The Study Of CaR In Abnormal Chondrocyte Differentiation In Osteoarthritic Cartilage Of TMJ Induced By Unilateral Anterior Crossbite

Temporomandibular disorder(TMD) is a common oral clinical disease, which could involve temporomandibular joint(TMJ) and muscles in maxillofacial and cervicoscapular region. The main symptoms of TMD are click, mandibular movement disorder and muscle pain. Temporomandibular joint osteoarthritis(TMJ OA) is an important subtype of TMD.OA is characterized by a progressive degradation of cartilage. In late stage,deposition of calcium-containing crystals in cartilage is common in OA patients.Calcium-containing crystals in OA cartilage include two types, basic calcium phosphate(BCP) and calcium pyrophosphate dehydrate(CPPD), both taking role in the process of OA. Mineral deposition was detected by μCT technology in ≈65% of patients attaining TMD as indicated by structural changes of their condyles. However, it is unclear whether the above crystals are existing in early stage of OA.During with the deposition of calcium-containing crystals, there is also the change of Ca2+ concentration in cartilage. Calcium-sensing receptor(CaR) is a membrane receptor,playing role on sensing the Ca2+ concentration in extracellular environment. It has been proved that CaR is important in development of skeletal system, but little is known about its role in OA. Only one study reported that CaR was up-regulated in osteoarthritic cartilage of knee joint.Recently, we have developed a unilateral anterior crossbite(UAC) procedure that could induce OA-like changes in the TMJ cartilage of rats and mice, characterized by chondrocytes dearth, a progressive degradation of cartilage matrix, attenuated proliferation,subchondral bone resorption and up-regulation of inflammatory factors and matrix metalloproteinase(MMPs). Using UAC model animals, we firstly detect whether calcium-containing crystals existed in TMJ OA of early stage and the expressive changes of molecules related to formation of crystals, in order to clarify the pathological role of calcium-containing crystals on cartilage degradation in TMJ OA. Secondly, we explored the expression of CaR in cartilage of TMJ OA and its possible relationship and molecular mechanisms with proliferation and differentiation of chondrocytes. Last, we inhibited the activity of CaR by NPS2143 or gene knockout to assess the therapic effect of TMJ OA.The main results:1. Abnormal mechanical force induced by UAC led the fragmentation of collagen fibers, making the Ca2+ binding site of the collagen fibers exposed. UAC stimulated cell death to produce matrix vesicles and apoptotic bodies to serve as mineral nucleation sites.UAC down-regulated the expression of inhibitors of mineralization accompanied with the up-regulation of TNAP and MMP13. The above changes contributed to the mineral deposition, especially BCP crystals. Mineral crystals served as a feed-forward signal to reduce production of matrix protein and enhanced the secretion of matrix degrading enzymes, thus further accelerating the cartilage degeneration.2. In TMJ cartialge of UAC rats, expression of CaR was up-regulated from 2-weeks,followed by up-regulated expression of PTHr P from 4-weeks, but expression of PPR, its unique receptor, was down-regulated from 2-weeks. Expression of Cdca8, Ccnb2 and Ki67(molecules related to proliferation) and Col-II, Aggrecan(phenotypical markers of chondrocytes) were decreased from 2-weeks, while Runx2、ALP and OCN(moleculesrelated to differentiation) were up-regulated from 4-weeks.3. Activation of CaR could facilitate phosphorylation of p38 and ERK. Inhibition of CaR or p38 could influence the expression of PTHr P and molecules related to proliferation.Inhibition of CaR, ERK or Runx2 could influence the expression of c and molecules related to differentiation in chondrcytes. But activation of CaR had no role in expression of PPR.4. Inhibiting the activation of CaR by locally injection of NPS2143 or knockout of CaR gene in UAC model animals could increase the thickness of cartilage and the proteoglycan content in matrix, as well as the expression of PPR and molecules related to proliferation and phenotypical markers of chondrocytes, but down-regulate the expression of CaR and molecules related to differentiation.Conclusions:1. Mechanical stimuli by UAC can enhance mineral accumulation of BCP-like minerals in the cartilage matrix in deep layers of TMJ condyle and this aberrant mineralization may contribute, at least in part, to the altered chondrocyte differentiation and the thinning of articular cartilage by promoting matrix degradation likely through the increased expression of matrix-degrading enzymes, as well as the expansion of the calcified cartilage.2. In early stage of TMJ OA, expression of CaR is up-regulated, accompanied with abnormal PTHrP signaling, leading to attenuated proliferative activity and enhanced differentiated process in cartilage. CaR plays regulative role in proliferation and differentiation via p38/PTHrP and ERK/Runx2 pathways respectively.3. Inhibiting the activation of CaR can ameliorate the osteoarthritic cartilage of TMJ through increasing the thickness and matrix content of cartilage, accompanied with enhanced cell proliferation and attenuated differentiated process. CaR might be a new target for OA therapy.