| BackgroundsHeterotopic ossification(HO)formation of Achilles tendon is a deliberating condition that results from traumatic injuries or abnormal recovery,for which the underlying cellular origin and regulating mechanisms remain unclear.Since no vascular vessels exists around Achilles tendon,the formation of new blood vessels should be the foundation of Achilles tendon injury recovery.The endothelial-mesenchymal transition(EndMT),which may be partially modulated by neuroendocrine cytokines is thought to play a major role in HO.Neurotrophin-3(NT-3),which has neuroendocrine characteristics is widely involved in the course and development of neurological diseases,cardiovascular diseases and skeletal diseases by binding to its highly specific receptor tyrosine kinase receptor(TrkC).In this study,we investigated the effect of NT-3 on EndMT as well as the function of NT-3-TrkC signaling on differentiation of tendon-derived stem cells(TDSC)throughout the formation of HO at injuried Achilles tendon in rats.Objective1.To investigate the mechanism of NT?3 in regulating EndMT during the formation of HO at injuried Achilles tendon.2.To illustrate the role and effect of NT-3-TrkC signalling on the differentiation of TDSC throughout HO formation,Materials and Methods1.The SD rats were employed to establish the animal models of HO at injuried Achilles tendon,and the rats were sacrificed at 4,8 and 12 weeks after surgery.The serum and specimens of Achilles tendon were collected for studies.Micro-CT was used for scanning the HO formation while expression of NT-3 and TrkC were detected by ELISA and qRT-PCR.The histological changes of HO were observed by HE and saffron O fast green(SOFG)staining,the expression sites of NT-3 and TrkC were detected by immunohistochemical staining,and the endothelial origin in heterotopic bone tissue was identified by immunofluorescence staining.2.The role and effect of NT-3 on EndMT:Primary ROAECs were separated and cultured,after that,CCK-8 was utilized to test the cell toxicity of NT-3 on RAOEC.Subsequently,EndMT was induced by NT-3,and then cell morphology was analyzed by photography.Cell migration and invasion were determined by scratch assay and Transwell assay.Expression of EndMT-specific proteins were detected by qRT-PCR and Western Blot.Osteoblast and chondroblast differentiation were induced after EndMT stimulation,and Alizarin red and Alizarin blue staining,qRT-PCR and Western Blot were employed for analyzing the osteogenesis and chondrogenesis.In vivo and in vitro experiments were repeated after the addition of EndMT specific inhibitor Dorsomorphin.3.To investigate the regulatory effect of NT-3-TrkC signaling on differentiation of TDSC:qRT-PCR was used to detect the expression of osteogenic and chondrogenic genes in rat Achilles tendon tissues;Primary ROAEC were separated and cultured,after that,CCK-8 was utilized to test the cell toxicity of NT-3 on RAOEC;Osteogenesis and chondrogenic differentiation were induced.Alialiin red and alcian blue staining,qRT?PCR and Western Blot were used to detect osteogenesis and chondrogenic differentiation,as well as activation of downstream ERK1/2 signaling pathway after NT-3-TrkC signaling.In vivo and in vitro experiments were repeated after the addition of TrkC specific inhibitor GNF5837.Results1.Micro-CT showed that the volume of heterotopic tissue of Achilles tendon increased significantly within time;HE staining revealed the invasion of new blood vessels in the Achilles tendon tissues of the experimental group,the expression of cartilage notch cells and osteoblasts,and the formation of new bone marrow cavities.Immunohistochemical staining showed that NT-3 and TrkC were highly expressed in neovascularization,cartilage notch cells and osteoblasts during the process of heterotopic ossification.ELISA confirmed the increased expression of NT-3 in serum as well.2.Immunofluorescence staining showed that the endothelial marker Tie-1 was positively expressed in heterotopic bone and eo-expressed with osteoblast marker OCN and chondrocyte marker SOX9.After NT-3 induced-EndMT in RAOEC,the cells morphology changed from cobblestone to long spindle,and the migration and invasion abilities of the cells were increased.The expression levels of endothelial marker Tie-1 and other genes and prote:ins were decreased,and the expression levels of mesenchymal marker FSP-1 and other genes and proteins were increased.After the induction of osteogenesis and chondrogenesis,alizarin red staining and toluidine blue staining were positive,and the gene and protein expression levels of osteogenesis and chondrogenesis were increased.In vitro experiments,the addition of Dorsomorphin reversed NT-3-induced EndMT,and the volume of heterotopic ossified tissue in the Dorsomorphin group was significantly reduced.3.qRT-PCR was used to detect the expression of osteogenic differentiation and chondrogenic differentiation genes in the Achilles tendon tissues in vivo;In vitro,after the addition of NT-3 into TDSC,osteogenic and chondrogenic differentiation was induced.Alizarin red and alcian blue staining were positive,and the expression of osteogenic and chondrogenic genes and proteins increased.The expression of critical proteins in the ERK1/2 signaling pathway was increased.After the addition of GNF5837,the positive staining rate of TDSC cells decreased,the expression levels of osteogenic and chondrogenic markers decreased,and the ERK1/2 signaling pathway was inhibited.In vivo experiments,GNF5837 injection group also significantly inhibited the formation of heterotopic bone.ConclusionNT-3 induces the EndMT and promote the pathogenesis of HO formation,and NT?3-TrkC signaling mediates the differentiation of TDSC to promote the progression of HO formation at injuried Achilles tendon.Inhibition the activation of EndMT and NT-3-TrkC signaling can effectively reduce the formation of NT-3-induced HO formation at Achilles tendon,offering a profound and novel sight for the prevention and treatment of HO at Achilles tendon in the future. |
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