The Study Of OA-like Alterations And Cx43Hemichannels Function In Cartilage Degradation Of Rat TMJ Induced By Experimentally Created Disordered Occlusion

Temporomandibular disorder （TMD） is a common oral clinical disease related withocclusion. Temporomandibular joint osteoarthritis （TMJ OA） is an important subtype ofTMD. OA is characterized by chondrocytes apoptosis, a progressive degradation ofcartilage, subchondral bone remodeling and so on. However, the process of condylarsubchondral bone remodeling and the initial mechanism of cartilage degeneration remainobscure.Based on our clinical experience, we have established a TMJ OA rat model inducedby experimentally created disordered occlusion. In present study, firstly, in vivo micro-CT,histomorphometric analysis, real time PCR and immunohistochemistry were used to observe the longitudinal condylar subchondral bone alterations and changes of theosteoblast and osteoclast activity in TMJ OA rat with posterior disordered occlusion.Secondly, we established a new TMJ OA rat model with unilateral anterior crossbite, andevaluated the TMJ OA like lesions with morphological method, in order to confirm thatthe OA like lesions in this new animal model were more serious than the former. Then,based on the rat model with unilateral anterior crossbite, methods of cell shear stress, dyeuptake, siRNA, ELISA and western blot were adopted to investigate the role of Cx43hemichannels of chondrocytes in TMJ OA cartilage degeneration induced by abnormalbiomechanical stimulation.The main results:1. Subchondral bone loss was detected from8weeks after dental occlusion alterationand reached to the maximum at12weeks, followed by a repair phase until32weeks.Although bone mass was increased at late stage, the poor mechanical structure and lowerbone mineral density were found in these rats. The number of TRAP-positive cells wasincreased at12weeks, while the number of osteocalcin expressing cells was increased atboth12and32weeks. Levels of mRNA expression of TRAP and cathepsin K wereincreased at12weeks, while levels of ALP and osteocalcin were increased at both12-and32weeks. These findings demonstrated that there is an active bone remodeling insubchondral bone in TMJs in response to alteration in occlusion although the new bonewas formed with lower BMD and poor mechanical property.2. To study the progressive degradation of condyle in TMJ OA, we established the ratmodel with unilateral anterior crossbite. Rats were sacrificed at2,4and8weeks aftersurgery for micro-CT scanning and real time PCR assay. Significant condylar subchondralbone loss could be observed at2weeks after surgery. Micro-CT analysis revealed thesignificant decreased BMD and BV/TV. The mRNA expression of RANKL、RANKL/OPG、RANK、TRAP and cathepsin K significantly increased. The expression ofosterix、RUNX2、ALP、osteocalcin decreased at2weeks, and increased at4and8weeksafter surgery. Depositions of calcium salt were found at the osteochondral interface indicating the aberrant calcification in mature chondrocyte layer.3. To study the molecular mechanism of TMJ OA like changes induced by abnormalocclusion, a series of in/ex vivo and in vitro experiments were performed on rat modelwith unilateral anterior crossbite. The in/ex vivo results showed that the expression ofCx43and the synthesis and secretion of PGE₂increased in rat condylar cartilage withunilateral anterior crossbite at2,4and8weeks after surgery. The in vitro results showedthat fluid flow shear stress promoted the expression of Cx43and synthesis of PGE₂inprimary chondrocyte of rat TMJ cartilage. Mechanical strain could also open Cx43hemichannels, and then the release of intracellular PGE₂increased. And the increasedsecretion of PGE₂could be blocked by the hemichannel inhibitor. The similarly changescould also be found in ATDC5cell line with the shear stress stimulation. In addition, thesecretion of PGE₂decreased due to the effect of siRNA on Cx43expression, while nosignificant change could be found in the synthesis of PGE₂.Conclusions:1. The present studies demonstrated a predominantly resorptive activity in thesubchondral bone of TMJ condyles in the initial phases and a reparative capability in laterstages in response to changes of dental occlusion. The newly formed subchondral bone hasa poor quality.2. The higher stimulation of abnormal occlusion could lead to earlier and aggravatingsubchondral bone loss in rat TMJ with unilateral anterior crossbite. In addition, aberrantcalcification was found at the osteochondral interface.3. One of the key mechanisms in TMJ condylar cartilage degradation induced bymalocclusion is that the abnormal biological force could increase the expression of Cx43and the synthesis of PGE₂, and open Cx43hemichannels in chondrocytes. Then theincreased release of PGE₂through Cx43hemichannels leads to cartilage degradation.