The Study On The Mechanotransduction Mechanism Of Mandibular Condylar Chondrocyte

Mechanotransduction is the process of translating mechanical stimulation into cellular reaction, by which the live tissues adapt to mechanical environmant all around and obtain extensive physiological functions. Mechanotransduction is involved in many important physiololgical processes of many kinds of tissues. Temporomandibular joint (TMJ) is the only joint maintaining lifelong reconstruction ability among all the joints of human. Biomechanical factors resulting from the functional activity of the mandibular joint are thought to influence the growth of condyle, especially of stress-sensitive articular cartilage. The chondrocytes can commendably receive the mechanical, ionic and osmotic signal and integrated with the effects of other simulation factors such as hormone, genie modulators and environment to regulate its metabolism, which is indispensable for the TMJ to maintein its normal structural relationship, as well as its regular function. For many years, the biomechanical studies of TMJ stress and the biochamical studies of TMJ reconstruction have always been the focus of attention in TMJ field. More and more studies showed that it is the load on the TMJ that results in the remodeling of it. However, how the condyle, the most important remodeling undertaker, especially the stress-sentive articular cartilage covered on it, translate the mechanical signals into essential life signals has not been highlightened. For the importance and particularity of TMJ and the character of secondary cartilage covering on it, it is necessary to study the mechanotransduction machanism of mandibular condylar chondrocytes(MCC) by cell culture in vitro. The content of this item includes three parts summarized as following. 1 .Establishment of in vitro culture model of MCCEnzyme digestion method was adopted for primary MCC culture in vitro.The cells were identified by type II collagen staining. The MCC obtained here showed favorable growth and function character. The phenotype expression of chondrocytes could be kept steadily for at least three passages. The growth curve which could reflect the proliferation ability of the cells was obtained. In vitro cultured MCC could also experienced the same mature process as they growed in vivo and had the similiar biological activity as those cells in vivo. The success in cell culture experiment provided us with a perfect live model to investigate further into the mechanotransduction mechanism of MCC.2. Establishment of hydraulic pressure unit and seletion of feasible pressure Following the method of Yousefian's, we established a hydraulic pressurecontrolled cellular strain unit to ensure exerting exact and adjustable static pressure to our in vitro cultured MCC. Cell proliferation and alkaline phosphatase(ALP) activity were adopted to choose the feasible pressure condition combined by different pressure time and pressure value. Together with the principle that the shorter is the time for the cells endured in vitro the better it is for their growth, we fix the short-period pressure on 90KPa for 60min, under which ALP activity could be promoted without affecting the proliferation of MCC. Also, the pressure condition of 90KPa for 360min was used for long-period pressure standard, under which the proliferation activity of MCC was distinctly prohibited. The further investigations of different kinds of signal molecules in mehcanotransdution process were all under the above-mentioned two different kinds of pressure conditions.3. Mechanotransduction mechanism of MCC (1) Signal pathway of mechanotransductionFor transmission pathway, In situ hybridization was used to test integrin α5 and integrin β1 mRNA expression in MCC.Coomassie BB staining was used to observe the characteristics of cytoskeletal structure in different groups and FITC- phalloidin was used for F-actin labelling. As to transduction pathway, the expression of G α q/11 protein was evaluated by Western Blot analyses. With IP3 channel blocking by 20g/L heparin or RYR channel blocking by 1g/L procaine. Fluo-3/AM Probe was loaded and then the intracellular calcium level with or without mechanical pressure were detected by laser scanning microscope.The expression and distribution of PKC was observed by immunocytochemical staining with different pressure and G protein inhibitor treatment. Expression of ERK₁ and JNK₁were evaluated by Western Blot analyses. The distributions of them, as well as FOS protein, were all observed by immunocytochemical staining.(2) Interaction between two classic signal pathwayDouble-marking immunofluorescence method was used for investigation of relationship between G α q/11 and F-actin. Coomassie BB staining was used to observe the role of intracellular calcium and its inositol triphosphate receptor channel in the recomposition process of MCC cytoskeleton undermechanical pressure.(3) Biological effects of MCC under mechanical pressureThe ultrastructure of MCC with or without mechanical pressure were observed under transmission electron microscope(TEM). The culture supernatant were collected. The spectrophotometric method was applied to determine the activity of nitric oxide synthase (NOS) and radioimmunology assay (RIA) was adopted for PGF₁ determination. The expression and distribution of IL-4 and IL-6 were observed by immunocytochemical staining. In situ hybridization method was used to test aggrecan mRNA expression in MCC.The item studied the immediate regulation mechanism of mandibular condylar cartilage under different mechanical enviroment from cellular and molecular level. The results showed as following.1. From the aspect of machanotransduction mechanism, we got two different signal pathway in MCC under pressure.One is transmission process initiated by integrin-cytoskeleton system. The other is transduction process initiated by G protein locating on cellular membrane. Feasible pressure (90KPa 60min) could cause a5 and(31 integrin mRNA upregulated, cytoskekleton and F-actin expression strengthened and stress fibers assembled. At the same time, mechanical pressure could also stimulate the expression of G protein, the basic signal molecule on the membrane. Along with that, the intracellular calcium increased and the protein kinase C was activated and translocated to the cytomembrane and nuclei. After that, the mitogen-activated protein kinases (MAPK) access was activated and the mechanical signal was transducted further into the nuclei, along with which the immediate early genes expressed. Besides, there were also some interactions between the two classic signal pathways, such as participation of calcium in the recomposition process of MCC cytoskeleton under mechanical pressure and co-distribution of Gαq/11 and F-actin. By the above-mentioned mechanotransduction ways, the mechanical simulation could cause extensive cytobiological effects...