Background: Heterotopic ossification (HO) refers to the development of bone tissue in areas other than the skeletal system. The development and maturation of the skeletal system are significantly influenced by macrophage migration inhibitory factor (MIF). The objective of this study was to examine the impact of MIF on the in vitro osteogenic differentiation and mineralization of tendon-derived stem cells (TDSCs), mediated by a positive feedback loop involving ROS/HIF-1 alpha/MIF. Methods: TDSCs were isolated and identified from the hind limbs of C57/BL6 mice. The functional and procedural roles of MIF in HO, focusing on the impact of MIF on the differentiation of TDSCs into bone-forming cells were investigated in vitro. Seventy-five mice were randomly assigned to five groups. Gene expression and histological analyses of MIF and its receptors, and determine the expression of osteogenic markers in vivo. Results: The results revealed a positive and concentration-dependent effect of MIF on the osteogenic differentiation of TDSCs. Furthermore, an ROS/HIF-1 alpha/MIF positive loop was detected in the simulated early trauma hypoxic microenvironment, resulting in a 3 to 4 folds increase in MIF expression levels. MIF was also found to enhance double the expression levels of markers associated with bone and cartilage at the site of injury, consequently facilitating the development of HO, which was thought to be associated with the activation of the Wnt/(3-catenin pathway. Conclusion: MIF, which mediates the ROS/HIF-1 alpha/MIF positive feedback loop during the hypoxic phase of HO, triggers the Wnt/(3-catenin signaling pathway to enhance the osteogenic differentiation and formation of HO in TDSCs.

Soft tissue trauma can cause immune system disturbance and neuropathological invasion, resulting in heterotopic ossification (HO) due to aberrant chondrogenic differentiation of mesenchymal stem cells (MSCs). However, the molecular mechanisms behind the interaction between the immune and nervous systems in promoting HO pathogenesis are unclear. In this study, we found that mast cell-specific deletion attenuated localized tissue inflammation, with marked inhibition of HO endochondral osteogenesis. Likewise, blockage of nerve growth factor (NGF) receptor, known as tropomyosin receptor kinase A (TrkA), led to similar attenuations in tissue inflammation and HO. Moreover, while NGF/TrkA signaling did not directly affect MSCs chondrogenic differentiation, it modulated mast cell activation in traumatic soft tissue. Mechanistically, lipid A in LPS binding to TrkA enhanced NGF-induced TrkA phosphorylation, synergistically stimulating mast cells to release neurotrophin-3 (NT3), thereby promoting MSC chondrogenic differentiation in situ. Finally, analysis of single-cell datasets and human pathological specimens confirmed the important role of mast cell-mediated neuroinflammation in HO pathogenesis. In conclusion, NGF regulates mast cells in soft tissue trauma and drives HO progression via paracrine NT3. Targeted early inhibition of mast cells holds substantial promise for treating traumatic HO.

Background Heterotopic ossification (HO) refers to the development of bone tissue in areas other than the skeletal system. The development and maturation of the skeletal system are significantly influenced by macrophage migration inhibitory factor (MIF). The objective of this study was to examine the impact of MIF on the in vitro osteogenic differentiation and mineralization of tendon-derived stem cells (TDSCs), mediated by a positive feedback loop involving ROS/HIF-1α/MIF. Methods TDSCs were isolated and identified from the hind limbs of C57/BL6 mice. The functional and procedural roles of MIF in HO, focusing on the impact of MIF on the differentiation of TDSCs into bone-forming cells were investigated in vitro. Seventy-five mice were randomly assigned to five groups. Gene expression and histological analyses of MIF and its receptors, and determine the expression of osteogenic markers in vivo. Results The results revealed a positive and concentration-dependent effect of MIF on the osteogenic differentiation of TDSCs. Furthermore, an ROS/HIF-1α/MIF positive loop was detected in the simulated early trauma hypoxic microenvironment, resulting in a 3 to 4 folds increase in MIF expression levels. MIF was also found to enhance double the expression levels of markers associated with bone and cartilage at the site of injury, consequently facilitating the development of HO, which was thought to be associated with the activation of the Wnt/β-catenin pathway. Conclusion MIF, which mediates the ROS/HIF-1α/MIF positive feedback loop during the hypoxic phase of HO, triggers the Wnt/β-catenin signaling pathway to enhance the osteogenic differentiation and formation of HO in TDSCs.

Background: As a class of promising bone augmentation materials, bone cements have attracted particular attention. Due to various limitations, the current bone cements are still imperfect. In this study, an injectable pH neutral bioactive bone cement (PSC/CSC) was developed by mixing phosphosilicate bioactive glass (PSC) and alpha-calcium sulfate hemihydrate (CSH), with the goal of optimizing bone defects repairs.Methods: A range of compositions (PSC/CSC: 10P/90C, 30P/70C, 50P/50C) were developed and their physico-chemical properties evaluated. Their bone regeneration ability was compared to those of two widely used bone cements as controls (calcium phosphate cement (CPC) and Genex (R)) in rabbit femoral condyle bone defect models for 4, 8 and 12 weeks. Based on physicochemical properties and in vivo bone regeneration ability, the PSC/CSC exhibited the best outcomes was selected. Then, in vitro, the effects of selected PSC/CSC, CPC and Genex (R) extracts on MC3T3-E1 cell proliferation, migration and osteogenesis as well as angiogenesis of HUVECs were examined.Results: Based on physicochemical properties, the 30P/70C formula exhibited suitable operability and compressive strength (3.5 +/- 0.3 MPa), which fulfilled the requirements for cancellous bone substitutes. In vivo, findings from micro-CT and histological analyses showed that the 30P/70C formula better promoted bone regeneration, compared to 10P/90C, 50P/50C, CPC and Genex (R). Hence, 30P/70C was selected as the ideal PSC-based cement. In vitro, the 30P/70C extracts showed better promotion of cell viability, alkaline phosphatase (ALP) activity, calcium mineral deposition, mRNA and protein expression levels of osteogenesis in MC3T3-E1 cells, further supporting its superi-ority. Meanwhile, the 30P/70C extracts also showed better stimulation of HUVECs proliferation and angiogenesis.Conclusion: The new composite cement, 30P/70C, is a favorable bioactive glass-based bone cement with suitable operability, compressive strength and bone regeneration ability.The translational potential of this article: Clinically, treatment of large bone defects is still a major challenge for orthopaedic trauma. We showed that 30P/70C has the potential to be clinically used as an injectable cement for rapid bone repairs and reconstruction of critical sized bone defects.

Heterotopic ossification (HO) is a pathological bone formation based on endochondral ossification distinguished by ossification within muscles, tendons, or other soft tissues. There has been growing studies focusing on the treatment with rapamycin to inhibit HO, but the mechanism of mTORC1 on HO remains unclear. Tendon cells (TDs) are the first cells to form during tendon heterotopic ossification. Here, we used an in vivo model of HO and an in vitro model of chondrogenesis induction to elucidate the effect and underlying mechanism of mTORC1 in HO. The current study highlights the effect of rapamycin on murine Achilles tenotomy-induced HO and the role of mTORC1 signaling pathway on TDs. Our result showed that mTORC1 was activation in the early stage of HO, whereas the mTORC1 maintained low expression in the mature ectopic cartilage tissue and the ectopic bone formation sites. The use of mTORC1-specific inhibitor (rapamycin) immediately after Achilles tendon injury could suppress the formation of HO; once ectopic cartilage and bone had formed, treatment with rapamycin could not significantly inhibit the progression of HO. Mechanistically, mTORC1 stimulation by silencing of TSC1 promoted the expression of the chondrogenic markers in TDs. In TDs, treated with mTORC1 stimulation by silencing of TSC1, mTORC1 increased the activation of the NF-κB signaling pathway. NF-κB selective inhibitor BAY11-7082 significantly suppressed the chondrogenesis of TDs that treated with mTORC1 stimulation by silencing of TSC1. Together, our findings demonstrated that mTORC1 promoted HO by regulating TDs chondrogenesis partly through the NF-κB signaling pathway; and rapamycin could be a viable HO therapeutic regimen.