Multiple Growth Factors-releasing Fibrin Gel Delivery System Stimulates Proliferation Of Human Embryonic Lung Fibroblasts In Vitro

Objective: To explore the proper way to promote the proliferation anddifferentiation of human embryonic lung fibroblasts, we compare several differentnovel local fibrin gel delivery systems, which could release multiple growth factorssteadily, in vitro.Methods：Multiple growth factors including platelet derived growthfactor，vascular endothelial growth factor，transforming growth factorand epidermal growth factor were added to fibrinogen solution. These fibrinogensolution with multiple growth factors in it were converted to fibrin gel, by addingthrombin to establish delivery system. The kinetics and dissolve curves of differentfibrin gel delivery systems were determined by measuring the volume ofdissolved collagen every day. Human embryonic lung fibroblasts were randomlydivided into three groups, namely growth factors group(groupA),non-growth factorsgroup(groupB) and medium without fibrin gel as control (groupC).According to theshape of fibrin gel, group A and group B ware randomly divided into foursub-groups, which are all covered fibrin gel group(A1, B1), all covered fibrinogengroup(A2, B2), island fibrin gel group(A3, B3), fragmented fibrin gel group(A4,B4).Control group (groupC) was divided into two groups: growth factors group(C1)and non-growth factors group(C2). The effects of different delivery systems on cellproliferation were compared among groups. Cell proliferation was evaluated by cellcounting. Cell viability was determined by MTT assay.Results：(1)The release kinetics of different delivery system: There was nostatistically difference among island fibrin gel group and fragmented fibrin gelgroup(P>0.05).Island fibrin gel group and fragmented fibrin gel group compared with the all covered fibrin gel group, the difference was statisticallysignificant(P<0.001).The gel releasing peak of all covered fibrin gel group wasbehind island and fragmented fibrin gel goups.(2) A1、A2、B1and B2group were noadherent cells, whereas A3、A4、B3、B4group cells grew well with activeproliferation.(3) Cells in the inner zone grew slower than the cells in the middle zone,and the cells in the middle zone grew slower than the cells in the external zone. Thenearer the fibrin gel, the slower cells grew.(4) Cells of growth factors groups grewsiginifantly faster than cells of non-growth factors groups (P<0.001).(5) In thegrowth factors group, the effect of A4group on cell growth and proliferation wassignificantly superior to A3group(P<0.001), while the effect of A3group on cellgrowth and proliferation was significantly superior to C1group(P<0.001). In thenon-growth factors group, the effect of B4group on cell growth and proliferationwas significantly superior to B3group(P<0.001), while the effect of C2group on cellgrowth and proliferation was significantly superior to B3group (P<0.001).(6)TheMTT colorimetric assay of cell proliferation and direct cell counting was positivelycorrelated (growth factor group: A3group r=0.929，P<0.001；A4group r=0.863，P<0.001；C1group r=0.716，P<0.005.Non-growth factor group：B3group r=0.706，P<0.005；B4group r=0.423，P<0.05；C2group r=0.512，P<0.05).Conclusion：(1)Multiple growth factors including PDGF, TGF, VEGF and EGFstimulate the human embryonic lung fibroblasts growth and proliferation.(2)Both island shape and fragmented shape fibrin gel multiple growth factorsreleasing system could benefit to maintain the growth factors bioactivity, and controlthe release rate of growth factors, stimulate the human embryonic lung fibroblastscontinuously in vitro.(3)The effect of fragmented fibrin gel multiple growth factorsreleasing system on cell growth and proliferation is superior to the island fibrin gelmultiple growth factors releasing system.