Experimental Study Of Heparin On Hyperplastic Scar Fiberblasts And Rabbit Ear Hyperplasic Scar Model

Introduction Hyperplastic scar is a common disease in burn and plastic surgery and its mechianism is complexity. The treatment is very difficulty about the hyperplasia scar. It was proved that collagen, Transforming Growth facter-β₁ (TGFβ₁) and basic fibroblast growth factor (bFGF) participate in the inflammation reaction process after injury and have close relation to cicatrisation Heparin affects both dermal fibroblast proliferation and collagen and may mediate these effects by altering the levels of basic fibroblast growth factor (bFGF) and transforming growth factor-beta1 (TGFβ1) production as a wound healing modulator. It is a new method to heal injury and restrain scar hyperplasia by heparin modulation.Purpose: this study is to probe the effect of heparin on bFGF and TGF-β1 production by hyperplastic scar. Using cell culture and the rabbit ear hyperplastic scar animal model, we probed the biological effect of heparin by hyperplastic scar from experimental animal and cell level, so we can find a new method to treat hyperplastic scar.Material/Methods: This research investigates the effect of heparin on bFGF and TGFβ1 production by human normal skin and hyperplastic scar fibroblasts with exposure to 0μg/ml, 100μg/ml, 300μg/ml, or 600μg/ml heparin for 24, 48, 72 or 96 hours in a serum-free in vitro model. Levels of bFGF,TGFβ1 in the supernatants and TGFβ1mRNA,bFGF mRNA in cells were determined by enzyme-linked immunosorbant assay (ELISA) and real time RT-PCR respectively. 48 health rabbit weighting between 2-2.5kg were employed as the model and were randomly divided into two groups: transplantation group (group A n=60) and normal group (group B, n=60). All the rabbit were anaesthetized with an intraperitoneal injection of sodium rabbit were randomly divided into 4 groups in 21 days after operation. the experiment groups ( BCD )intervenous drop infusion heparin (100u/kg,200u/kg,400u/kg).A groupis control group.we draw the rabbit ear hyperplastic scar after heparin modulatin in 1,3,7,14,2l,30,40days. Then, immunohistochernistry combined with computer image analysis was used to determine collagenIII-expression index , collagen I -expression index and I/III . Levels of bFGF and TGFβ1 in the rabbit ear hyperplastic scar were determined by enzyme-linked immunosorbant assay (ELISA).. Data was collected andexpressed as mean±standard error of mean. The significance of any differences wereevaluated by student's t-test by using a software called Microsoft Excel. The level of significance was set at P < 0.05.Results: All doses of heparin significantly stimulated production of bFGF by normal skin (393% to 1019% increase) and hyperplastic scar fibroblasts (405% to 899% increase) at all time points (p<0.05). Heparin (300μg/ml and 600μg/ml) also stimulated TGFβ1 production by normal skin (26% to 83%) and hyperplastic scar fibroblasts (63% to 85%) with statistical significance (p<0.05) at various time points. Heparin (100μg/ml, 300μg/ml and 600μg/ml) stimulated bFGF mRNA expression by normal skin (251% to 683%) and hyperplastic scar fibroblasts (412% to 799%) with statistical significance (p<0.05) at various time points. All doses of heparin significantly stimulated production of TGFβ1mRNA by normal skin(51% to 68% increase) and hyperplastic scar fibroblasts (12% to 79% increase) at all time points (p<0.05).Heparin cause significant changes in hyperplasy index level by the hyperplastic scar. B(100u/kg)C(200u/kg) D(400u/kg) group control to A group, the hyperplasy index is similar in 1-7 days. A samples had a peak in hyperplasy index at 14 days and the levels dropped and were maintained at a plateau. However, B(100u/kg)C(200u/kg) D(400u/kg) group had a peak in hyperplasy index at 14 days and the levels dropped and were maintained at a plateau. The decreases in hyperplasy index levels for the hyperplastic scar treated with B(100u/kg)C(200u/kg) D(400u/kg) at 14,21,30,40days were greater than control group A (P<0.05 ) .The collagenI expression level in group CDis low than AB (P<0.05) at 14,21,30,40day. All groups had a peak in collagenI expression index at 14 days and the levels dropped and were maintained at a plateau and the collagenl expression index is similar in 1 -7 days. The collagenIII expression level in group CDis low than AB (P<0.05 ) at 14,21,30,40day. All groups had a peak in collagenlll expression index at 14 days and the levels dropped and were maintained at a plateau and the collagenl expression index is similar in 1-7 days. Control to the A group, the bFGF of hyperplastic scar in B (153%-294%) C (210%-372%) D (208%-374%) all increased (p<0.05). the bFGF of all samples had a peak at 24h and the levels dropped and were maintained at a plateau in 21 days. Control to the A group the TGFβ1 of hyperplastic scar in C and D group increase 95%, 93%, 52% and 96%, 94% repectively p<0.05). the TGFβ1 of all samples had a peak at 24h and the levels dropped and were maintained at a plateau in 21 days.Conclution: Using cell culture and the rabbit ear hyperplastic scar model after difference density heparin modulation, we probe the effection of heparin on production of basic fibroblast growth factor and transforming growth factor-beta1 by human normal skin and hyperplastic scar fibroblasts and bFGF,TGF-β1,collagen production in hyperplastic scar. These effects of heparin on normal skin, hyperplastic scar fibroblasts and rabbit ear hyperplastic scar may have implications for hyperplastic scar formation and wound healing in vitro.