Triple-layered Cell Sheet For Tissue-engineering The Synovial Membrane Of The Temporomandibular Joint

Temporomandibular disorder (TMD) causes degenerative changes of the articular cartilage and the disc of temporomandibular joint (TMJ). During this process, fibroblast-like synoviocytes (FLSs) which exist in the lining layer (LL) of synovial membrane (SM) dysfunction or suffer apoptosis, causing the loss of the normal secretion functions of SM. FLSs are predominant in the lining layer (LL) of synovial membrane (SM), and arranged in3to4layers. In a normal state, hyaluronic acid (HA) is synthesized mainly by hyaluronic acid synthetase2(HAS2) which exists on the membrane of FLSs. FLSs are responsible for the normal secretion functions of SM and providing nutrients for joint cartilage. However, during TMD, the secretion abilities dysfunction (such as secreting inflammatory factors and matrix metallopeptidases) or suffer apoptosis, causing the loss of the normal secretion functions of SM and subsequently the joint cartilage lose nutrient and suffer degradation. Therefore, it might be a new method to treat TMD if we construct a tissue-engineered SM possessing the normal HA secretion functions in order to replace the pathological SM.The FLSs cell sheet (CS) engineered by thermo-sensitive CS technique cannot pricisely imitate the extracellular matrix of the synoviocytes cell sheet, because the CS technique relies on the extracullar matrix of the high-density cells to connect them. However, FLSs secrete HA mainly, rather than type Ⅰ collagen. Therefore, the study created a new method to create CS with the purpose of imitating the multiple-layered structure of the LL of SM. This study includes four sections:Section one Cell culture and identificationExperiment one In vitro culture of human fibroblast-like synoviocytes (FLSs) of TMJ and dermal fibroblast Objective:To obtain normal FLSs of human TMJ and normal human dermal fibroblasts (DFs) for constructing CS.Methods:Normal SM specimens were harvested from three patients who suffered the fracture of condylar process. The dermis specimens were harvested from the patients who suffered cleft lip and palate. The explants method was applied for cell culture.Results:FLSs grew out of the SM specimen after3-5days, showing a spindle shape.2weeks later, it came to80%confluence. After the first passage, it needed4days to conduct the next passage. DFs showed a good activity. After the first passage, it needed2-3days to conduct the next passage.Conclusion:The explants method for culturing these two kinds of cells is easy and highly repeatable. Cells showed a good activity and could be used for subsequent experiments.Experiment two Immunocytochemistry method for identifying FLSs and DFsObjective:To identify FLSs and DFsMethods:Immunocytochemistry method to identify these two kinds of cells.Results:FLSs expressed vimentin, CD44, Hsp27, and negatively express CD14. DFs express vimentin, and negatively express cytokeratin15.Conclusion:The FLSs harvested from explants method were type B synoviocytes. DFs possessed a dermis origin, rather than epithelium origin.Section two Construction of CS and morphological comparison with natural SMExperiment three Preparation of methylcellulose (MC) solutions and the measurement of their gelation temperature (GT) Objective:To testing the thermo-sensitive features of MCMethods:Prepare MC mixed solution which included different concentrations of phosphate buffered saline (PBS) and MC. The GT of all these specimens was measured by method of inclining the test tube.Results:GT was decreased with the increase of MC or PBS concentration. The GT of experiment group "5%MC,8%PBS" was20.5℃.Conclusion:The GT of experiment group "5%MC,8%PBS" was preferable and could be used in subsequent experiments.Experiment four Construction of triple-layered CSObjective:To construct triple-layered CS using a novel method.Methods:The first method combined type Ⅰ collagen and FLSs using an original method. The second method was to add MC in the center of culturing surface and then use the first method to construct CS.Results:The first method was stable and possessed a high success rate. However, the second method was not as stable as the first method to construct CS. Detachment between type Ⅰ collagen and well wall was occasionally observed.Conclusion:The first method of creating triple-layered CS presented the advantages of simple materials, easy way of harvesting and high repeatability. Therefore it could be used in subsequent experiments.Experiment five Morphological observations for CS and human normal SMObjective:To find the morphological similarities between triple-layered FLSs sheet and normal human SM.Methods:Triple-layered FLSs sheet was constructed using the method without MC. Normal SM specimens were obtained. HE staining and transmission electron microscope were applied to analyze them.Results:Both CS and normal SM possessed3-4layers of spindle-shaped cells with high concentration. Desmosome, semi-desmosome, or gap junction was not observed between adjacent cells.Conclusion:Triple-layered FLSs sheet and normal human SM were morphologically similar.Experiment six Morphological observation for normal SM and TMJ degenerative changed SMObjective:To compare morphological features of normal SM and TMJ degenerative changed SM.Methods:Normal SM specimens were obtained from three patients who suffered the fracture of condylar process. Pathological SM specimens were obtained from10osteoarthritis (OA) patients. All specimens were treated with conventional histological methods and stained by haematoxylin and eosin (HE).Results:The LL of normal SM was composed of3-4layers of spindle-shaped synoviocytes. During the degenerative changes of TMJ, the SM presented several different kinds of observations. The LL could either increase to10layers or maintain3-4layers. The synoviocytes could also suffer apoptosis and decrease obviously in numbers. The blood vessels in the sublining layer of SM were increased in most cases, but remained normal in a few cases.Conclusion:During the OA of TMJ, the LL of SM was abnormal in a morphological aspect, needing the replacement of tissue-engineered SM.Section three Functional assessments of the CSExperiment seven ELISA assay for CS Objective:To measure the HA secretion amount of different CS.Methods:It was divided into four groups:ordinary culturing of FLSs group, single-layered FLSs sheet, triple-layered FLSs sheet, and triple-layered DFs sheet. The HA secretion amounts were measured using ELIS A method.Results:HA secretion amount in triple-layered FLSs sheet was higher than other groups. The secretion amounts in this group were2.53and3.27times that of single-layered FLSs sheet on day1and day3, respectively.Conclusion:FLSs possessed the HA secretion function. FLSs cultured on type Ⅰ collagen were able to produce more HA. Triple-layered FLSs sheet was able to produce3folds of HA.Experiment eight Assay for measuring proliferative abilityObjective:To compare the proliferative ability of FLSs with different culturing fashions.Methods:It was divided into three groups:ordinary culturing of FLSs group, single-layered FLSs sheet, and triple-layered FLSs sheet. The proliferative abilities of these groups were measured by CCK-8method.Results:The OD value of single-layered FLSs sheet was higher than ordinary culturing of FLSs group on day3. The OD values in triple-layered FLSs sheet were higher than other two groups on both day1and day3.Conclusion:FLSs cultured on type Ⅰ collagen presented higher proliferative ability than FLSs cultured on ordinary dish. FLSs in triple-layered sheet possessed proliferative ability.Experiment nine Realtime-PCR for measuring the HAS2gene expressions in different CSObjective:To compare HAS2expressions in different kinds of CS.Methods:It was divided into four groups:ordinary culturing for FLSs group, single-layered FLSs sheet, triple-layered FLSs sheet, and triple-layered DFs sheet. HAS2gene expressions in these groups were assessed by realtime-PCR.Results:HAS2expression in single-layered FLSs sheet was higher than that of ordinary culturing for FLSs group. HAS2expression in triple-layered FLSs sheet was highest in all groups.Conclusion:Type Ι collagen was able to promote FLSs to up-regulate the expression of HAS2gene. The culturing fashion of triple-layered CS facilitated the highest HAS2expression in FLSs.Experiment ten RT-PCR for measuring HAS2gene expressions in natural and artificial SMObjective:To compare the HAS2gene expressions in normal human SM and triple-layered FLSs sheet (artificial SM).Methods:Collect the triple-layered FLSs sheet and the SM specimens of the patients who suffered from the fracture of condylar process. HAS2gene expressions were measured by RT-PCR method.Results:HAS2expression in artificial SM was slightly higher than that of the normal human SM, without statistical differences.Conclusion:HAS2expressions in artificial and natural SM were not different.Section four Transplantation in nude mice subcutaneouslyExperiment eleven Assay of transplantation of CS subcutaneously in nude miceObjective:To investigate the survival and function of triple-layered FLSs sheet subcutaneously in nude mice.Methods:Type Ⅰ collagen, acellular dermal matrix, and triple-layered FLSs sheet were transplanted subcutaneously in nude mice.1week and2weeks later, animals were executed. Specimens were treated with histological and immunohistological methods.Results:1week later, type I collagen and acellular dermal matrix were suffering from collagens degradation. Triple-layered FLSs detached from the supporting collagen.2week later, the implantation materials in the two control groups were degraded completely. In CS implantation group, multiple-layered cells of a parallel arrangement were lying on the surface of the supporting collagen which was invaded by host fibroblasts and blood vessels. CD44and HAS2were expressed in the cells existing in the surface of the supporting collagen. HA was expression in the ECM of the CD44-positive cells.Conclusion:Triple-layered FLSs sheet was more capable of resisting collagen degradation subcutaneously in nude mice, compared with control groups. In the subcutaneous environment, FLSs remained the differentiated feature and possessed the function of synthesizing and secreting HA.Summary for the researchThis research for the first time constructed the SM of TMJ through an original method. Triple-layered FLSs sheet was able to imitate the natural LL of SM morphologically. Functional assessments confirmed that triple-layered FLSs sheet was capable of synthesizing and secreting HA. The assay of nude mice transplantation confirmed that triple-layered FLSs sheet could resist the degradation and maintain the HA secretion function.