Effects Of Staurosporine On Contraction Of Self-assembled Construct Of Goat Tempromandibular Joint Disc Cells

Objective:The purpose was to examine the effects of Staurosporine on the contraction and extracellular matrix synthesis of the self-assembled constructs of goat tempromandibular joint disc in two weeks?.?Methods:1.Goat temporomandibular articular disc was isolated in vitro,and digested by type I collagenase.Cells were collected and cultured.Toluidine blue staining and type I collagen immunohistochemical staining were performed to identify the distrubution of extracelluar matrix.2.Cylindrical agarose molds with a 5 mm diameter and a 10 mm depth were produced in a24-well plate.The culture medium in each mold was refreshed each day for one week.3.5.5×10~6 cells were placed in each mold,and control and experimental groups were set up,corresponding to nothing and different concentrations of staurosporine treatment(0.1 nM,1 nM,10 nM,100 nM).The culture medium was refreshed every day and cells were cultured for one or two weeks.4.The morphology changes of constructs were observed.Picrosirius red staining was used to analyze collagen distribution,and Safranin-O staining was used to examine GAGs distribution.Immunohistochemical analysis was performed to analyse the expression of Type?colleage and?-SMA.The ELISA and Blyscan kit were used to detect the contents of extracellular matrix.Results:1.Light microscope showed that the cells morphology of P0,P1,P2 and P3 generations were different.With the increase of passage times,the cell became more flat,especially for P3generation.Toluidine blue and type I collagen immunohistochemical staining of P3 generation cells were positive.2.Goat temporomandibular joint disc cells in each group were self-assembled into a round shape.Constructs exhibited contractility in 1 week and became stable in 2 weeks.The 10 nM group showed a significant increase of the diameter and wet weight in the first week and second week(P<0.01).The wet weight of constructs was increased in the second week,but the diameter change was not obvious.3.The staining of frozen sections in the first week and second week showed that the Picrosirius red staining,Safranin-O staining and Type?collagen?immunohistochemical staining were positive in each group.Compared with the control group,the?-SMA immunohistochemical staining was weakly positive in the Staurosporine group.4.Using ELISA and Blyscan kit,Type?collagen and GAGs contents of Staurosporine group were higher than the control group,especaially for the 10 nM group(P<0.01).In addition,the content of extracellular matrix of each group in the second week were higher than that in the first week(P<0.01).Conclusion:Staurosporine has a certain effect on the shinkage of self-assembled construct,but it is not very prominent.It is more contribute to the synthesis of extracellular matrix of construction.