一种新型真皮替代物--人发角蛋白-胶原海绵的制备及其生物活性研究

目的制备一种人发角蛋白-胶原海绵的三维立体多孔状结构材料,探讨将其作为真皮替代物的可行性。方法将人发在体内具有慢(Z)、中(B)、快(F)三种吸收速度的HHK组分材料编织成网孔为1 mm×1 mm的网格,以酸溶法从牛跟腱中抽提胶原原液,用离心、盐析、透析等方法制备Ⅰ型胶原蛋白溶液,向溶液中滴加胶原总量8％的6-硫酸软骨素(6-GAG)进行初交联。制备好的Ⅰ型胶原溶液与HHK网格混合后放入模具,真空冷冻干燥成海绵状膜,于0．25%戊二醛溶液中浸泡再交联。将制备好的HHK-胶原海绵膜切块(1 cm×1 cm)移植于21只大鼠的真皮与皮下组织之间(实验组),并与单纯胶原海绵膜组(阴性对照)、单纯全层皮肤切开组(空白对照)作对照。于术后3 d和1、2、4、6、8、12周7 个不同时间点取材料及其周围组织,行石蜡切片的HE染色、醛复红弹性纤维染色观察及扫描电镜观察检测其组织相容性、血管化能力及材料的体内降解情况。结果人发角蛋白呈棕黄色细丝状。胶原海绵膜为微黄色。半透明状,孔径为 50至300μm的三维立体状结构。皮下移植后3 d,各组均表现为中度炎症反应,以中性粒细胞和单核细胞为主。术后1 周,炎症反应稍减轻,材料周围可见少量成纤维细胞及新生血管,实验组较对照组明显。术后2周,实验组及阴性对照组,炎性细胞中巨噬细胞增多,实验组成纤维细胞增生活跃,分泌少量胶原物,材料周围新生血管数量增多。HHK及胶原海绵开始降解,创口表皮细胞增殖移行。电镜下可见实验组材料周围包绕的胶原纤维和弹性纤维,人发角蛋白毛小皮降解、剥离;术后4周,成纤维细胞分泌大量粗大的胶原物。创口的表皮细胞增殖移行明显,实验组基本愈合。两种材料崩解明显,胶原海绵网孔内可见巨噬细胞侵入及少量小血管长入。醛复红弹性纤维染色可见短棒或细长条状弹性纤维; 术后6至8周,成纤维细胞数量相对减少,分泌胶原物融合成均质粗大,实验组其排列趋于整齐,表皮层愈合良好。人发角蛋白材料中部出现降解裂隙,胶原海绵基本降解完全。实验组醛复红弹性纤维染色可见细长条状弹性纤维;术后12 周,人发角蛋白材料降解完全。结论人发角蛋白-胶原海绵膜是物理及生物学性能良好的材料,其组织相容性好,血管化能力强,能刺激细胞增殖,促进受损皮肤愈合,可作为真皮替代物。

Preparation and bioactivity of human hair keratin-collagen sponge, a new type of dermal analogue

Objective To develop a three-dimensional porous film of human hair keratin (HHK)-collagen sponge complex for use as a dermal substitute. Methods The three components F, B, and Z derived from healthy human hair were weaved into a meshwork and integrated with purified soluble type I collagen extracted from bovine tendons to prepare a highly porous film with vacuum freeze-drying followed by secondary cross-linking with glutaraldehyde. The film was grafted beneath the dorsal skin in 21 SD rats (experimental group), with simple collagen sponge serving as the negative control. The rats receiving surgical operation but without graft served as the blank control. The graft and its surrounding tissue were harvested on days 3, 7 and at weeks 2, 4, 6, 8, 12 after implantation for evaluation of tissue compatibility, vascularization and degradation. Results The prepared collagen sponge film was semitransparent and porous. Three to 7 days after grafting, inflammatory reaction was relieved gradually, and several fibroblasts and blood vessels were found adherent to the grafts in the experimental groups. At week 4, the wounds healed in the experimental groups, and the fibroblasts were actively secreting collagen and the film degraded obviously with the appearance of elastic fibers. At weeks 6 and 8, new collagen fibers thickened and assumed regular arrangement, and the collagen sponge films disappeared completely. In the control groups, the changes were less obvious and total HHK degradation occurred till week 12. Conclusion The degradable and absorbable HHK-collagen sponge film has relatively satisfactory tissue compatibility and can accelerate wound healing by stimulating cell proliferation and vascularization, showing the potential as an optimal dermal substitute.