| Osteochondrosis(OC)is a developmental orthopedic disease that seriously affects the movement in human and animals.The lesion was characterized by the formation of a focal disturbance in the epiphyseal growth cartilage in the process of endochondral ossification.The individuals of OC usually demonstrate inconspicuous clinical signs in the early stage,leading to their difficult diagnosis.With the development of the disease,it has been reported to cause clinical signs of joint pain,effusion,and dysfunction in the middle and late stage.OC is also difficult to be treated when developing into these stages.Studies on the etiology of OC have focused on ischemia in growth cartilage,mainly manifested as cell necrosis or apoptosis,and decreased collagen and proteoglycan(PG)in extracellular matrix(ECM).However,the changes of the main components and microstructure of the matrix,and the underlying biological mechanism of OC are still unclear.The majority of studies focused on the molecular change in the surgically removed osteochondral fragments to investigate the pathogenesis of OC in human.However,these fragments could not represent for the primary disease.There are high similarities in OC between dog and human,including clinical manifestations,predilection sites,imaging changes and histological lesions,indicating that they may have a potential common pathogenesis.Therefore,based on establishing ischemic OC in dogs,the purpose of this study is to understand the OC pathogenesis at the early stage of onset so as to seek out diagnostic biomarkers and new preventive or therapeutic strategies in animals and humans at their early stage.1.Ischemia induced canine osteochondrosisGrowth cartilage has a blood supply that runs within cartilage canals and are present for a limited period of time during the early phases of development in human and animals.During the growth process,the interruption of the cartilage canal blood supply occurs at the junction between cartilage and bone where anastomoses are formed between cartilage canal vessels and vessels from the bone marrow.When endochondral ossification develops,the blood vessels in the cartilage canals would change,involving the formation of vascular anastomoses in epiphyseal growth cartilage.Once the vascular anastomosis is disordered,the OC may occur.Most cases have been found in the late stage.Therefore,establishing OC-like lesions by artificially transecting the vascular supply of epiphyseal growth cartilage to carry out the related research at the early stage.The methods of ischemia induced OC in growth cartilage depend on the direction of cartilage canal vessels.Based on the results of microvascular perfusion in dogs from our lab,the canine cartilage canal vessels in the femoral condyle were longer and less than those in the femoral trochlea,while the vessels of medial condyle were longer and less than those of lateral femoral condyle.Therefore,the medial femoral condyle may be an appropriate site to induce cartilage ischemic lesion compared to the trochlear and lateral condyle in dogs.In this study,in order to establish the OC model,a full thickness cartilage incision on the rim of abaxial aspect of the medial femoral condyle was made to interrupt the blood supply to the epiphyseal growth cartilage.The medial femoral condyle of the contralateral limb was used as a healthy control.X plain radiography before surgery(day0)and at day 6,10,20,and 30 postoperatively were performed to evaluate the calcification of epiphyseal growth cartilage.The cartilage slabs from the femoral condyle of knee joint from six dog at day 6 and 30 postoperatively were routinely paraffined,sectioned and stained with haematoxylin and eosin(H&E),safranin-fast green and toluidine blue staining for histological evaluation.The chondrocytes apoptosis rates were measured by TUNEL staining.The changes of collagen morphology and cartilage components were detected by scanning electron microscope(SEM)and Raman spectroscopy.The results showed that the distal medial femoral condyle showed a local defect in a time-dependent manner from day6,10,20 to 30 after surgery.The histological observation of OC group indicated typical ischemic chondronecrosis.The cartilage canal vessels were incomplete with the endothelial cells detached and the intraluminal erythrocytes disappeared,displaying severe necrosis and degeneration.Surrounding the necrotic cartilage canal vessels were chondrocytes with disappeared nuclei or pyknotic nuclei and condensed or eosinophilic cytoplasm,forming a vermiculate-like appearance zone.The safranin-fast green and toluidine blue staining showed that the vascular intervention caused lighter staining in ECM surrounding the necrotic cartilage canal vessels at day 6 after surgery.All these lesions were more observed at day 30 after operation.The chondrocytes in the middle and deep zones showed apoptosis by TUNEL staining at day 6 after surgery.This chondrocyte apoptosis gradually developed from deep zone to superficial zone at day 30 after operation.The results of Raman analysis showed that the changes of proteoglycan and collagen related peaks in OC group were mainly occur in the middle and deep zones.Therefore,it can be seen that chondrocyte apoptosis and matrix disorder would promote OC.2.Analysis of differentially expressed proteins by Label-free proteomics technology in canine OCThere is a normal physiological regression of these canals in the growth plate cartilage and/or epiphyseal cartilage with maturity in human and animals.The physiological regression is characterized histologically by chondrification: the vessels regress,disappear and the mesenchymal cells of the canals differentiate into chondrocytes that eventually fill the canal with matrix.The adjacent chondrocytes remain viable.Under the pathological condition,the ischemic OC mainly manifested as chondrocyte necrosis or apoptosis and ECM disorder around the cartilage canal at the early stage,which could eventually develop into osteochondrosis dissecans(OCD)or self-healing at its late stage.This suggests that some specific biological substances within growth cartilage may be involved in the regulation of the occurrence and development of ischemic OC.In this study,proteomic technique of Label free was used to identify differentially expressed proteins,exploring the potential molecular biological mechanism of OC.In this study,at day 30 after operation,these full-thickness cartilage samples(about 3mm)were collected from the epiphyseal growth cartilage in OC and control groups,and the differentially expressed proteins were analyzed by Label free technique.These results of proteomics were verified by ELISA.A total of 1369 proteins identified by mass spectrometry were differentially screened,78 differential proteins of them were obtained.Among them,31 proteins were significantly increased,while 47 proteins were significantly down-regulated.The results of ELISA were highly consistent with those of proteomic sequencing.From them,some differential proteins(Htr A1,POSTN,S100A4,IGFBP5,PEDF,ALP and F??,etc.)were involved in the regulation of extracellular matrix disorders.Some differential proteins(Htr A1,S100A4 and Fis,etc.)were involved in apoptosis.Importantly,Htr A1,a secretory serine protease,was significantly increased in OC cartilage.It has been reported that Htr A1 participates in inducing apoptosis and disordering the ECM.Therefore,we speculate that Htr A1 may play an important role in this disease by inducing chondrocyte apoptosis and matrix disorder.3.The role of Htr A1 in the pathogenesis of OCThe primary chondrocytes were isolated from the growth plate cartilage in dogs and identified by immunofluorescence.The over expression carrier of pc DNA3.1(+)-Htr A1 plasmid was constructed.Different doses of Htr A1 overexpression plasmids(1,3 and 6?g/m L)were used to transfect into canine primary chondrocytes.Then,the apoptosis rate of chondrocytes induced by Htr A1 overexpression was detected by flow cytometry.These proteins related to apoptosis in XIAP/caspase 9/caspase 3 signaling pathway were detected at gene and protein levels.The gene expressions of components in ECM(i.e.collagen II and aggrecan)and matrix metalloproteinases(i.e.ADAMTS4 and ADAMTS5)were detected.Furthermore,different doses of Htr A1 recombinant protein(0.1,1 and 5 ?g/m L)were used to treat the explants from epiphyseal growth plate cartilage for 24 h.The cartilage explants from control and Htr A1 overexpression treatments were routinely paraffined,sectioned and stained with H&E,safranin-fast green and toluidine blue staining for histological evaluation.The changes of collagen morphology and matrix components in cartilage were detected by SEM and Raman spectroscopy.The expression of genes and proteins related to cell apoptosis and matrix disorder were detected.These studies confirmed the role of Htr A1 in inducing chondrocyte apoptosis and matrix disorder,and elucidated the molecular biological mechanism of the occurrence and development of OC.The results showed that overexpressed Htr A1 at different doses(1,3 and 6 ?g/m L)can induce apoptosis of the canine primary chondrocyte by activating XIAP/caspase9/caspase 3 signaling pathway in a dose-dependent manner.Htr A1 overexpression significantly increased the gene expression of ADAMTS4 and ADAMTS5,and significantly decreased the gene and protein expression of collagen II and aggrecan.These results indicate that Htr A1 overexpression can induce chondrocyte apoptosis and ECM disorder.Meanwhile,in the study of cartilage explants,it was showed that recombinant Htr A1 protein at medium and high doses(1 and 5 ?g/m L)could induce OC-like lesions in cartilage explants in a dose-dependent manner.Histological analysis showed that necrotic cartilage canal and its surrounding necrotic chondrocyte could be observed in medium-and high-dose recombinant Htr A1 protein treatment group.Meanwhile,the staining of ECM of cartilage explants was light,especially in the area surrounding the necrotic cartilage canal vessels.It was showed that the collagen distribution was disordered and even obvious fracture in the full-thickness of cartilage(superficial,middle and deep zones)in Htr A1 recombinant protein group with SEM.This microscopic change is similar to those in the middle and deep zones of cartilage in OC group.Furthermore,the changes of PG and collagen were also detected by Raman spectroscopy.Therefore,it was confirmed that abnormally elevated Htr A1 can induce chondrocyte apoptosis and ECM disorder in vitro,participating in the process of OC development.In conclusion,transecting the local blood vessels within epiphyseal growth cartilage canals of canine distal femur can successfully induce ischemic chondronecrosis.The specific biological substance of Htr A1 in growth plate cartilage was found by using label free proteomics.Based on primary chondrocytes and epiphyseal growth cartilage explants,the role of Htr A1 in inducing chondrocyte apoptosis and matrix disorder(collagen and PG)was determined,and the molecular biological mechanism of OC occurrence and development was elucidated. |
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