林蛙皮胶原/壳聚糖复合海绵的制备及性质

Preparation and properties of collagen/chitosan composite sponge from forest frog skin

背景:林蛙是中国珍稀的药用动物,林蛙皮是林蛙取油后的废弃物,其胶原蛋白含量丰富且量大、无污染和疫病危险,具有开发成为胶原海绵的潜力,但目前尚未见林蛙皮胶原海绵制备工艺方面的研究。目的:对林蛙皮胶原海绵的制备工艺进行优化,考察林蛙皮胶原海绵的物理性质及体外细胞相容性。方法:利用壳聚糖和戊二醛对林蛙皮胶原蛋白海绵进行改性,以壳聚糖与胶原蛋白质量比(1∶1、1∶2、1∶4)、戊二醛浓度(1%、1.5%、2%)为实验因子,以具有显著性差异的吸水倍数、机械性能和热变性温度为指标,采用正交实验设计对林蛙皮胶原蛋白海绵的制备工艺进行优化,观察林蛙皮胶原蛋白海绵表观形貌。将9种林蛙皮胶原蛋白海绵分别与人包皮成纤维细胞共培养,检测细胞增殖。结果与结论:(1)确定林蛙皮胶原海绵的最佳工艺条件为:壳聚糖与林蛙胶原蛋白质量为1∶1,戊二醛浓度为1%,在该条件下,林蛙皮胶原海绵的吸水倍数为5.22,断裂伸长率为10.96%,热变性温度为81.24℃;此条件下的林蛙皮胶原海绵孔径200-400μm,网孔大小基本一致,排列有序;(2)除壳聚糖与胶原蛋白为1∶4、戊二醛浓度为1%或5%的林蛙皮胶原海绵外,其他林蛙皮胶原海绵对人包皮成纤维细胞的细胞活性均有促进作用,有利于细胞的活性和生长;(3)结果表明,林蛙皮胶原海绵具有良好的生物相容性。

BACKGROUND： Forest frog is a rare medicinal animal in China, but the skin of forest frog is waste after Oviductus Ranae production. The forest frog skin is rich of collagen, and is large in quantity without pollution and disease risk. So the forest frog skin has potential to be developed into collagen sponge; however, there is no research on collagen sponge preparation as yet. OBJECTIVE： To optimize the preparation of collagen sponge from forest frog skin, and to investigate the physical properties and in vitro cell compatibility. METHODS： Chitosan and glutaraldehyde were used to modify collagen sponge from forest frog skin. Chitosan/collagen （w/w） （1：1, 1：2, 1：4） and glutaraldehyde concentration （1%, 1.5% and 2%） were selected as the experimental factors. The significant water absorbency, mechanical properties and thermal denaturation temperature were chosen as the indexes. Using the orthogonal experimental design, we optimized collagen sponge preparation process. We also investigated the in vitro cell compatibility and surface morphology of the collagen sponge. The nine kinds of collagen sponges from forest frog skins were co-cultured with human foreskin fibroblasts to detect cell proliferation. RESULTS AND CONCLUSION： When the chitosan/collagen was 1：1 and the glutaraldehyde concentration was 1%, we could get the collagen sponge with ideal water absorbency （water absorption capacity=5.22）, mechanical properties （elongation at break=10.96%） and thermal denaturation temperature （81.24 ℃）. The aperture of the forest frog skin collagen sponge was 200-400 pro, and the pores were consistent in the size and arranged regularly. Except the chitosan/collagen of 1：4 and the glutaraldehyde concentration of 1% or 5%, all kinds of forest frog skin collagen sponges could promote the viability of human foreskin fibroblasts and exerted benefits to cell viability and growth. To conclude, the forest frog skin collagen sponge has good biocompatibility and apparent morphology, in aggreement with the requirements of biological materials.