Self-assembly Fibrocartilage Model Of TMJ Disc And The Effects Of Hydrostatic Pressure On Matrix Synthesis Of The Self-assembled Constructs

Objective1. To construct self-assembly fibrocartilage model of goat temporomandibular joint disc and observe the biological characteristics of the self-assembled fibrocartilage constructs, further to provide a condition for tissue engineering of the temporomandibular joint disc and other fibrocartilage.2. To evaluate the effects of dynamic hydrostatic pressure and static hydrostatic pressure on extracelluar matrix synthesis of self-assembled constructs of goat tempromandibular joint disc.Methods1. Cells were harvested and cultured. 5.5×106 cells were seeded in each agarose well with diameter 5mm×depth 10mm, daily exchange of medium, cultured for 2 weeks and observed the constructs. Samples were frozen and sectioned. HE staining was detected histology of the constructs, Safranin-O/fast green staining was examined for GAG distribution, picrosirius red and immunohistochemical staining were observed for type I collagen distribution.2. Using the self-assembled constructs that made in method 1 and dividing them into three groups,that is,control group,dynamic compression group(0.2MPa, 1Hz) and static compression group (0.2MPa, OHz).After culturing for one week,the two experimental groups were exposed to hydrostatic pressure loading for one hour every day and last for one week,control group were continued to culture. Samples of three groups were evaluated at 3 weeks for gross morphological and frozen and sectioned to detect histology of constructs by HE staining, GAG distribution was examined by Safranin-O/fast green staining, type I collagen distribution were observed by picrosirius red and immunohistochemical staining. All samples were frozen-dry and disgeted, the cell numbers were conversed by DAN contents with a Picogreen reagent kit, GAG contents were measured by Blyscan GAG assay kit,and type I collagen contents were tested by indirect method of ELISA.Results1. After one day seeded, goat temporomandibular joint disc cells in agarose wells gathered and began to self-assemble into a disc-shaped base, then gradually turned into a round shape. When cultured for 2 weeks, hematoxylin-eosin staining can be observed that cells were round and wrapped around by the substrate. Positive Safranin-O/fast green staining for glycosaminoglycans were hardly observed throughout the constructs. picro-sirius red staining were examined and found distribution of numerous type I collagen. Immunohistochemistry staining were exhibited brown yellow particles in cytoplasm and around extracellular matrix, which shows self-assembly fibrocartilage construct can produce type I collagen as native temporomandibular joint disc tissues.2. Compared with control group,dynamic and static hydrostatic pressure groups showed the cell nucleus were more larger and staining stronger,cells shape were irregular and extracelluar matix were more dense by HE staining, staining were negative by Safranin-O/fast green staining in all groups, larger collagen were observed in all groups by picro-sirius red and IHC staining, showed strong positive, particular in the outer part. The dry wets,cells numbers,typeⅠcollagen contents and GAG contents in dynamic and static hydrostatic pressure group were more than control group,and the expressions of type I collagen in dynamic group were higher than static group.Conclusion1. Formation of extracellular matrix in self-assembly model as native temporomandibular joint disc tissue indicates that the use of agarose wells to construct engineered temporomandibular joint disc will be possible and practicable.2. Both dynamic hydrostatic pressure and static hydrostatic pressure up-regulate the extracelluar matrix synthesis of TMJ disc constructs, and the effects of up-regulation was more obvious.