胶原-壳聚糖/胶原-纳米羟基磷灰石仿生支架材料的制备及其生物相容性检测

目的制备胶原-壳聚糖/胶原-纳米羟基磷灰石(collagen-chitosan/nano-hydroxyapatite-collagen-polylactic acid,Col-CS/nHAC-PLA)仿生支架材料,检测生物相容性,为其在骨软骨缺损修复中的应用奠定基础。方法以1,4二氧六环为溶剂,按8%质量浓度加入PLA和等量nHAC溶解,制备nHAC-PLA;用2%醋酸溶液配制浓度为2%的CS纯溶液及1%的Col纯溶液,以质量比(M/M)20∶1混合,倒入含nHAC-PLA的模具中,冷冻干燥成形制备Col-CS/nHAC-PLA仿生支架。行急性全身毒性实验、皮内刺激实验、热源实验、溶血实验、体外细胞毒实验、骨植入实验,评价其生物相容性。结果 Col-CS/nHAC-PLA仿生支架无急性全身毒性;皮内刺激实验示原发刺激指数为0分,为极轻微刺激;热原实验示3只实验动物体温升高均<0.6℃,体温升高总和<1.3℃,符合规定标准;溶血实验示平均溶血率为1.38%,符合≤5%标准。体外细胞毒性实验示材料浸提液不影响兔BMSCs增殖,毒性分级为Ⅰ级。骨植入实验显示支架材料与周边组织相容性好。结论 Col-CS/nHAC-PLA仿生支架材料具有良好生物相容性,有望成为较理想的组织工程骨软骨支架。

PREPARATION AND BIOCOMPATIBILITY OF A NOVEL BIOMIMETIC OSTEOCHONDRAL SCAFFOLD:COLLAGEN-CHITOSAN/NANO-HYDROXYAPATITE-COLLAGEN-POLYLACTIC ACID

Objective To prepare collagen-chitosan/nano-hydroxyapatite-collagen-polylactic acid (Col-CS/nHAC-PLA) biomimetic scaffold and to examine its biocompatibility so as to lay the foundation for its application on the treatment of osteochondral defect. Methods PLA was dissolved in dioxane for getting final concentration of 8%, and the nHAC power was added at a weight ratio of nHAC to PLA, 1∶1. The solution was poured into a mold and frozen. CS and Col were dissolved in 2% acetum for getting the final concentrations of 2% and 1% respectively, then compounded at a weight ratio of CS to Col, 20∶1. The solution was poured into the frozen mold containing nHAC-PLA, and then biomimetic osteochondral scaffold of Col-CS/nHAC-PLA was prepared by freeze-drying. Acute systemic toxicity test, intracutaneous stimulation test, pyrogen test, hemolysis test, cytotoxicity test, and bone implant test were performed to evaluate its biocompatibility. Results Col-CS/nHAC-PLA had no acute systemic toxicity. Primary irritation index was 0, indicating that Col-CS/nHAC-PLA had very slight skin irritation. In pyrogen test, the increasing temperature of each rabbit was less than 0.6℃, and the increasing temperature sum of 3 rabbits was less than 1.3℃, which was consistent with the evaluation criteria. Hemolytic rate of Col-CS/nHAC-PLA was 1.38% (far less than 5%). The toxicity grade of Col-CS/nHAC-PLA was classified as grade I. Bone implant test showed that Col-CS/nHAC-PLA had good biocompatibility with the surrounding tissue. Conclusion Col-CS/ nHAC-PLA scaffold has good biocompatibility, which can be used as an alternative osteochondral scaffold.