Experimental Study Of Rat Bone Marrow Mesenchymal Cells And Tenocytes Indirect Cocultured In Vitro

Objective:How to treat tendinopathy is a Clinical difficulty. The application of bone marrow mesenchymal cells(BMSCs) in treating tendinopathy is a novel method which has already lead to some positive results. BMSCs have the multipotentials of differentiation into various cell types,but both of the mechanism of differentiation and the role BMSCs plays in the therapeutic process are poorly understood and because of which the scientific and Clinical application of BMSCs has been restricted. We were trying to build an indirect coculture system to investigate differentiation of BMSCs induced by indirect coculturing with tenocytes. comparison was also made between the simple coculture group and the coculture group with platelet-rich plasma added to study the effects of platelet-rich plasma to BMSCs on secretion of extracellular matrix when being inducted by indirect coculture with tenocytes. On this basis we could explore the mechanism of differentiation and making a reference for Clinical application of BMSCs in treatment of tendinopathy.Methods:Tenocytes of SD rats were isolated using explant cuture method.BMSCs were collected using adherence screening method. Autolo-gous platelet-rich plasma was prepared. An indirect coculture system was established using transwell(0.4μm pore size) to inhibiting migration of cells and direct contact.The BMSCs(P4) and tenocytes (P3) were collected, BMSCs were placed in the outer chamber of this system and tenocytes in the inner chamber, one additional PRP added indirect coculture group was set up. The mRNA expression of TNMD and typeⅠcollagen were measured by RT-PCR and the corresponding protein levels of TNMD and typeⅠcollagen by technique of immunohistochemistry at various time points (3d,7d,10d) to study the secretion of extracellular matrix and differentiation of BMSCs induced by the coculture envioroment.Results:Cells were isolated using tendon explant cuture and proved to be tenocytes.There is no significant TNMD nor typeⅠcollagen expresssion observed 3days after coculture. Indistinc bands were observed on the PCR image detecting typeⅠcollagen expression by BMSCs in coculture enciroment and some of BMSCs were stained by immunohistochemical staining 7days later.TNMD expression of BMSCs in coculture enciroment was not observed.On the 10th day after coculture,distinct bands were observed on the PCR image detecting typeⅠcollagen expression by BMSCs in coculture enciroment and BMSCs of experimental groups were widely stained by immunohistochemical staining.Mean optical density(MOD)of the results of immunohistochemical staining were measured for statistical analysis.The statistical data showed that experimental groups had a higher typeⅠcollagen expression than the negative control but lower than the positive control.During the experimental groups, there is no marked qualitative difference between the simple coculture group and PRP group.There is indistinct bands on the PCR image detecting TNMD expression by BMSCs in coculture enciroment,the result of TNMD immuno-histochemical staining is negative.Conclusions:Tenocytes of SD rats could be successfully isolated using explant cuture method.The typeⅠCollagen expression of BMSCs could be induced by the enviroment of being cocultured with tenocytes.No significant TNMD expression of BMSCs was observed after coculture process.There is no significant effect of PRP to the secretion of extracellular matrix of BMSCs in the enviroment of coculture.There is not enough evidence to prove that BMSCs could differentiate into tenocytes after being induced by the inviroment of coculture.