| Research backgroundHeterotopic ossification(HO)is defined as the formation of bone tissue in extraskeletal tissues such as muscle and soft tissue.Nearly 40% of patients with heterotopic ossification are accompanied by local pain and severely limited motor function.At present,the mechanism of heterotopic ossification has not been fully elucidated.The mainstream view is that the formation of heterotopic ossification requires three necessary conditions: 1.Inducible progenitor cells;2.A suitable soft tissue environment for induction;3.Signaling pathways for inducing ossification.The current clinical treatment methods for heterotopic ossification mainly include radiation and non-steroidal anti-inflammatory drugs,but both of them have side effects such as affecting bone healing and gastrointestinal reactions,so new treatment methods are urgently needed.Studies have shown that during osteogenic differentiation,osteogenic progenitor cells need to be in a low-concentration endogenous retinoic acid environment.Therefore,artificially increasing the concentration of retinoic acid may have the effect of treating heterotopic ossification.Research objectiveFocusing on the three elements of heterotopic ossification,the identity of the heterotopic ossification progenitor cells of tendon stem cells(TSCs)was determined,the role of inflammatory microenvironment(IM)in heterotopic ossification was explored..And the relationship between nuclear factor-kappa B(NF-κB)and Smad signaling pathway with heterotopic ossification was explored,at last,explore the mechanism of Palovarotene in the treatment of heterotopic ossification.Research methodsIn vitro,we constructed a co-culture model of tendon stem cells and inflammatory cells.In vivo,we constructed a SD rat model of heterotopic ossification.Through Western Blot and RT-q PCR,tissue sections,immunohistochemistry,immunofluorescence and Mirco CT and other experimental techniques to explore in vitro and in vivo ectopic bone formation,the origin of ectopic bone and the occurrence of ectopic ossification of osteogenesis-related proteins,Expression of inflammation-related proteins.Focus on the activation of target signaling pathways,and observe the blocking effect of Palovarotene on heterotopic ossification in the above process.Next,the overexpression and knockdown vectors of Smad signaling pathway and Nf-kb signaling pathway were respectively constructed to further explore the synergistic effect of Nf-kb and Smad signaling pathways in the osteogenic differentiation of tendon stem cells,and to explore the mechanism of Palovarotene in the treatment of heterotopic ossification.Results1.Tendon stem cells were located in the rat Achilles tendon tissue,and the cells extracted from the rat Achilles tendon were identified as tendon stem cells.2.Under inflammatory microenvironment and trauma stimulation,alizarin red staining(ARS)and alkaline phosphatase staining(ALP)showed increased calcium deposition and alkaline phosphatase content in tendon stem cells.Micro CT showed increased ectopic bone in the model group;Western Blot,RT-q PCR,immunofluorescence and immunohistochemistry indicated that the osteogenic genes OCN,RUNX2,SOX9 in tendon stem cells and HO model rats under inflammatory microenvironment and trauma stimulation The levels of inflammatory factors such as TNF-α,TGF-β,and IFN-γ were up-regulated,and the levels of Nf-kb signaling pathway core molecule P65 and Smad signaling pathway core molecule Smad5 were up-regulated.The above process can be blocked by pararotene.3.The constructed overexpression and knockdown vector transfection experiments showed that further overexpression of Nf-kb and Smad signaling pathway further enhanced the osteogenic differentiation trend,which could be inhibited by Palovarotene.Knockdown of Nf-kb and Smad signaling pathways could inhibit the osteogenic differentiation of tendon stem cells,and the osteogenic differentiation trend was similar to that of the Palovarotene-administered group.Conclusion1.Tendon stem cells are one of the progenitor cells of heterotopic ossification.2.The inflammatory microenvironment plays an important role in the process of heterotopic ossification by activating Nf-kb and Smad signaling pathways.3.Palovarotene can treat heterotopic ossification by deactivating Nf-kb and Smad signaling pathways.Figure 22 Table 1 Reference 105... |
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