Study on Human Temporomandibular Joints Synovial Mesenchymal Stem Cells Neurogenesis and Pain
|Keywords||Tempotomandibular joints synovial mesenchymal stem cells neurogenesis pain neuropeptides|
Adult stem cells can be differentiated into various types of cells. For instance, mesenchymal stem cells can be differentiated into chondrogenic, osteogenic, neurogenic, myogenic and vascular of blood cells. Skin stem cells can be differentiated into neurons, adipose cells or muscle cells. Some researcher suggested that ability of tissue-derived stem cells to differentiate into other stem cells. And neural stem cells to derive from more primitive cells that have the capacity to generate neural stem cells and stem cells of other tissues. The neural stem cell is used to describe that can generate neural tissue or derived from the various system; have some capability for self-renewal; can give rise to cells other than themselves through asymmetric cell division. In the last century, some researchers were separated and cultured embryonic stem cells, and induced the stem cells differentiated into neurons, oligodendrocytes and astrocytes. Due to the ethical and tissue origin restrictions, more and more other tissue-derived mesenchymal stem cells were found. Bone marrow mesenchymal cells, adipose tissue derived mesenchymal stem cells can be differentiated into neuron-like cells, Schwann’s cells and glial cells in growth factors and microenvironment. The subintimal tissue contains fibroblasts, macrophages and mast cells, and richly supplied with blood vessels, lymphatics and the nerve fibres being those on the adventitia of the blood vessels. In our group, synovial mesenchymal stem cells (SMSCs) were separated and cultured in2001, and the cells can be differentiated into chondrocytes and osteocytes in some growth factors and microenvironment.Tempotomandibular disorders (TMD) are the common diseases, which result from psychosocial disorders, social psychic factor, trauma and occluding relation disorders, among human being with the incidence of20%-40%. The clinical symptoms of TMD are pain, clicking and souffle. The chief complaint of patients is limited of mouth opening and pain of TMJ. Some researchers reported that one of the common symptoms of TMD is pain with relation to inflammation, trauma and psychosocial disorders. Interleukin-1p (IL-1β), Interleukin-6,(IL-6), Tumor necrosis factor-a,(TNF-α) are three important inflammatory cytokines in pain of TMD. During pain processing in TMD, substance P (SP) and calcitonin gene-related peptide (CGRP) are the main neuropeptides released from nerve endings. In the pain of rat animal model, some report showed that Complete Freund’s adjuvant (CFA) and never grown factor (NGF) can lead to nerve endings neurogenesis and the protein level of PGP-9.5and CGRP higher than control group in the rat TMJ disk synovial membrane. It is showed that neurogenesis of nerve endings or neurongenic SMSCs were related to pain of TMD. High molecular weight hyaluronic acid (HA) is used to treat TMD. The common symptom of these diseases is pain. The treatments called viscosupplementation are administered as a course of injections into the TMJ, and are believed to supply the viscosity of the joint fluid, thereby lubricating the joint and producing an analgesic effect. Therefore, in this study we were investigated the capability of HA to inhibit the expression of SP&CGRP, to provide a series of research basis to understand the pain of TMD.In this study, we cultured SMSCs derived from the TMJ synovial membrane of candylar hyperplasia patients, and used bFGF induced SMSCs to differentiate into neuronal cells, to investigate SMSCs and neurogenic SMSCs secreted the level of neuropeptides expression under the inflammatory cytokines microenvironment, and to detect the relationship neurogenic SMSCs between pain of TMD, to measure the capability of HA to inhibit the expression of SP&CGRP. In this study, including two parts as follow:Part One:Differentiation of temporomandibular joint synovial mesenchymal stem cells into neuronal cells in vitro:an in vitro studyObjective:To isolated and cultured tempotomandibular joints (TMJ) synovial mesenchymal stem cells (SMSCs) and to induce neuronal cells, and to explore the differentiation potential of SMSCs into neuronal cells.Methods:This study cultured SMSCs derived from the TMJ synovial membrane of condylar hyperplasia patients. A certain concentration of fetal calf serum (FCS) and dulbecco’s modified eagle’s medium (DMEM) in vitro amplification. Through the use of25ng/ml of basic fibroblast growth factor (bFGF) induced synovial mesenchymal stem cells differentiated into neuronal cells. Inverted microscopy, scanning electron microscope, immnuocytochemical,RT-PCR and flow cytoMeter (FCM) were used for oberserved the change of the induced cells.Results:FCS and DMEM were used to culture SMSCs which morphological were most spindle, small part of a polygon. However, undifferentiated SMSCs showed the fibroblast-like cells characterization in inverted microscopy. Furthermore, after the bFGF induced being induced by bFGF, SMSCs can be found a unique long extension from the cell body in scanning electron microscope. RT-PCR, mmnuocytochemical analysis and FCM confirm the nestin (neural stem cells marker) and NF-L (mature nerve cells marker) expression in SMSCs.Conclusion:In this study, SMSCs can be isolated from synovial membrane in TMJ. The SMSCs can be differentiated into neuronal cells using induced by bFGF. The bFGF induced SMSCs are not only changed into neural-like cells but also expressing the specific markers of neuronal cells and neural stem cells. Part Two:SP and CGRP from neurogenic SMSCs of human temporomandibular joint mediated by inflammatory cytokinesObjective:To investigate the capability of SP and CGRP to stimulate SMSCs and neurogenic SMSCs secreted inflammatory cytokines on pain processing of TMD in-vitro, and evaluate the synergistic effect about inflammatory cytokines and neuropeptides, and detecte the analgesic effect of HA.Methods:In this study, the SMSCs and neurogenic SMSCs secreted levels of neuropeptides SP&CGRP were measured by immunocytochemistry and the levels of IL-1β, IL-6and TNF-a during neuropeptides environment were measured by ELISA. SP and CGRP produced by those cells were determined by RT-PCR and western blotting to measure analgesic effect of HA.Results:The capability of expression SP and CGRP of neurogenic SMSCs were confirmed by immunocytochemistry. The expression levels of SP and CGRP were significantly enhanced in the neurogenic SMSCs in response to IL-1β，IL-6and TNF-a, while HA remarkably inhibited the enhancing effect of inflammatory cytokines.Conclusion:These findings suggest that SP&CGRP and IL-1β, IL-6, TNF-a can act on neurogenic SMSCs to reciprocally enhance each other in an in-vitro pain processing of TMD. The analgesic effect mediated by HA may be through involvement inhibition of SP and CGRP expression in neurogenic SMSCs.