Effect of injectable composites of calcium sulfate and hyaluronate in enhancing osteogenesis

Objective: To fabricate an injectable composite bone substitute with hyaluronic acid (HA) and calcium sulfate and to evaluate the biocompatibility and effect of the composite on cell proliferation, osteogenic differentiation in vitro and osteogenic capability in vivo. 方法: 将硫酸钙分别与透明质酸钠溶液、交联透明质酸钠溶液、PBS 溶液，按照 2∶1（ W/ V）比例混合，制备 3 种复合材料（记作 CA+HA、CA+HAC 以及 CA）。通过将 3 种复合材料浸泡于 PBS 溶液中观察其形变并进行 X 线衍射分析，观察材料稳定性。参照 ISO10993-5 制备 3 种复合材料浸提液，用于培养小鼠前成骨细胞（MC3T3-E1），并采用细胞增殖-毒性检测试剂盒（cell counting kit-8，CCK-8）方法检测材料的生物相容性和不同浓度浸提液促进细胞增殖的能力，以单纯培养基培养细胞作为对照组。采用成骨分化培养基制作促进细胞增殖的最佳浓度浸提液，用于培养 MC3T3-E1 细胞，ELISA 法检测成骨分化相关蛋白 ALP、Ⅰ型胶原（collagen typeⅠ，COL-Ⅰ）及骨钙蛋白（osteocalcin，OCN）浓度。取新西兰大白兔制作股骨髁骨缺损模型，分别植入 CA+HA、CA+HAC 以及 CA 材料，于 6、12 周取标本行 Micro-CT 扫描并计算骨组织占组织体积百分比（bone volume/tissue volume，BV/TV），然后标本切片行HE染色，观察缺损区新骨形成情况。. Results: CA+HA and CA+HAC were better in injectability and stability in PBS than CA. The biocompatibility test showed that absorbance ( A) value of CA group was significantly lower than that of control group ( P 0.05). The proliferation test showed 25% and 50% extract of all 3 materials had significantly higher A value than control group ( P 0.05). HE staining revealed that a little bone tissue was seen in CA+HA group and CA+HAC group, but there was no bone formation in CA group at 6 weeks; more streak bone tissue in CA+HA group and CA+HAC group than CA group at 12 weeks. Conclusion: Composites prepared with calcium sulfate and HA or with cross-linked HA are stable, injectable, and biocompatible. The materials have excellent effect on proliferation and differentiation of mouse MC3T3-E1 cells. They also show good osteogenic capability in vivo. So it is a potential bone substitutes for bone defective diseases.