Comparison Of Proliferation And Differentiation Ability Of Mesenchymal Stem Cells From Traumatic Temporomandibular Joint Fibrous And Bony Ankylosis

Temporomandibular joint ankylosis is a long-term difficulty opening mouth disease caused by the fusion of the condyle with the glenoid fossa,which seriously affects the quality of life.Because of its difficult treatment technology and high recurrence rate,it is still a huge challenge for clinicians.Histologically,it can be divided into fibrous,fibrous-bony and bony ankylosis.It is generally believed that fibrous ankylosis can further calcify to the degree of bony ankylosis.Clinical and experimental studies have shown that the fibrous and bony ankylosis is not the same pathological process,bony ankylosis can only be transformed from fiber-bony ankylosis,and fibro-bony ankylosis is the early manifestation of bony ankylosis.The main difference between the two is the existence of fibrocartilage stage.In the animal model,it was found that sufficiently severe TMJ wound can lead to bony ankylosis,and a relatively mild TMJ wound results in fibrous ankylosis.However,how the TMJ trauma affects the tissue differentiation of the joint gap and results in a different outcome is not clear.In the early stage of our group,Wnt signaling and angiogenesis were used as the entry points to regulate the key signaling pathways of bone healing,and the difference of gene expression between fibrous and bony ankylosis was preliminarily explored.The results suggested that:(1)Enhanced BMP,Wnt signal promotes the occurrence of bony ankylosis.(2)Enhanced angiogenesis in the joint gap,especially in the early stage,promotes the formation of bony ankylosis.Recent studies have found that mesenchymal stem cells(MSCs)with multidirectional differentiation potential in the area of TMJ ankylosing radiolucent zone cause bone formation and decrease of osteogenesis ability in this region.We believe that the differentiation of tissue in the joint gap after TMJ trauma is closely related to the proliferation and differentiation of MSCs itself.This article is divided into two parts to explore this problem.Part 1: Establishment of animal model traumatic TMJ ankylosis Objective:The purpose of this study is to construct an animal model of traumatic TMJ fibrous and bony ankylosis and to explore the histologic manifestations in the early stage of ankylosis.Methods : In this experiment,12 young healthy male sheep were selected as experimental animals,and 12 sheep operated on the left and right side at the same time.Left control group(fibrous ankylosis induced side): condylar fracture + excision of the lateral 2/3 disc + removal of the fibrous zone of the glenoid fossa.Right experimental group(bony ankylosis induced side): condylar fracture + excision of the lateral 2/3 disc + glenoid fossa bone destruction.12 sheep were sacrificed and sampled at 1、2、4and 8 weeks after surgery respectively.3 sheep were killed at each time point.Histological analysis by HE staining(n=3).The records of mouth opening and body weight were measured before and after operation.Result:1.There was no significant change in the mouth opening and body weight of sheep at1 and 2 weeks postoperatively(P > 0.05),The degree of mouth opening of the sheep at 4 and 8 weeks postoperatively was significantly lower than that before surgery,and the body weight increased(P<0.05).2.Histologic analysis showed that at 1 week postoperatively,the bilateral joints of the three sheep showed an inflammatory response,which was filled with a large amount of granulation tissue,fibrin scaffold,hematoma residual and tissue debris.At 2 weeks postoperatively,the histological features of fibrous ankylosis induced side and bony ankylosis induced side of 3 sheep were different.Although the bilateral joint gap was occupied by fibrous connective tissue without cartilage.the fibrous tissue of the fibrous ankylosis induced side is more disorderly and coarse,and the hematoma remains,while the bony ankylosis induced side has no hematoma residual.At 4 and 8weeks postoperatively,the bony ankylosis induced side was fibrous-bony ankylosis.The fibrous ankylosis induced side was fibrous ankylosis.Conclusion:We successfully established a model of TMJ bony ankylosis by condylar fracture,2/3 disc removed and glenoid fossa bone destruction in sheep.and TMJ fibrous ankylosis by condylar fracture,2/3 disc and the fibrous zone of the glenoid fossa removed in sheep.The animal model is stable and can be repeated,and provides a good carrier for further exploring the pathogenesis of TMJ ankylosis.Part 2:Comparison of proliferation and differentiation of Mesenchymal Stem cells from traumatic TMJ fibrous and Bony ankylosis Objective : The aim of this study was to comparison of the proliferation and multidirectional differentiation of mesenchymal stem cells(MSCs)from traumatic TMJ fibrous and bony ankylosis.Methods : 12 young male sheep were selected as experimental animals to induce fibrous ankylosis on the left side and bony ankylosis on the right side according to the experimental 1 method.Three sheep were sacrificed at 1,2,4 and 8 weeks after surgery respectively,and obtain the ankylosing callus tissue of the joint gap.In addition,3 young male sheep were selected to obtain normal condylar cancellous bone tissue.Enzyme digestion method and improved tissue adherent method were used to isolate,culture and identify MSCs from different sources,and the proliferation and differentiation ability of MSCs in each group were compared according to the time point of sampling.The MSCs from fibrous ankylosis was collectively called FA-MSCs,and the FA-MSCs from 1,2,4 and 8 weeks after operation was called FA-MSC-1W group,FA-MSC-2W group,FA-MSC-4W group and FA-MSC-8W group,respectively.The MSCs from bony ankylosis was collectively called BA-MSCs,and the BA-MSCs from 1,2,4 and 8 weeks after operation was called BA-MSC-1W group,BA-MSC-2W group,BA-MSC-4W group and BA-MSC-8W group,respectively.MSCs from cancellous bone of normal condyle in sheep is called NC-MSCs group.Total 9 groups of MSCs from different sources.(1)flow cytometry was used to identify and detect the surface immunophenotypes of the cells in each group.(2)the proliferation ability of cells in each group was compared by cell colony-forming-unit and CCK-8 assay.(3)the differentiation ability of cells in vitro was compared by inducing osteogenesis,lipogenesis and chondrogenesis in vitro.Result:1.“Fibroblasts-like”stromal cells were successfully isolated and cultured from sheep’s normal condylar cancellous bone tissue and TMJ ankylosis callus tissue in,and could be highly proliferated in vitro.2.flow cytometry :MSCs from different sources positively expressed CD29,CD44,CD166 MSCs specific surface antigen,did not express CD31,CD45 hematopoietic stem cell surface antigen,immunofluorescence results showed that all three groups of cells can simultaneously express two positive antigens on one cell(CD29,CD44).3.colony-forming-unit : The colony forming ability of BA-MSC-1W group and BA-MSC-2W group tends to be higher than FA-MSC-1W group and FA-MSC-2W group,respectively(P > 0.05).The clone formation ability of BA-MSC-4W group and BA-MSC-8W group was higher than that of FA-MSC-4W group and FA-MSC-8W group,respectively,and lower than that of NC-MSCs group.(P < 0.05).4.CCK-8: The proliferative activity of the BA-MSC-1W group was higher than that of the FA-MSC-1W group from the third day(P < 0.05).The proliferation activities of BA-MSC-2W group and FA-MSC-2W group,BA-MSC-4W group and FA-MSC-4W group,BA-MSC-8W group and FA-MSC-8W group were similar(P > 0.05).However,from day 3 to day 7,the proliferation activities of FA-MSCs and BA-MSCs were significantly lower than those of NC-MSCs(P < 0.05).5.The results of alizarin red,oil red O,alexin blue staining and immunohistochemistry showed that MSCs of different groups could differentiate into osteoblasts,adipocytes and chondrocytes in vitro.6.Real-Time PCR detected the expression of relevant osteogenic marker genes(ALP,Runx2,OCN,OSX),and the results showed that when osteogenic induction was performed for 7 days in vitro,the expression of ALP,Runx2,OCN,OSX in BA-MSC-1W group tended to be higher than that in FA-MSC-1W group,but there was no significant difference.The expression of ALP in BA-MSC-2W group was higher than that in FA-MSC-2W group(P < 0.05),but the expression of Runx2,OCN,OSX in BA-MSC-2W group was similar to that in FA-MSC-2W group(P > 0.05).The expression of Runx2 in BA-MSC-4W group was higher than that in FA-MSC-4W group(P < 0.05).However,the expression of ALP,OCN,OSX in BA-MSC-4W group was similar to that in FA-MSC-4W group,(P > 0.05).The expression of Runx2,OCN and OSX in BA-MSC-8W group was higher than that in FA-MSC-8W group(P < 0.05).The expression of osteogenesis related gene in BA-MSCs group and FA-MSCs group tended to be lower than that in NC-MSCs group,(P > 0.05).On the 14 th day after osteogenic induction in vitro,the expressionof ALP,Runx2,OCN,OSX in BA-MSC-1W group,BA-MSC-2W group and BA-MSC-4W group was similar to that in FA-MSC-1W group,FA-MSC-2W group and FA-MSC-4W group(P > 0.05).The expression of ALP,Runx2,OCN in BA-MSC-8W group was higher than that in FA-MSC-8W group(P < 0 05).The expression of osteogenesis related gene in BA-MSCs group and FA-MSCs group tended to be lower than that in NC-MSCs group,and the expression of ALP in BA-MSC-1W group and BA-MSC-2W group was lower than that in NC-MSCs group(P < 0 05).The expression of Runx2 in FA-MSC-2W group was significantly lower than that in NC-MSCs group(P < 0 05).7.Real-Time PCR detection of the expression of the related adipogenic marker gene(PPARγ)showed that the expression of PPAR γ in FA-MSC-1W and FA-MSC-4W groups was higher than that in BA-MSC-1W group,BA-MSC-4W group and NC-MSCs group,respectively.(P < 0 05).The expression of PPARγ in BA-MSCs group was similar to that in NC-MSCs group at each time point,and there was no statistical difference.8.Real-Time PCR detection of cartilage-like marker genes(COL2A-1,SOX9)showed that The expression of COL2A-1 in BA-MSC-1W group and BA-MSC-4W group was higher than that in FA-MSC-1W group and FA-MSC-4W group and NC-MSCs group,respectively(P < 0 05).The expression of COL2A-1 in BA-MSC-2W group tends to be higher than that in FA-MSC-2W group(P > 0.05).The expressions of COL2A-1 in BA-MSC-8W group,FA-MSC-8W group and NC-MSCs group were similar(P > 0.05).The expression of SOX9 in BA-MSC-1W group and BA-MSC-4W group was higher than that in FA-MSC-1W group and FA-MSC-4W group,respectively(P < 0 05).The expression of SOX9 in BA-MSC-2W group and BA-MSC-8W group was higher than that in FA-MSC-2W group and FA-MSC-8W group,respectively(P > 0.05).BA-MSCs group and FA-MSCs group were lower than NC-MSCs at each time point(P > 0.05).Conclusion:1.MSCs were found in the regions of traumatic TMJ fibrous and bony ankylosis in sheep.The MSCs may be involved in the tissue differentiation of the joint gap and the progression of ankylosis.2.Compared with traumatic TMJ fibrous ankylosis,the MSCs in the bony ankylosis have stronger ability to proliferate,osteogenesis and cartilage,but the ability of lipogenesis decreased.This indicates that the enhanced osteogenesis and cartilage ability of MSCs in TMJ joint gap tissue promotes the occurrence of TMJ sbony ankylosis.3.The differentiation ability of MSCs in the early joint gap after TMJ trauma may be related to the outcome of TMJ after trauma.