Dynamic And Static Compression Alters The Extracellular Matrix Synthesis Of Goat Temporomandibular Joint Disc Cells Cultured In Agrose Gel

Objective:The purpose of this study was to search a novel method suitable for proliferation of temporomandibular joint (temporomandibular joint, TMJ) disc cells, to observe the biological characteristics of cultured TMJ disc cells and to investigate how dynamic and static compression influences extracellular matrix synthesis of goat temporomandibular joint disc cells cultured in agrose gel.Methods:1. The small pieces (1.0 mm3 in size) of isolated and chopped TMJ disc tissues, which were dissected from two one-month-goats under sterile conditions, were digested with 0.25% trypsin and 0.1% typeâ… collagenase, then put into 6-well plate to be cultured with DMEM. The phase contrast microscope was used to observe the morphological changes and attachment efficiency of TMJ cells. Immunohistochemical staining for type I collagen and toluidine blue staining for proteoglycans were used for identifying extracellular matrix characterization. The cell growth curve was plotted.2. Temporomandibular joint disc cells were mixed with agarose, serum and medium to form agrose-gel constructs. After cultured for 3d in static state, the constructs were divided into three groups:dynamic compression group, static compression group and static comparison group. The compression unit was set up followed the method of Sharam et al. The constructs were exposed to 0.2MPa, 0.1Hz cyclic loading and 0.2MPa static loading. Each group was cultured for 2 weeks.2 weeks later, the TMJ disc cells were identified with Safranin-O,Toluidine blue and immunohistochemistry of collagen type I staining,and the histochemistry analysis was used to examine distribution of extracellular matrix in constructs.Results:1. On the 4th day of culture, primary TMJ disc cells (fibrochondrocytes) could be seen in adherence to the dishes. After the 7th days, the number of adhesive fibrochondrocytes was increased. On the day 10, the cells were spindle-shaped and some of them were polygon-shaped, started to contact with each other to form a monolayer on which cells covering the bottom of the 6-well plate. At the 12th hour after passage, the seeding efficiency of the fibrochondrocytes was 90%. From the 5th to 6th day after passage, the bottom of the culture bottle was filled with these cells. Immunohistochemical staining for type I collagen and toluidine blue staining for GAGs exhibited positive results.2. Agarose gel was found to provide a good microenvironment for the formation of extracellular matrix. The amount of the extracellular matrix in dynamic compression group and static compression group increased compared with the static comparison group, however, there was no significant difference between the dynamic compression group and the static compression group.Conclusions:1. The goat TMJ disc cells present proliferous ability in vitro culture using enzyme digestion with explant culture method. It shows that the method is a practical way to obtain numberous primary cells for the TMJ disc tissue engineering.2. The compression has an important effect on metabolism of constructs composed of TMJ disc cells and agarose gel. Both the dynamic and static compression can promote the production of main extracellular matrix in native TMJ disc tissue such as GAGs and type I collagen.