The Study Of Tendon Derived Cells For Tendon Regeneration

Human tendon derived cells were cultured in vitro in different concentrations of fetal bovine serum (FBS) and combinations of growth factors (PDGFBB, IGF-1, bFGF and TGFβ-3) supplemented in a-MEM medium. Fractional factorial design was utilized to select the combinations of growth factors that support (1) cell proliferation in less differentiated states with the least amount of FBS;(2) cell differentiation without FBS. Cells were cultured with a-MEM supplemented with FBS (0%,1%,5%and10%) and combinations of different concentrations of PDGFBB (0ng/ml,5ng/ml,10ng/ml and50ng/ml), IGF-1(0ng/ml,10ng/ml and50ng/ml), bFGF (0ng/ml,5ng/ml,10ng/ml and50ng/ml) and TGFP-3(0ng/ml,1ng/ml and1Ong/ml). The result showed that (1) the cell number of the cells cultured for14days in culture medium supplemented with1%FBS,50ng/ml PDGFBB and50ng/ml bFGF matched that of the10%FBS supplement. The collagen synthesized and the mRN A expression of the differentiation markers were significantly down regulated compared to the10%FBS counterparts. IGF-1did not promote significant cell proliferation in low serum conditions but did promote cell differentiation. Cell morphology also confirmed less differentiated cells cultured with1%FBS,50ng/ml PDGFBB and50ng/ml bFGF;(2) the cells maintained survival for14days in serum free a-MEM supplemented with50ng/ml IGF-1and10ng/ml TGF β-3and cell differentiation was achieved as shown by collagen formation and real time RT-PCR of the cell differentiation markers.(3) By combining the low/no serum culture mediums sequentially in2-D culture for14days each (totally28days), dense collagen construct formation was observed and pictured, whereas in10%FBS group (control group), no collagen construct formation was observed even after prolonged culture of45days.(4) By combining the low/no serum culture mediums sequentially in3-D culture with degummed silk as scaffold for28days, tendon like structures which resemble the microstructure of the human hamstring tendon were generated.These works show for the first time that (1) it is possible to achieve long term in vitro tendon derived cell expansion with low serum concentration down to1%that is favourable to future tendon tissue engineering;(2) tendon derived cells could maintain cell survival over14days period under FBS free conditions and differentiation can be induced by specific combination of growth factors supplemented in culture medium.(3) Tendon derived cells cultured with sequential application of above methods showed significant cell differentiation in2-D and3-D culture.(4) Tendon derived cell cultured in our designed culture medium showed significant differentiation potential in vivo.