基于生物3D打印技术的芍药苷-海藻酸钠-明胶皮肤支架体外生物相容性研究

Bio-3D printing technique-based paeoniflorin-sodium alginate-gelatin skin scaffold:an experimental study of its biocompatibility in vitro

目的 探讨采用生物3D打印技术制备的芍药苷-海藻酸钠(SA)-明胶皮肤支架的体外生物相容性.方法 通过生物3D打印技术制备质量百分比分别为0、1、3、5、10%的芍药苷-SA-明胶皮肤支架.采用扫描电子显微镜(SEM)观察其表面形态结构,高效液相色谱仪检测芍药苷累积释放浓度.将大鼠皮肤成纤维细胞与其共培养3 d,行Live/Dead染色,观察支架表面细胞存活和生长状态,检测1、2、3 d时细胞增殖、羟脯氨酸和白细胞介素(IL)-6表达水平.结果 SEM显示5组皮肤支架均呈多孔网状立体结构,孔隙均匀.3%组皮肤支架表面较其他4组光滑.药物累积释放量随着时间延长逐渐增加,10%皮肤支架组在各时点的释放均高于其他4组.Live/Dead染色显示细胞在5组皮肤支架表面均能较好地黏附,生长良好,SEM显示3%组皮肤支架活细胞数多于其他4组.3%组皮肤支架的细胞增殖率、羟脯氨酸分泌水平显著高于其他4组(均P<0.05),IL-6表达水平显著低于0和1%组(P<0.01),但高于10%组(P<0.01).结论 3%芍药苷-SA-明胶皮肤支架实现了生物效应与细胞毒性的平衡,具有良好的生物相容性和胶原蛋白分泌效应,可有效抑制炎症反应发生,有望为糖尿病足溃疡创面修复提供一种可降解的生物皮肤支架.

Objective To investigate the biocompatibility of paeoniflorin-sodium alginate(SA)-gelatin skin scaffold that is prepared based on bio-3D printing technique through experiment in vitro. Methods By using bio-3D printing technique the skin scaffolds with the mass fraction of paeoniflorin-SA-gelatin of 0%,1%, 3%, 5% and 10%(five groups in total) were separately prepared. Scanning electron microscope(SEM)was used to observe the surface morphology of the skin scaffold, and the cumulative release concentration of paeoniflorin was determined by high performance liquid chromatography(HPLC). The rat skin fibroblasts were co-cultured with the scaffold for 3 days. Live/Dead staining was performed on the 3rd day to observe the survival and growth status of the cells on the scaffold surface. The cell proliferation, hydroxyproline and IL-6levels were tested on the 1st, 2nd and 3rd day separately. Results SEM demonstrated that the skin scaffold assumed a porous network stereoscopic structure appearance with uniform pores in all five groups. The scaffold surface of 3% group was smoother than that of all the other four groups. The cumulative release concentration of paeoniflorin was gradually increased with the time passing, the cumulative release concentration at each time-point in 10% group was higher than that in the other four groups. Live/Dead staining revealed that in all the five groups the cells could well adhere to the skin scaffold surface and grew well. SEM showed that the number of survival cells on the scaffold surface in 3% group was larger than that in the other four groups. The cell proliferation rate and the hydroxyproline secretion level in 3% group were significantly higher than those in the other four groups(P<0.05), while IL-6 expression level in 3% group was remarkably lower than that in 0% group and 1% group(P<0.01) but prominently higher than that in 10% group(P<0.01). Conclusion The paeoniflorinSA-gelatin skin scaffold with 3% mass fraction achieves a balance between biological effect and cytotoxicity,and it carries good biocompatibility and collagen secretion effect, which can effectively inhibit the inflammatory reaction. It is expected that bio-3D printing technique-based paeoniflorin-SA-gelatin skin scaffolds with 3% mass fraction will become a biodegradable skin scaffold for the treatment of diabetic foot ulcer wound.