咬合异常致颞颌关节骨关节炎动物模型的创建及其软骨退变的细胞与分子机理研究

Occlusal loading is a natural biomechanical stimulation to the temporomandibular joints (TMJs). Changes in occlusion contact relation will lead to a loading alteration of TMJs. In recent, we developed a unilateral anterior crossbite (UAC) relation which can induce ultrastructural lesions of the hypertrophic chondrocytes and also calcium deposition in matrix which increased the thickness of the calcific cartilage zone of the mandibular condylar cartilage. There was also enhanced chondrocytes differentiation activity so that the hypertrophic and prehypertrophic zones grew thinning. Many reports stated that calcium influx is one of the early responses of the chondrocytes to the mechanical stimulation which immediately activates CaMKⅡ-Ihh signaling to stimulate chondrocytes differentiation, and upregulates PTHrP-PPR signaling to negatively modulate Ihh. In the present project, we will deliver UAC stimulation to the Tamoxifen inducible cartilage-specific Smo knockout mouse, the Tamoxifen inducible cartilage-specific Ppr knockout mouse and constitutively active Ppr transgenic mouse. The changes in TMJs will be investigated with the methods of Micro-CT, histology, and molecular biology. We will also conduct in vitro experiments via Flexcell loading on the isolated chondrocytes and in vivo experiments by injecting relative inhibitors or activators to TMJ region. The aim of this project is to verify that the calcification directed ultrastructural lesions of the hypertrophic chondrocytes in the deep zone of the articular cartilage and the aberrant chondrocyte differentiate due to the unbalanced CaMKⅡ-Ihh and PTHrP-PPR signalings are the principle cellular and molecular mechanisms of the UAC induced TMJ OA. The superficial invasion of the calcific cartilage zone is expected to be diagnostic and evaluative for OA progress. Elimination of the abnormal biomechanical stimulation and blocking the aberrant chondrocyte differentiation will be the principle therapeutic strategy of TMJ OA.

咬合力是天然生物力，上下牙接触关系异常可致咬合力异常。课题组最新研究发现，单侧前牙反合（UAC）可致下颌髁突深层软骨细胞超微结构损伤、钙化软骨带增厚，软骨细胞异常分化、软骨变薄。钙内流是细胞受力刺激后的初始反应之一，可激活CaMKⅡ-Ihh信号促软骨细胞分化，同时激活PTHrP-PPR信号负反馈调节Ihh的分泌。本项目拟对Ihh受体Smo基因敲除鼠、PTHrP受体Ppr基因敲除鼠及Ppr过表达鼠施以UAC刺激，从影像学、组织学、分子生物学等方面，评价颞颌关节变化，结合离体加载实验和局部注射相应药物的在体实验，论证：深层软骨细胞超微结构损伤相关的软骨钙化及CaMKⅡ-Ihh和PTHrP-PPR信号失衡所致软骨细胞过度分化，是UAC导致OA的重要细胞与分子病理机理；钙化软骨带向软骨浅层推进程度可作为OA软骨退变的诊断和评价指标；改善生物力环境、阻断软骨细胞异常分化是治疗颞颌关节OA的主要策略。

针对咬合所致颞下颌关节骨关节炎（TMJ-OA）样退行性变的问题，本项目：（1）构建了大鼠、小鼠单侧前牙反合（UAC）导致TMJ-OA的动物模型，论证了该模型中关节软骨退变随刺激时间延长而加重、随UAC的撤除而改善的特点；论证了该模型中关节盘组织瘢痕样退变的特点；论证了该模型中软骨下骨的皮质骨矿化活动增强、小梁骨吸收增强的病变规律；论证了该模型外周学白细胞Egr1、Ephx1和Il10基因表达变化的诊断意义。（2）运用该模型论证了UAC导致TMJ-OA的中枢机制：UAC通过兴奋三叉神经中脑核，导致三叉神经运动核、面神经核、舌下神经核等运动神经核团的兴奋，促进相关骨骼肌收缩， UAC还可以兴奋外侧僵核，通过外侧僵核-三叉神经中脑核神经通路，进一步加剧三叉神经中脑核的兴奋活动，最终可导致咬肌的损伤甚至萎缩。（3）运用该模型论证了OA软骨退变的细胞学机制，即：颞下颌关节髁突深层软骨细胞超微结构损伤、钙化软骨带增厚，软骨细胞异常分化，OA病变由深层逐渐向前层推进，最终导致软骨变薄、耗尽，采用终止UAC刺激的方法证实了所提出的相应治疗策略：改善异常生物力环境，抑制细胞异常终末分化。（4）采用Ihh受体Smo基因敲除鼠、PTHrP受体Ppr基因敲除鼠、Ppr过表达鼠、mTORC1基因敲除鼠以及mTORC1抑制性上游基因TSC1基因敲除鼠，分别施以UAC刺激，结合流体剪切力对软骨细胞进行体外加载实验，论证了钙内流是细胞受力刺激后的初始反应之一，通过激活CaMKⅡ、mTOR、Ihh信号促进软骨细胞分化，从而减少了PPR阳性细胞的数量，导致PTHrP-PPR信号减弱，在此过程中mTORC1是软骨细胞从自噬转向凋亡的关键信号分子。（5）构建了促进髁突软骨增厚的双侧咬合增高动物模型，为进一步研究髁突软骨增殖性活动奠定了动物模型基础。