Preconditioning of murine mesenchymal stem cells synergistically enhanced immunomodulation and osteogenesis

Abstract Background Mesenchymal 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. Methods In 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. Results Preconditioned 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. Conclusions The 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

Effect of Aging on the Macrophage Response to Titanium Particles

Macrophage-mediated inflammatory reaction to implant wear particles drives bone loss around total joint replacements (TJR). Although most TJR recipients are elderly, studies linking wear particle-activated macrophages and peri-implant osteolysis have not taken into account the multiple effects that aging has on the innate immune system and, in particular, on macrophages. To address this, we compared the wear particle responses of bone marrow macrophages obtained from young (2-month) and aged (18-month) mice. Macrophages were polarized to M0, M1, or M2 phenotypes in vitro, challenged with titanium particles, and their inflammatory response was characterized at multiple time points by quantitative reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, age-dependent changes in activation of transcription factor nuclear factor-kappa B were analyzed by a lentiviral vector-based luciferase reporter system. The particle stimulation experiment was further repeated using human primary macrophages isolated from blood donors of different ages. We found that the pro-inflammatory responses were generally higher in macrophages obtained from young mice, but differences between the age groups remained small and of uncertain biological significance. Noteworthily, M2 polarization effectively suppressed the particle-induced inflammation in both young and aged macrophages. These results suggest that aging of the innate immune system per se plays no significant role in the response of macrophages to titanium particles, whereas induction of M2 polarization appears a promising strategy to limit macrophage-mediated inflammation regardless of age.Peer reviewe

Tumor Necrosis Factor Primes and Metal Particles Activate the NLRP3 Inflammasome in Human Primary Macrophages

Aseptic loosening of total joint replacements is driven by a macrophage-mediated inflammatory reaction to implant-derived wear particles. Phagocytosis of implant debris has been suggested to activate the NLRP3 inflammasome leading to secretion of interleukin (IL)-1β. However, factors and molecular mechanisms driving the particle-induced inflammasome activation are yet to be fully elucidated. In this study, we investigated the inflammasome response of human primary macrophages to titanium, chromium, and molybdenum particles in vitro. We observed that particles alone were not sufficient to induce IL-1β secretion, but an additional priming signal—such as bacterial lipopolysaccharide (LPS)—was required to license the inflammasome activation. By using specific inhibitors against the inflammasome signaling pathway, we demonstrate that the particle-induced IL-1β secretion depended upon activation of the NLRP3 inflammasome. We further hypothesized that tumor necrosis factor (TNF) could substitute for LPS as a priming signal, and found that particle stimulation together with preceding TNF treatment resulted in inflammasome-dependent IL-1β production as well. Our results show that the NLRP3 inflammasome mediates wear particle responses in human primary macrophages, and its activation does not necessarily require the presence of bacterial components, but can be induced under aseptic conditions by TNF priming. Statement of Significance This study was conducted to elucidate the molecular mechanisms of metal particle-induced IL-1β secretion in human primary macrophages. Production of this pro-inflammatory mediator from wear particle-activated macrophages has been associated with increased bone loss around total joint replacements—a condition eventually requiring revision surgery. Our results confirm that together with a co-stimulatory priming signal, particles of common implant metals elicit macrophage-mediated IL-1β secretion through activation of the NLRP3 inflammasome pathway. We also present a concept of TNF priming in this context, demonstrating that the particle-related IL-1β secretion can take place in a truly sterile environment. Thus, inhibition of inflammasome signaling appears a means to prevent wear particle-induced inflammation and development of peri-prosthetic osteolysis.Peer reviewe

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 coculture, 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 antiinflammatory drugs on fracture healing and bone regeneration

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 (TJRs) 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

Additional file 1: Figure S1. of Preconditioning of murine mesenchymal stem cells synergistically enhanced immunomodulation and osteogenesis

Showing immunophenotypes of murine bone marrow-derived MSCs. Surface marker expression in murine MSCs at passage 4 analyzed by flow cytometry. Figure S2. showing titration of LPS dose for MSC preconditioning with TNF-α to modulate murine macrophage polarization. Murine MSCs were preconditioned with TNF-α (20 ng/ml) plus LPS (1–20 μg/ml) for 3 days, and cocultured with M1 macrophages for 24 hours. M2 (Arg1 and CD206) and M1 (TNF-α/IL-1Ra) macrophage marker expression in macrophages measured by quantitative PCR. Data presented as fold-change compared to macrophages cocultured with unstimulated control MSCs. *p < 0.05, **p < 0.01, ***p < 0.005. Figure S3. showing increased osteogenic differentiation in the MSCs with temporal inhibition of COX2 signaling. MSCs were treated with 25 μM Celecoxib in osteogenic media at indicated time points. Osteogenic differentiation ability examined by bone mineralization (Alizarin Red staining) at week 3. *p < 0.05, ***p < 0.005. Figure S4. showing increased iNOS expression in MSCs preconditioned with TNF-α and/or LPS. Murine MSCs were preconditioned with TNF-α (20 ng/ml) plus LPS (1–20 μg/ml) for 3 days, and washed out for 24 hours. Expression of iNOS determined by quantitative PCR. Data presented as fold-change compared to macrophages cocultured with unstimulated control MSCs. ***p < 0.005. (PDF 3973 kb

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