition of interleukin-4 (IL-4) facilitates osteogenesis; however, the molecular mediators underlying this phenomenon remain unknown. This study characterizes the IL-4-dependent paracrine crosstalk between macrophages and osteoprogenitors and its effect on osteogenesis in vitro. Primary murine M1 were co-cultured with MC3T3 cells (M1-MC3T3) in both transwell plates and direct co-cultures. To modulate M1 to M2, M1-MC3T3 were treated with IL-4 (20 ng/mL) at day 3 after seeding (M1 + IL-4-MC3T3). Selected molecular targets were assessed at days 3 and 6 after seeding at protein and mRNA levels. Mineralization was assessed at day 21. Transwell M1 + IL-4-MC3T3 significantly enhanced the secretion of CCL2/MCP-1, IGF-1 and to a lesser degree, CCL5/RANTES at day 6. At day 3, alkaline phosphatase (Alpl) was upregulated in direct M1-MC3T3. At day 6, Smurf2 and Insulin growth factor-1 (IGF-1) were downregulated and upregulated, respectively, in direct M1 + IL-4-MC3T3. Finally, M1 + IL-4-MC3T3 increased bone matrix mineralization compared with MC3T3 cells in transwell, but this was significantly less than M1-MC3T3. Taken together, macrophage subtypes enhanced the osteogenesis in transwell setting and the transition from M1 to M2 was associated with an increase in bone anabolic factors CCL2/MCP-1, CCL5/RANTES and IGF-1 in vitro. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3069-3076, 2017.

Orthopaedic wear particle-induced bone loss and exogenous macrophage infiltration is mitigated by local infusion of NF-κB decoy oligodeoxynucleotide.

Excessive production of wear particles from total joint replacements induces chronic inflammation, macrophage infiltration, and consequent bone loss (periprosthetic osteolysis). This inflammation and bone remodeling are critically regulated by the transcription factor NF-κB. We previously demonstrated that inhibition of NF-κB signaling by using the decoy oligodeoxynucleotide (ODN) mitigates polyethylene wear particle-induced bone loss using in vitro and in vivo models. However, the mechanisms of NF-κB decoy ODN action, and in particular its impact on systemic macrophage recruitment, remain unknown. In the current study, this systemic macrophage infiltration was examined in our established murine femoral continuous particle infusion model. RAW264.7 murine macrophages expressing a luciferase reporter gene were injected into the systemic circulation. Quantification of bioluminescence showed that NF-κB decoy ODN reduced the homing of these reporter macrophages into the distal femurs exposed to continuous particle delivery. Particle-induced reduction in bone mineral density at the distal diaphysis of the femur was also mitigated by infusion of decoy ODN. Histological staining showed that the decoy ODN infusion decreased osteoclast and macrophage numbers, but had no significant effects on osteoblasts. Local infusion of NF-κB decoy ODN reduced systemic macrophage infiltration and mitigated particle-induced bone loss, thus providing a potential strategy to treat periprosthetic osteolysis. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3169-3175, 2017.

Pro-inflammatory M1 macrophages promote Osteogenesis by mesenchymal stem cells via the COX-2-prostaglandin E2 pathway.

Bone fractures are among the most common orthopaedic problems that affect individuals of all ages. Immediately after injury, activated macrophages dynamically contribute to and regulate an acute inflammatory response that involves other cells at the injury site, including mesenchymal stem cells (MSCs). These macrophages and MSCs work in concert to modulate bone healing. In this study, we co-cultured undifferentiated M0, pro-inflammatory M1, and anti-inflammatory M2 macrophages with primary murine MSCs in vitro to determine the cross-talk between polarized macrophages and MSCs and their effects on osteogenesis. After 4 weeks of co-culture, MSCs grown with macrophages, especially M1 macrophages, had enhanced bone mineralization compared to MSCs grown alone. The level of bone formation after 4 weeks of culture was closely associated with prostaglandin E2 (PGE2) secretion early in osteogenesis. Treatment with celecoxib, a cyclooxygenase-2 (COX-2) selective inhibitor, significantly reduced bone mineralization in all co-cultures but most dramatically in the M1-MSC co-culture. We also found that the presence of macrophages reduced the secretion of osteoprotegerin (OPG), the decoy RANKL receptor, suggesting that macrophages may indirectly modulate osteoclast activity in addition to enhancing bone formation. Taken together, these findings suggest that an initial pro-inflammatory phase modulated by M1 macrophages promotes osteogenesis in MSCs via the COX-2-PGE2 pathway. Understanding the complex interactions between macrophages and MSCs provide opportunities to optimize bone healing and other regenerative processes via modulation of the inflammatory response. This study provides one possible biological mechanism for the adverse effects of non-steroidal anti-inflammatory drugs on fracture healing and bone regeneration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2378-2385, 2017.

The biological response to orthopaedic implants for joint replacement: Part I: Metals.

Joint replacement is a commonly performed, highly successful orthopaedic procedure, for which surgeons have a large choice of different materials and implant designs. The materials used for joint replacement must be both biologically acceptable to minimize adverse local tissue reactions, and robust enough to support weight bearing during common activities of daily living. Modern joint replacements are made from metals and their alloys, polymers, ceramics, and composites. This review focuses on the biological response to the different biomaterials used for joint replacement. In general, modern materials for joint replacement are well tolerated by the body as long as they are in bulk (rather than in particulate or ionic) form, are mechanically stable and noninfected. If the latter conditions are not met, the prosthesis will be associated with an acute/chronic inflammatory reaction, peri-prosthetic osteolysis, loosening and failure. This article (Part 1 of 2) is dedicated to the use of metallic devices in orthopaedic surgery including the associated biological response to metallic byproducts is a review of the basic science literature regarding this topic. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2162-2173, 2017.

Mutant CCL2 protein coating mitigates wear particle-induced bone loss in a murine continuous polyethylene infusion model.

Wear particle-induced osteolysis limits the long-term survivorship of total joint replacement (TJR). Monocyte/macrophages are the key cells of this adverse reaction. Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) is the most important chemokine regulating trafficking of monocyte/macrophages in particle-induced inflammation. 7ND recombinant protein is a mutant of CCL2 that inhibits CCL2 signaling. We have recently developed a layer-by-layer (LBL) coating platform on implant surfaces that can release biologically active 7ND. In this study, we investigated the effect of 7ND on wear particle-induced bone loss using the murine continuous polyethylene (PE) particle infusion model with 7ND coating of a titanium rod as a local drug delivery device. PE particles were infused into hollow titanium rods with or without 7ND coating implanted in the distal femur for 4 weeks. Specific groups were also injected with RAW 264.7 as the reporter macrophages. Wear particle-induced bone loss and the effects of 7ND were evaluated by microCT, immunohistochemical staining, and bioluminescence imaging. Local delivery of 7ND using the LBL coating decreased systemic macrophage recruitment, the number of osteoclasts and wear particle-induced bone loss. The development of a novel orthopaedic implant coating with anti-CCL2 protein may be a promising strategy to mitigate peri-prosthetic osteolysis.

Decreased osteogenesis in mesenchymal stem cells derived from the aged mouse is associated with enhanced NF-κB activity.

Aging is associated with significant bone loss and delayed fracture healing. NF-κB activation is highly correlated with inflammatory-associated bone diseases including infection, wear particle exposure, and chronic inflammation during natural aging processes. The critical roles of NF-κB in both the pro-inflammatory response and osteoclast-mediated bone resorption have been well defined. However, the biological effects of NF-κB activation in mesenchymal stem cell (MSC)-mediated bone formation remain largely unknown. In the current study, bone marrow-MSCs were isolated from young (8 weeks old) and aged (72 weeks old) mice. NF-κB activity in MSCs at basal levels and under different biological conditions were determined by our recently established lentiviral vector-based luciferase reporter assay. We found that NF-κB activity was increased in aged MSCs at basal levels or when exposed to low dose (10 or 100 ng/ml) lipopolysaccharide (LPS); this effect was not seen when the cells were exposed to higher dose (1 µg/ml) LPS. During osteogenesis, NF-κB activity was increased in aged MSCs at weeks 1 and 2, but showed no significant difference at week 3. Both Smurf2 and TAZ, the NF-κB target genes that regulate osteogenic differentiation, were increased in aged MSCs. In addition, the expression of RANKL was dramatically increased, and OPG was decreased in aged MSCs. Our findings suggest that targeting NF-κB activity in MSCs has the potential to modulate aging-associated bone loss, or enhance bone-healing in aged patients. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:281-288, 2017.

Mechanoreceptor Reinnervation of Autografts Versus Allografts After Anterior Cruciate Ligament Reconstruction.

BACKGROUND: Loss of proprioceptive function occurs after anterior cruciate ligament (ACL) rupture. Clinical, motor, and proprioceptive function is known to improve after ACL reconstruction but does not return to normal. While histological studies of human ACL allografts have been unable to demonstrate mechanoreceptor reinnervation, animal data suggest that reinnervation may occur when an autograft is used. PURPOSE: To compare the presence or absence of mechanoreceptors between allograft versus autograft after ACL reconstruction in humans. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: Ten patients with previous ACL reconstruction presenting for either revision ACL surgery or knee arthroscopy for other reasons were enrolled in a prospective, comparative study. Five patients had a previous autograft ACL and 5 patients had an allograft. Biopsies, either from intact or ruptured grafts, were taken from identical locations as close to the femoral and tibial insertions as possible. Specimens were stained with hematoxylin-eosin (H-E) and monoclonal antibodies against neurofilament protein (NFP), known to be present in mechanoreceptor tissue. Immunohistochemical examination was carried out, and the number of NFP+ neural tissue analogs was counted and compared with that of native ACL tissue. RESULTS: The mean time between original graft and biopsy was 6.9 years (range, 0.5-15 years). Histological examination showed significantly less NFP+ neural analogs in allograft and autograft patients than control tissue (mean number of NFP+ analogs per high-power field, 0.7 ± 0.9 [allograft] and 0.5 ± 0.8 [autograft] vs 4.7 ± 0.9 [controls]; P < .0001). There was no significant difference in NFP analogs between autograft and allograft tissue. CONCLUSION: We found a reduced concentration of NFP+ neural analogs in ACL grafts compared with native ACL tissue. This deficit exists irrespective of whether allograft or autograft is used. These findings may explain the continued proprioceptive deficits seen clinically after ACL reconstruction.

NF-κB decoy oligodeoxynucleotide mitigates wear particle-associated bone loss in the murine continuous infusion model.

UNLABELLED: Total joint replacement is a cost-effective surgical procedure for patients with end-stage arthritis. Wear particle-induced chronic inflammation is associated with the development of periprosthetic osteolysis. Modulation of NF-κB signaling in macrophages, osteoclasts, and mesenchymal stem cells could potentially mitigate this disease. In the current study, we examined the effects of local delivery of decoy NF-κB oligo-deoxynucleotide (ODN) on wear particle-induced bone loss in a murine continuous femoral particle infusion model. Ultra-high molecular weight polyethylene particles (UHMWPE) with or without lipopolysaccharide (LPS) were infused via osmotic pumps into hollow titanium rods placed in the distal femur of mice for 4weeks. Particle-induced bone loss was evaluated by µCT, and immunohistochemical analysis of sections from the femur. Particle infusion alone resulted in reduced bone mineral density and trabecular bone volume fraction in the distal femur. The decoy ODN reversed the particle-associated bone volume fraction loss around the implant, irrespective of the presence of LPS. Particle-infusion with LPS increased bone mineral density in the distal femur compared with particle-infusion alone. NF-κB decoy ODN reversed or further increased the bone mineral density in the femur (3-6mm from the distal end) exposed to particles alone or particles plus LPS. NF-κB decoy ODN also inhibited macrophage infiltration and osteoclast number, but had no significant effects on osteoblast numbers in femurs exposed to wear particles and LPS. Our study suggests that targeting NF-κB activity via local delivery of decoy ODN has great potential to mitigate wear particle-induced osteolysis. STATEMENT OF SIGNIFICANCE: Total joint replacement is a cost-effective surgical procedure for patients with end-stage arthritis. Chronic inflammation is crucial for the development of wear particle-associated bone loss. Modulation of NF-κB signaling in macrophages (pro-inflammatory cells), osteoclasts (bone-resorbing cells), and osteoblasts (bone-forming cells) could potentially mitigate this disease. Here we demonstrated that local delivery of decoy NF-κB oligo-deoxynucleotide (ODN) mitigated ultra-high molecular weight polyethylene (UHMWPE) wear particle induced bone loss in a clinically relevant murine model. The protective effects of decoy ODN was associated with reduced macrophage infiltration and osteoclast activation, but had no significant effects on osteoblast numbers. Our study suggests that targeting NF-κB activity via local delivery of decoy ODN has great potential to mitigate wear particle-induced bone loss.

The effect of local IL-4 delivery or CCL2 blockade on implant fixation and bone structural properties in a mouse model of wear particle induced osteolysis.

Modulation of macrophage polarization and prevention of CCL2-induced macrophage chemotaxis are emerging strategies to reduce wear particle induced osteolysis and aseptic total joint replacement loosening. In this study, the effect of continuous IL-4 delivery or bioactive implant coating that constitutively releases a protein inhibitor of CCL2 signaling (7ND) on particle induced osteolysis were studied in the murine continuous femoral intramedullary particle infusion model. Polyethylene particles with or without IL-4 were infused into mouse distal femurs implanted with hollow titanium rods using subcutaneous infusion pumps. In another experimental group, particles were infused into the femur through a 7ND coated rod. After 4 weeks, fixation of the implant was assessed using a pullout test. The volume of trabecular bone and the geometry of the local cortical bone were assessed by µCT and the corresponding structural properties of the cortical bone determined by torsional testing. Continuous IL-4 delivery led to increased trabecular bone volume as well as enhanced local bone geometry and structural properties, while 7ND implant coating did not have effect on these parameters. The results suggest that local IL-4 treatment is a promising strategy to mitigate wear particle induced osteolysis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2255-2262, 2016.

Inflammation, fracture and bone repair.

The reconstitution of lost bone is a subject that is germane to many orthopedic conditions including fractures and non-unions, infection, inflammatory arthritis, osteoporosis, osteonecrosis, metabolic bone disease, tumors, and periprosthetic particle-associated osteolysis. In this regard, the processes of acute and chronic inflammation play an integral role. Acute inflammation is initiated by endogenous or exogenous adverse stimuli, and can become chronic in nature if not resolved by normal homeostatic mechanisms. Dysregulated inflammation leads to increased bone resorption and suppressed bone formation. Crosstalk among inflammatory cells (polymorphonuclear leukocytes and cells of the monocyte-macrophage-osteoclast lineage) and cells related to bone healing (cells of the mesenchymal stem cell-osteoblast lineage and vascular lineage) is essential to the formation, repair and remodeling of bone. In this review, the authors provide a comprehensive summary of the literature related to inflammation and bone repair. Special emphasis is placed on the underlying cellular and molecular mechanisms, and potential interventions that can favorably modulate the outcome of clinical conditions that involve bone repair.

The effects of immunomodulation by macrophage subsets on osteogenesis in vitro.

BACKGROUND: Bone formation and remodeling are influenced by the inflammatory state of the local microenvironment. In this regard, macrophages are postulated to play a crucial role in modulating osteogenesis. However, the differential effects of macrophage subsets and their plasticity on bone formation are currently unknown. METHODS: Polarized primary murine macrophages and preosteoblastic MC3T3 cells were co-cultured to investigate the effect of non-activated M0, pro-inflammatory M1, and tissue-regenerative M2 macrophages on the osteogenic ability of MC3T3-E1 cells in vitro. Furthermore, to model the physiological transition from inflammation to tissue regeneration, M1-MC3T3 co-cultures were treated with interleukin-4 (IL-4) at different time points to modulate the M1 phenotype towards M2. Macrophage phenotypic markers were assessed by flow cytometry and enzyme-linked immunosorbent assay. A time course study of osteogenic markers at different time points was conducted: alkaline phosphatase (ALP) mRNA levels were evaluated at week 1, ALP activity and osteocalcin and osteopontin mRNA levels at week 2, and matrix mineralization and osteocalcin and osteopontin protein concentrations at week 3. Supernatant collected 72 hours after seeding or IL-4 treatment, whichever was later, was analyzed for oncostatin M, a cytokine released by macrophages that has been recognized to enhance osteogenesis. Unpaired t test or one-way ANOVA with Tukey's or Dunnett's post hoc tests were used for statistical comparison of the groups. RESULTS: Co-culture with any of the macrophage subtypes increased the osteogenic ability of MC3T3 cells as indicated by increases in ALP activity and matrix mineralization. Increased ALP activity, osteocalcin concentration, and matrix mineralization demonstrated that osteogenesis by M1-MC3T3 co-cultures was further enhanced by macrophage phenotype modulation to M2 via IL-4 treatment 72 hours after seeding. Increased oncostatin M protein concentration in untreated M1-MC3T3 co-cultures and M1-MC3T3 co-cultures treated with IL-4 at 72 hours correlated with greater ALP activity and matrix mineralization. CONCLUSIONS: These results suggest that a transient inflammatory phase is crucial for enhanced bone formation. Macrophage plasticity may offer new strategies for modulating the local inflammatory microenvironment with the aim of potentially enhancing bone repair.

Local delivery of mutant CCL2 protein-reduced orthopaedic implant wear particle-induced osteolysis and inflammation in vivo.

Total joint replacement (TJR) has been widely used as a standard treatment for late-stage arthritis. One challenge for long-term efficacy of TJR is the generation of ultra-high molecular weight polyethylene wear particles from the implant surface that activates an inflammatory cascade which may lead to bone loss, prosthetic loosening and eventual failure of the procedure. Here, we investigate the efficacy of local administration of mutant CCL2 proteins, such as 7ND, on reducing wear particle-induced inflammation and osteolysis in vivo using a mouse calvarial model. Mice were treated with local injection of 7ND or phosphate buffered saline (PBS) every other day for up to 14 days. Wear particle-induced osteolysis and the effects of 7ND treatment were evaluated using micro-CT, histology, and immunofluorescence staining. Compared with the PBS control, 7ND treatment significantly decreased wear particle-induced osteolysis, which led to a higher bone volume fraction and bone mineral density. Furthermore, immunofluorescence staining showed 7ND treatment decreased the number of recruited inflammatory cells and osteoclasts. Together, our results support the feasibility of local delivery of 7ND for mitigating wear particle-induced inflammation and osteolysis, which may offer a promising strategy for extending the life time of TJRs.

Establishment of Green Fluorescent Protein and Firefly Luciferase Expressing Mouse Primary Macrophages for In Vivo Bioluminescence Imaging.

Macrophages play a key role in tissue homeostasis as well as in a range of pathological conditions including atherosclerosis, cancer, and autoimmunity. Many aspects of their in vivo behavior are, however, poorly understood. Bioluminescence imaging (BLI) with green fluorescent protein (GFP) and firefly luciferase (FLUC) labelled autologous reporter macrophages could potentially offer a powerful tool to study macrophage biology, but this approach has been hindered by the relative difficulty of efficient gene transfer into primary macrophages. Here we describe a straightforward method for producing large numbers of GFP/FLUC expressing mouse primary macrophages utilizing lentivirus vector, cyclosporine, and a double infection strategy. Using this method we achieved up to 60% of macrophages to express GFP with correspondingly high FLUC signal. When injected into the circulation using a mouse model of local biomaterial induced inflammation and osteolysis, macrophages were initially detectable within the lungs, followed by systemic homing to the local area of chronic inflammation in the distal femur. In addition, transduced macrophages maintained their ability to assume M1 and M2 phenotypes although the GFP/FLUC expression was altered by the polarizing signals. These reporter macrophages could prove to be valuable tools to study the role of macrophages in health and disease.

NF-κB decoy oligodeoxynucleotide inhibits wear particle-induced inflammation in a murine calvarial model.

Wear particles induce periprosthetic inflammation and osteolysis through activation of nuclear factor kappa B (NF-κB), which up-regulates the downstream target gene expression for proinflammatory cytokines in macrophages. It was hypothesized that direct suppression of NF-κB activity in the early phases of this disorder could be a therapeutic strategy for preventing the inflammatory response to wear particles, potentially mitigating osteolysis. NF-κB activity can be suppressed via competitive binding with double stranded NF-κB decoy oligodeoxynucleotides (ODNs) that blocks this transcription factor from binding to the promoter regions of targeted genes. In this murine calvarial study, clinically relevant polyethylene particles (PEs) with/without ODN were subcutaneously injected over the calvarial bone. In the presence of PE particles, macrophages migrated to the inflammatory site and induced tumor necrosis factor alpha (TNF-α) and receptor activator of nuclear factor kappa B ligand (RANKL) expression, resulting in an increase in the number of osteoclasts. Local injections of ODN mitigated the expression of TNF-α, RANKL, and induced the expression of two anti-inflammatory, antiresorptive cytokines: interleukin-1 receptor antagonist and osteoprotegerin. Local intervention with NF-κB decoy ODN in early cases of particle-induced inflammation in which the prosthesis is still salvageable may potentially preserve periprosthetic bone stock.

NF-κB decoy oligodeoxynucleotide enhanced osteogenesis in mesenchymal stem cells exposed to polyethylene particle.

Excessive generation of wear particles after total joint replacement may lead to local inflammation and periprosthetic osteolysis. Modulation of the key transcription factor NF-κB in immune cells could potentially mitigate the osteolytic process. We previously showed that local delivery of ultrahigh-molecular-weight polyethylene (UHMWPE) particles recruited osteoprogenitor cells and reduced osteolysis. However, the biological effects of modulating the NF-κB signaling pathway on osteoprogenitor/mesenchymal stem cells (MSCs) remain unclear. Here we showed that decoy oligodeoxynucleotide (ODN) increased cell viability when primary murine MSCs were exposed to UHMWPE particles, but had no effects on cellular apoptosis. Decoy ODN increased transforming growth factor-beta 1 (TGF-ß1) and osteoprotegerin (OPG) in MSCs exposed to UHMWPE particles. Mechanistic studies showed that decoy ODN upregulated OPG expression through a TGF-ß1-dependent pathway. By measuring the alkaline phosphatase activity, osteocalcin levels, Runx2 and osteopontin expression, and performing a bone mineralization assay, we found that decoy ODN increased MSC osteogenic ability when the cells were exposed to UHMWPE particles. Furthermore, the cellular response to decoy ODN and UHMWPE particles with regard to cell phenotype, cell viability, and osteogenic ability was confirmed using primary human MSCs. Our results suggest that modulation of wear particle-induced inflammation by NF-κB decoy ODN had no adverse effects on MSCs and may potentially further mitigate periprosthetic osteolysis by protecting MSC viability and osteogenic ability.

Exposure of polyethylene particles induces interferon-γ expression in a natural killer T lymphocyte and dendritic cell coculture system in vitro: a preliminary study.

Two major issues in total joint arthroplasty are loosening of implants and osteolysis caused by wear particle-induced inflammation. Wear particles stimulate the release of pro-inflammatory cytokines, chemokines, and other inflammatory mediators from macrophages and other cells. Although the biological response of macrophages to wear debris is well established, the role of other cell types such as natural killer T lymphocytes (NKT) and dendritic cells (DCs) is limited. Here we show that ultra-high molecular weight polyethylene (UHMWPE) particles stimulate NKT cells to secrete Interferon-γ (IFN-γ); coculture with DCs further enhanced IFN-γ secretion. Furthermore, UHMWPE particles did not stimulate NKT cells to secrete IL-4, while the NKT cell natural ligand α-galactosylceramide (α-GalCer) treatment in the coculture system significantly enhanced both IFN-γ and IL-4 expression by NKT cells. Comparatively, NKT cells and/or DCs exposed to polymethylmethacrylate particles did not stimulate IFN-γ or IL-4 expression. Mouse bone marrow derived macrophage polarization by lipopolysaccharide and conditioned medium from NKT cells and/or DCs exposed to UHMWPE particles increased tumor necrosis factor-α (TNF-α), but reduced arginase-1 expression in macrophages. The current findings indicate that UHMWPE particles stimulate NKT cells/DCs to produce pro-inflammatory cytokines; this pathway is a novel therapeutic target to mitigate wear particle induced peri-prosthetic osteolysis.