The optimal treatment for complex fractures and large bone defects is an important unsolved issue in orthopedics and related specialties. Approximately 5-10% of fractures fail to heal and develop non-unions. Bone healing can be characterized by three partially overlapping phases: the inflammatory phase, the repair phase, and the remodeling phase. Eventual healing is highly dependent on the initial inflammatory phase, which is affected by both the local and systemic responses to the injurious stimulus. Furthermore, immune cells and mesenchymal stromal cells (MSCs) participate in critical inter-cellular communication or crosstalk to modulate bone healing. Deficiencies in this inter-cellular exchange, inhibition of the natural processes of acute inflammation, and its resolution, or chronic inflammation due to a persistent adverse stimulus can lead to impaired fracture healing. Thus, an initial and optimal transient stage of acute inflammation is one of the key factors for successful, robust bone healing. Recent studies demonstrated the therapeutic potential of immunomodulation for bone healing by the preconditioning of MSCs to empower their immunosuppressive properties. Preconditioned MSCs (also known as "primed/ licensed/ activated" MSCs) are cultured first with pro-inflammatory cytokines (e.g., TNFα and IL17A) or exposed to hypoxic conditions to mimic the inflammatory environment prior to their intended application. Another approach of immunomodulation for bone healing is the resolution of inflammation with anti-inflammatory cytokines such as IL4, IL10, and IL13. In this review, we summarize the principles of inflammation and bone healing and provide an update on cellular interactions and immunomodulation for optimal bone healing.

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Fracture healing is a well-orchestrated and coordinated process and begins with the inflammatory stage involving the infiltration of immune cells and the release of cytokines, including tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10). Low-magnitude high-frequency vibration (LMHFV) stimulation is effective in promoting fracture healing. The study hypothesis was that the innate immune response was impaired in osteoporotic fracture and LMHFV could positively modulate it. 9-month-old ovariectomy (OVX)-induced osteoporotic rats were randomised into sham (SHAM), OVX control (OVX), OVX-vibration (OVX-VT) or OVX vibration plus administration of COX-2 specific non-steroid anti-inflammatory drugs (OVX-VT-NSAID). LMHFV (35 Hz, 0.3 ×g) was given 20 min/d and 5 d/week to the treatment groups. Healing and innate immune response were evaluated by weekly radiographs, endpoint micro-computed tomography (µCT), enzyme-linked immunosorbent assay (ELISA) and histomorphometry at weeks 1, 2, 4 and 8 post-treatment.Results showed that OVX slightly elevated systemic inflammation but impaired the innate immune response locally at the fracture site, with significantly lower expressions of TNF-α and IL-6 but higher IL-10 expression during the early stage of healing. LMHFV was effective in accelerating the delayed fracture healing in OVX bones by partly restoring the impaired innate immune response at the fracture site, accompanied by promoted progression of macrophage polarisation from M1 (pro-inflammatory) to M2 (anti-inflammatory) phenotype. In conclusion, vibration treatment could positively modulate the impaired innate immune response and promote macrophage polarisation in osteoporotic-fracture healing.

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Mesenchymal stem cell (MSC)‐based therapy is a promising strategy for bone repair. Furthermore, the innate immune system, and specifically macrophages, plays a crucial role in the differentiation and activation of MSCs. The anti‐inflammatory cytokine Interleukin‐4 (IL‐4) converts pro‐inflammatory M1 macrophages into a tissue regenerative M2 phenotype, which enhances MSC differentiation and function. We developed lentivirus‐transduced IL‐4 overexpressing MSCs (IL‐4 MSCs) that continuously produce IL‐4 and polarize macrophages toward an M2 phenotype. In the current study, we investigated the potential of IL‐4 MSCs delivered using a macroporous gelatin‐based microribbon (μRB) scaffold for healing of critical‐size long bone defects in Mice. IL‐4 MSCs within μRBs enhanced M2 marker expression without inhibiting M1 marker expression in the early phase, and increased macrophage migration into the scaffold. Six weeks after establishing the bone defect, IL‐4 MSCs within μRBs enhanced bone formation and helped bridge the long bone defect. IL‐4 MSCs delivered using macroporous μRB scaffold is potentially a valuable strategy for the treatment of critical‐size long bone defects.

Wear particle‐associated bone loss (periprosthetic osteolysis) constrains the longevity of total joint arthroplasty (TJA). Wear particles induce a prolonged upregulation of nuclear factor kappa B (NF‐κB) signaling in macrophages and osteoclasts. Synthetic double‐stranded oligodeoxynucleotides (ODNs) can prevent the binding of NF‐κB to the promoter regions of targeted genes and inhibit genetic activation. We tested the hypothesis that polyethylene‐particle induced chronic inflammatory bone loss could be suppressed by local delivery of NF‐κB decoy ODNs in murine in vivo model. Polyethylene particles were continuously infused into the medullary cavity of the distal femur for 6 weeks to induce chronic inflammation, and micro‐computational tomography and immunohistochemical analysis were performed. Particle‐induced chronic inflammation resulted in lower BMD values, an increase in osteoclastogenesis and nuclear translocation of p65, a prolonged M1 pro‐inflammatory macrophage phenotype, and a decrease of M2 anti‐inflammatory macrophage phenotype. Delayed timing of local infusion of NF‐κB decoy ODN for the last 3 weeks reversed polyethylene‐particle associated chronic inflammatory bone loss and facilitated bone healing. This study demonstrated that polyethylene‐particle associated chronic inflammatory osteolysis can be effectively modulated via interference with the NF‐κB pathway; this minimally invasive intervention could potentially be an efficacious therapeutic strategy for periprosthetic osteolysis after TJA.

Background Mesenchymal stem cell (MSC)-based therapy has the potential for immunomodulation and enhancement of tissue regeneration. Genetically modified MSCs that over-express specific cytokines, growth factors, or chemokines have shown great promise in pre-clinical studies. In this regard, the anti-inflammatory cytokine interleukin (IL)-4 converts pro-inflammatory M1 macrophages into an anti-inflammatory M2 phenotype; M2 macrophages mitigate chronic inflammation and enhance osteogenesis by MSC lineage cells. However, exposure to IL-4 prematurely inhibits osteogenesis of MSCs in vitro; furthermore, IL-4 overexpressing MSCs inhibit osteogenesis in vivo during the acute inflammatory period. Platelet-derived growth factor (PDGF)-BB has been shown to enhance osteogenesis of MSCs with a dose-dependent effect. Methods In this study, we generated a lentiviral vector that produces PDGF-BB under a weak promoter (phosphoglycerate kinase, PGK) and lentiviral vector producing IL-4 under a strong promoter (cytomegalovirus, CMV). We infected MSCs with PDGF-BB and IL-4-producing lentiviral vectors separately or in combination to investigate cell proliferation and viability, protein expression, and the capability for osteogenesis. Results PDGF-BB and IL-4 co-overexpression was observed in the co-infected MSCs and shown to enhance cell proliferation and viability, and osteogenesis compared to IL-4 overexpressing MSCs alone. Conclusions Overexpression of PDGF-BB together with IL-4 mitigates the inhibitory effect of IL-4 on osteogenesis by IL-4 overexpressing MSCS. PDGF-BB and IL-4 overexpressing MSCs may be a potential strategy to facilitate osteogenesis in scenarios of both acute and chronic inflammation.

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