Mechanism Of Chondrocytes-mediated Differentiation Of Synovial-derived Osteoclasts To TMJ OA

Objective:In this experiment,by constructing a model of TMJ OA caused by decreased occlusal height in rats,exploring the changes of articular cartilage and subchondral bone and osteoclasts in the inflammatory microenvironment,further studying the role of Wnt(LRP5/6)signaling pathway in TNF-α-induced synovial-derived osteoclast differentiation,and exploring its deep molecular mechanism,so as to reveal the mechanism of TMJ OA occurrence and development in vivo,aiming to seek new therapeutic targets for the treatment of TMJ OA.Methods:1.The model of reduced posterior teeth height in rats was established,and the condylar cartilage specimens were collected at 3 time points after the successful establishment of the model,namely,2,4 and 8 weeks.2.Using Micro-CT,a comparison of condyles between the experimental and control groups was made,as well as morphological and histological changes in cartilage and subchondral bone,and hematoxylin/eosin(HE)staining was used to detect alterations in the temporomandibular joint’s synovium.Safranin O-Fast Green staining was also employed to detect modifications in the cartilage matrix Immunohistochemistry and Immunofluorescence technique to detect chondroblast related genes such as COLX,PCNA,and osteoclast differentiation related genes such as RANK.Chondrocyte apoptosis detected by TUNEL staining.3.Screening and study on differentiation of osteoclast progenitor RAW264.7 in vitro under different concentrations of TNF-α.RAW264.7+Inducer(RANKL),RAW264.7+Inducer(RANKL)+TNF-α(10ng/ml),RAW264.7+Inducer(RANKL)+TN F-α(25ng/ml)were compared with RAW264.7 cultured alone.Osteoclast differentiation was detected by TRAP staining,RANK related mRNA expression was detected by qRT-PCR,and RANK protein expression was detected by Western-blot.Annexin V-FITC/PI was used to detect the apoptosis rate of osteoclast progenitor cells,and the experimental group with the most obvious osteoclast differentiation was selected as the optimal concentration of TNF-α.4.To investigate the role of the classical Wnt signaling pathway in TNF-α-induced osteoclastic differentiation.During osteoclast differentiation of RAW264.7,WNT3 a and WNT5 a inhibitors were administered respectively.TRAP staining was utilized to differentiate osteoclast,qRT-PCR was employed to detect RANK related mRNA expression,and Western-blot was employed to detect RANK protein expression in comparison to the control group.5.To investigate the effect of Wnt/(LRP5/6)signaling pathway on osteoclast differentiation induced by TNF-α.DDK-1 and MDC were given respectively during osteoclast differentiation of RAW264.7.TRAP staining was utilized to differentiate osteoclast,qRT-PCR to detect RANK related mRNA expression,and Western-blot to identify RANK protein expression,in comparison to the blank control group.Results:1.HE staining showed that the condyle of rats in the experimental group had degenerative changes,among which,the sequence of cartilage trabeculae was abnormal,and the density of chondrocytes was significantly reduced,accompanied by the increase of time.The degree of such changes gradually intensified until the condyle structure damage reached its peak at the 8th week.2.Saffron O-Fast green staining showed that the loss of cartilage matrix in condylar cartilage increased with time and reached its peak at 8 weeks.3.Micro-CT showed that compared with the control group,the trabecular thickness(Tb.Th),trabecular number(Tb.N),bone volume/tissue volume(BV/TV)and bone mineral density(BMD)of the experimental group were significantly reduced,and the bone destruction was more with the extension of time.4.Immunohistochemistry showed that compared with the control group,the number of positive cells of COLX,TUNEL,TNF-α in cartilage in the experimental group increased,and the number of RANK-positive cells in the synovial membrane increased.However,PCNA-stained positive cells in cartilage showed no significant changes.5.Compared with the control group,there was no significant change in PCNA expression in COLⅡ-PCNA-TNF-α in the experimental group,while the expression of TNF-α increased significantly.Immunofluorescence staining of COLⅡ-COLX-TNF-α showed that COLX and TNF-α in cartilage increased significantly in the experimental group compared with the control group.Compared with the control group,the expression of RANK in the synovial membrane of the experimental group was greatly increased,while the expression of Wnt3 a and LRP5/6 was relatively reduced.6.Annexin V-FITC/PI results showed that apoptosis rate of RAW264.7+ Inducer(RANKL)+TNF-α(25ng/ml)group was significantly increased compared with RAW264.7+ Inducer(RANKL)+TNF-α(10ng/ml)group and control group.7.qRT-PCR and Western-blot results showed that RANK-related mRNA and protein expressions in RAW264.7+ Inducer(RANKL)+TNF-α(10ng/ml)group were significantly higher than those in RAW264.7+ Inducer(RANKL)+TNF-α(25ng/ml)group.The mRNA and protein expressions of RANK in Wnt5 a inhibitor group were significantly lower than those in Wnt3 a inhibitor group.The mRNA and protein expressions of RANK in DDK1 group were significantly higher than those in control group and MDC group.Conclusions:During the occurrence and development of TMJ OA caused by the change of joint compressive stress caused by the decrease of occlusal height,TNF-α secreted by hypertrophic chondrocytes plays an important role in regulating the differentiation of osteoclast progenitor cells in joint synovium.This study proves that TNF-α inhibits osteoclastic differentiation by Wnt/(LRP5/6)signaling pathway,that,the occlusal height decreases by inhibiting the activation of Wnt/(LRP5/6)signaling pathway and promotes the occurrence and development of TMJ OA.The deep molecular mechanism of TMJ OA induced by stress stimulation was revealed,which provided a new idea for clarifying the cellular and molecular mechanism of TMJ OA therapy and a new theoretical basis for clinical treatment of TMJ OA.