CS/HACC/GP温敏水凝胶复合MSCs的实验研究

Experimental research on CS/HACC/GP temperature-sensitive hydrogel loaded with MSCs

目的制备壳聚糖(CS)/壳聚糖季铵盐(HACC)/甘油磷酸钠(GP)温敏性水凝胶,检测其成胶时间、超微结构、渗透压、生物安全性等性质,将其和骨髓间充质干细胞(MSCs)复合培养,观察MSCs的生长情况。方法制备壳聚糖季铵盐,将壳聚糖/壳聚糖季铵盐/甘油磷酸钠按一定配比制备温敏性水凝胶,观察其成胶情况、内部超微结构,测量孔隙率、孔径大小,评估其降解情况,进行生物安全性检测。将MSCs与支架复合培养,倒置显微镜观察凝胶表面细胞生长情况,扫描电镜观察凝胶内部细胞的生长、增殖情况。结果成功制备HACC,水凝胶37℃时10分钟可成胶,生物安全性好,渗透压为290~310 mmol/kg,水凝胶无细胞毒性,其浸提液对小鼠体重增加无影响。扫描电镜见内部结构多空疏松的三维网状结构,孔径50~100um,孔隙率≥85%,降解时间约12周。MSCs在水凝胶中能正常生长、增殖。结论本实验证明CS/HACC/GP温敏水凝胶孔隙率、孔径大小合适,无细胞毒性,适合MSCs生长、增殖,具有较大的潜力成为神经组织工程中的细胞支架。

Objective To fabricate a temperature-sensitive hydrogel and identify its characteristics such as porosity, degradation rate, microscopic structure and biocompatibility of the cell scaffold. To observe growth and proliferation of BMSCs in the scaffold. Methods In this experimental, we modified Cs to obtain a new derivative of Cs, HACC. The hydrogel was fabricated by proper riato of CS/HACC/GP mixed solution prompted by temperature. The gelation time was decided. The cross-section morphology andporosity of the scaffolds was examined. The weight loss was calculated and the change of its aspect and shape was measured at set time. The biological safety of the hydrogel was evaluated. Then MSCs were seeded into the hydrogel. Cell adhesion and morphology of MSCs were analysed by SEM. Results HACC and CS/HACC/GP Temperature-sensitive Hydrogel wassuccessfully synthesized. The osmolality of the hydrogel was range of 290-310 mmol/kg. Gelation time of CS/HACC/GP hydrogel was around 10 minutes. Macroporous internal morphology with pore size ranging from50 to 100 um was observed in CS/HACC/GP scaffold. It had a high macro porosity of greater than 85 percent. The time of CS/HACC/GP scaffold degradation completely was 12 weeks in vitro. From the SEM observation, the proliferation and metabolism of MSCs cultured on the three-dimensional hydrogel cell culture grew well. Conclusion The hydrogel with high porosity and a moderate degradation rate, without cytotoxicity, was suited for MSCs togrow and multiply. The hydrogel have the huge potential to be used as an optional carrier of transplanted cells for nerve tissue engineering.