明胶/纳米羟基磷灰石三维多孔骨支架的制备及其生物安全性评价

Study on preparation of gelatin/nanohydroxypatite scaffold with three-dimension porous structure and evaluation of its biocompatibility

[目的]探讨冷冻干燥法制备明胶/纳米羟基磷灰石(nano-hydroxyapatite,nHA)三维多孔支架的可行性并评价材料的生物安全性。[方法]取明胶水溶液,将其分别与10 wt%、20 wt%、30 wt%的nHA混合,交联后采用冷冻干燥法制备明胶/nHA三维多孔支架。评价材料的理化性质,通过测定支架孔径、孔隙率、吸水率、抗压强度及降解pH值优化制备条件;MTT法分析支架浸提液毒性;共培养观察细胞在材料表面及周围生长情况;材料埋入背部肌肉内,组织学染色观察评价其生物安全性。[结果]体视显微镜及电镜显示,制备的支架均呈三维多孔隙结构。随着nHA含量的增加,材料成孔效果变差,孔径、孔隙率、吸水率变小,而抗压强度增加。其中以nHA含量为20 wt%的材料表现更为适宜,并选其进行生物相容性实验。MTT法显示不同浓度支架浸提液与对照DMEM培养液吸光度值比较,差异无统计学意义(P>0.05)。细胞与材料共培养生长状态良好。HE染色观察材料与周围肌肉组织无排斥反应,组织相容性良好。[结论]制备的明胶/nHA支架材料具有三维孔隙结构,具备合适的孔径和孔隙率,无毒,生物相容性良好,可作为骨组织工程支架使用。

[ Objective]To develop a scaffold with three dimension porous structure, composed of nano-hydruxyapatite and gelatin and evaluate its biocompatibility. [ Method] Three different kinds of porous scaffolds were prepared by gelatin in combi- nation with 10wt% ,20wt% ,30wt% nanohydroxyapatite using freeze-drying method. The differences among three kinds of porous scaffolds in porosity, pore size, PH value and mechanical strength were detected. And the optimal scaffold was chosen. MTr test was also done to assess cytotoxicity of the scaffolds. The effects of the scaffolds on BMSCs growth were also detected using co- culture method. The materials was buried in the back muscles of New Zealand rabbits. Histological staining was used to evaluate the biological safety of the scaffolds. [ Result] Stereomicroscope and SEM observation showed that the scaffold had three dimen- sion porous structure. As the content of nHA increased, the diameter of the pore, porosity and water absportion of scaffolds de- creased, but the strength of anti-compression enhanced. The scaffold composed with 20 wt% nHA was chosen as the optimal. The intrinsic cytotoxicity assessment of scaffolds using MTT test and co-culture method showed that the scaffolds had no cytotoxic effect on BMSCs. liE staining showed that the bistocompatibility between scaffolds and surrounding muscle tissue was good and no immune rejection was found. [ Conclusion] The prepared gelatila/nHA scaffold has three dimension porous structure, good pore diameter and porosity, non-toxicity and good biocompatibility, which make it a suitable candidate as an alternative carrier for bone tissue engineering.