Effect Of Basic Fibroblast Growth Factor In Different Concentration On Proliferation Of Tendon Cells Cultured In Vitro

Objective:With the developing of molecular biology and the depth study of tendon healingmechanism,at present,we have founded that there were several growth factors and their receptorsplayed a very crucial role in the regulation of tendon healing process，because the basic fibroblastgrowth factor have been widely used in wound repair and the clinical efficacy had beenconfirmed,it have becomed research focus in promoting tendon healing by exogenous factors. Thepurpose of our study in this experiment was to research the effects of basic fibroblast growthfactor(bFGF) in different concentration on proliferation of tendon cells cultured in vitro and toestimate the best bFGF concentration in promoting proliferation of tendon cells cultured in vitro.Then compared the proliferation difference between cryopreserved and not cryopreserved cell,bothwere cultured in vitro by the best bFGF concentration.It can provide important underlyingparameter for tendon injury repairing and the seed cell culturing in tissue engineering tendon.Methods:1、The flexor tendons of New Zealand rabbit were cut under sterile conditions,theperitenon of flexor tendon were removed by microsurgical technique, isolated tendon cells with theHenderson-step enzymatic digestion.The cell were cultured in a complete medium consisting ofDMEM/F12and20％fetal bovine serum for primary culture、passage and cryopreserved thesecond generation tendon cells;2、Choosed the second generation tendon cells, observed themorphology under inverted microscope and the collagen types which were detected byimmunocytochemical staining under the fluorescence microscope;3、The second generatedtendon cell were cultured in vitro with different concentrations (0.5ng/ml、1ng/ml、2ng/ml、5ng/ml、10ng/ml、20ng/ml、30ng/ml、40ng/ml and50ng/ml) of bFGF for48hours,thendetected their optical density (OD) of each groups by MTT test,then estimated the best bFGFconcentration in promoting proliferation of tendon cells cultured in vitro;4、Resuscitated thesecond generation tendon cell which were cryopreserved one month ago,then cultured by bestbFGF concentration in vitro, detected their optical density by MTT, compared the proliferationdifference between cryopreserved and not cryopreserved cell;5、Statistic package SPSS18.0was employed for the measurements analysis,P <0.05as statistically significant standard to judge thedifference.Results:1、The tendon cell can be successfully isolated by trypsin and collagenaserespectively, and the cell can be proliferated and passaged in vitro;2、The cell stained with mouseanti-rabbit collagen I、Ⅲ antibody, colored with DAB kit, The tendon cell showed positivereaction;3、Compared to control group:5ng/ml、10ng/ml、20ng/ml、30ng/ml、40ng/mland50ng/ml bFGF groups had significant difference(P<0.05);When bFGF concentration varyingfrom0.5ng/ml to20ng/ml,the OD average values increase gradually,while varying from20ng/ml to50ng/ml,it decreased step by step;4、After the cell(cryopreserved and notcryopreserved cell)cultured by best bFGF concentration in vitro,the cell proliferation had nosignificant difference（P>0.05）.Conclusions:1、Tendon cell can be isolated、proliferated and passaged in vitro,thesefindings provided a reliable experimental basis for seed cell requiring in the tendon tissueengineering;2、bFGF can significant promote the proliferation of tendon cells in vitro culturedand depend on its proper concentration;5ng/ml bFGF is the initial concentration and20ng/ml isthe best concentration in promoting proliferation of tendon cells cultured in vitro;3、There haveno diference between cryopreserved and not cryopreserved cell proliferation cultured in vitro.