Eemeli Jämsen

Characterization of macrophage polarizing cytokines in the aseptic loosening of total hip replacements

Aseptic loosening of hip replacements is driven by the macrophage reaction to wear particles. The extent of particle‐induced macrophage activation is dependent on the state of macrophage polarization, which is dictated by the local cytokine microenvironment. The aim of the study was to characterize cytokine microenvironment surrounding failed, loose hip replacements with an emphasis on identification of cytokines that regulate macrophage polarization. Using qRT‐PCR, the expression of interferon gamma (IFN‐γ), interleukin‐4 (IL‐4), granulocyte–macrophage colony‐stimulating factor (GM‐CSF), IL‐13, and IL‐17A was low and similar to the expression in control synovial tissues of patients undergoing primary hip replacement. Using immunostaining, no definite source of IFN‐γ or IL‐4 could be identified. IL‐17A positive cells, identified as mast cells by double staining, were detected but their number was significantly reduced in interface tissues compared to the controls. Significant up‐regulation of IL‐10, M‐CSF, IL‐8, CCL2‐4, CXCL9‐10, CCL22, TRAP, cathepsin K, and down regulation of OPG was seen in the interface tissues, while expression of TNF‐α, IL‐1β, and CD206 were similar between the conditions. It is concluded that at the time of the revision surgery the peri‐implant macrophage phenotype has both M1 and M2 characteristics and that the phenotype is regulated by other local and systemic factors than traditional macrophage polarizing cytokines.

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.

Correlations between macrophage polarizing cytokines, inflammatory mediators, osteoclast activity, and Toll-like receptors in tissues around aseptically loosened hip implants.

Aseptic loosening and osteolysis of joint replacements are driven by macrophage-mediated inflammatory reactions to implant-derived wear debris, but many aspects of these events remain poorly characterized. To better understand the relationships among inflammatory and chemotactic mediators, macrophage phenotype and polarizing cytokines, osteoclast activity, and Toll-like receptors (TLRs) in the pathogenesis of aseptic loosening, we determined how the relative expressions of these factors in the peri-implant tissues correlate to each other and to the life span of the implants using Pearson correlation. The expression of pro-inflammatory mediators and chemokines showed positive correlations among themselves, and with TLR4. Furthermore, M1-polarizing IFN-γ showed positive correlations with a number of pro-inflammatory and chemotactic mediators, whereas M2-polarizing IL-4 showed no such association. IL-8 expression significantly correlated with early time to revision. Similar trends were observed for TNF-α, IFN-γ, and CCL3, while the opposite was detected for IL-4. However, none of the inflammatory mediators correlated with the markers of osteoclast activity or the RANKL/OPG ratio. The results highlight the importance of the inflammatory mediators, IFN-γ and TLR4, in the pathogenesis of aseptic loosening; increased pro-inflammatory status was associated with early time to revision, whereas IL-4 correlated with longer implant survival.

Mesenchymal stem cells in the aseptic loosening of total joint replacements

Peri-prosthetic osteolysis remains as the main long-term complication of total joint replacement surgery. Research over four decades has established implant wear as the main culprit for chronic inflammation in the peri-implant tissues and macrophages as the key cells mediating the host reaction to implant-derived wear particles. Wear debris activated macrophages secrete inflammatory mediators that stimulate bone resorbing osteoclasts; thus bone loss in the peri-implant tissues is increased. However, the balance of bone turnover is not only dictated by osteoclast-mediated bone resorption but also by the formation of new bone by osteoblasts; under physiological conditions these two processes are tightly coupled. Increasing interest has been placed on the effects of wear debris on the cells of the bone-forming lineage. These cells are derived primarily from multipotent mesenchymal stem cells (MSCs) residing in bone marrow and the walls of the microvasculature. Accumulating evidence indicates that wear debris significantly impairs MSC-to-osteoblast differentiation and subsequent bone formation. In this review, we summarize the current understanding of the effects of biomaterial implant wear debris on MSCs. Emerging treatment options to improve initial implant integration and treat developing osteolytic lesions by utilizing or targeting MSCs are also discussed.

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.

Preconditioning of murine mesenchymal stem cells synergistically enhanced immunomodulation and osteogenesis

BackgroundMesenchymal stem cells (MSCs) are capable of immunomodulation and tissue regeneration, highlighting their potential translational application for treating inflammatory bone disorders. MSC-mediated immunomodulation is regulated by proinflammatory cytokines and pathogen-associated molecular patterns such as lipopolysaccharide (LPS). Previous studies showed that MSCs exposed to interferon gamma (IFN-γ) and the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) synergistically suppressed T-cell activation.MethodsIn the current study, we developed a novel preconditioning strategy for MSCs using LPS plus TNF-α to optimize the immunomodulating ability of MSCs on macrophage polarization.ResultsPreconditioned MSCs enhanced anti-inflammatory M2 macrophage marker expression (Arginase 1 and CD206) and decreased inflammatory M1 macrophage marker (TNF-α/IL-1Ra) expression using an in-vitro coculture model. Immunomodulation of MSCs on macrophages was significantly increased compared to the combination of IFN-γ plus TNF-α or single treatment controls. Increased osteogenic differentiation including alkaline phosphate activity and matrix mineralization was only observed in the LPS plus TNF-α preconditioned MSCs. Mechanistic studies showed that increased prostaglandin E2 (PGE2) production was associated with enhanced Arginase 1 expression. Selective cyclooxygenase-2 inhibition by Celecoxib decreased PGE2 production and Arginase 1 expression in cocultured macrophages.ConclusionsThe novel preconditioned MSCs have increased immunomodulation and bone regeneration potential and could be applied to the treatment of inflammatory bone disorders including periprosthetic osteolysis, fracture healing/nonunions, and osteonecrosis.

Aseptic Loosening of Total Hip Arthroplasty as a Result of Local Failure of Tissue Homeostasis

Total hip arthoplasty (THA) is the most effective and safest method for treating severe degenerative, post-traumatic and other diseases of the hip joint. It is estimated that more than 1,000,000 THAs are performed each year globally. The incidence of primary THA increased in the period 1990 to 2002 in the USA from 47/100,000 to 69/100,000 (Kurtz et al. 2007). A similar study in Denmark reported an increased rate of THAs from 101/100,000 to 131/100,000 during the period 1996 to 2002 (Pedersen et al. 2005). More importantly, modelled future projections expect further increase in the need for THAs. It is believed that THA can reliably relieve pain and improve function in the majority of patients for a period of 15 to 20 years or more postoperatively. On this basis, with the extension of THAs to a younger and generally more active population, the expected time of service of THAs would be insufficient and the number of revision surgeries would therefore increase during this time. The Kaplan-Meier ten-year revision-free survival estimates for younger patients range from 72% (95%CI: 67-76) in Finland to 86% (95%CI: 84,5-88,2) in Sweden (Corbett et al. 2010). Hence, 14% to 28% of such patients on average did not achieve a 10-year THA functioning without revision. The main reason for late failure of THA is aseptic loosening accompanied by osteolysis followed by infection and instability of the THA that compromise more the early postoperative

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.

Biomaterial–Host Interactions in Aseptic and Septic Conditions

Implanted total joint replacement is initially osteointegrated via successive steps of inflammation, resorption of necrotic bone, bone matrix production and ultimately bone remodeling, and is largely mediated by the coordinated action of osteoclasts and osteoblasts. Years later, a total joint replacement that has previously been well osteointergrated, can become loosened necessitating technically difficult and costly revision operations. Two different modes of prosthesis loosening have traditionally been distinguished, namely prosthesis infection, also known as septic loosening, and aseptic prosthesis loosening. Septic and aseptic prosthesis loosening have long been considered as two separate entities where septic loosening is due to chronic inflammation and accompanied osteolysis caused by bacterial infection of prosthesis components while aseptic loosening is driven by macrophages inflammatory foreign body reaction against biomaterial wear particles that are generated due to unavoidable abrasion between prosthesis components. During last decade this strict dichotomy between septic and aseptic prosthesis loosening has been questioned by observations that subclinical bacterial biofilms are present at last in some cases of aseptic prosthesis loosening and that the pro-inflammatory and osteolytic properties of wear particles are largely dependent on the presence of bacterial structural components adhering to their surfaces. The recognition of such bacterial product coated wear particles and subsequent activation of macrophages to inflammatory phenotype is possibly mediated by Toll-like receptors expressed in the interface tissues. Further studies are warranted to better characterize the role of subclinical bacterial biofilms in the aseptic prosthesis loosening. Peri-implant B lymphocyte and plasma cell infiltrates might provide additional diagnostic tools to detect such, low-grade, biofilm hidden implant infections.

Modulating Innate Inflammatory Reactions in the Application of Orthopedic Biomaterials

Orthopaedic biomaterials are used in a wide variety of surgical procedures including total joint replacement, spine reconstruction, and fracture repair. Despite the development of materials with enhanced mechanical and biological properties, the attachment of an implant to the surrounding bone is still occasionally lost and revision surgery is required in some of the patients with prolonged implantation of orthopaedic biomaterials. The macrophage-associated innate immune response plays a crucial role both in the successful integration and potential rejection of the implant. Acute inflammation is essential for the successful osseointegration and bone regeneration around the implants. Chronic inflammation, on the other hand, is associated with impaired bone formation and osteolysis in the presence of excessive macrophage infiltration and pro-inflammatory cytokine secretion. Here we summarize the current development of immunomodulating strategies to improve the application of orthopaedic biomaterials. The potential drug delivery and controlled release methods that could be applied to administrate immunomodulatory biomolecules are also discussed. In summary, modulations of the innate immune response with the goal of sequential transition from a pro-inflammatory to an anti-inflammatory reaction provides a promising strategy for successful bone regeneration and implant osseointegration.