Abstract:

Objectives: To construct a double-layer composite by tissue engineering strategy, which could sequentially release the activators of bone morphogenetic protein (BMP) and WNT signaling pathway and investigate the influence on the differentiation of osteoblasts in vitro. Methods: CCK-8 assay, alkaline phosphatase (ALP) staining and alizarin red staining were used to detect the effects of BMP signaling activator tacrolimus (FK506) and WNT signaling activator 6-bromoindirubin-3-oxim(BIO) on the cell viability and osteogenic differentiation of pre-osteoblast cell line MC3T3-E1. Then the optimal drug concentrations were screened. Chitosan/sodium alginate scaffold was prepared and the degradation curve of the material was measured by weighing method. The scaffold with inner chitosan layer loaded with BIO and outer sodium alginate layer loaded with FK506 was prepared and the drug release was determined by ultraviolet spectrophotometry. ALP staining and alizarin red staining were used to detect the ability of the drug-loading composites to promote the osteogenic differentiation of the pre-osteoblast cell line MC3T3-E1. Real-time quantitative polymerase chain reaction(RT-qPCR) was used to detect the effects of the drug-loaded scaffolds on the expressions of osteogenic related genes in the pre-osteoblast cell line MC3T3-E1. Results: FK506 could promote ALP activity at an earlier stage during osteogenic differentiation of pre-osteoblast cell line MC3T3-E1, compared with BIO. The optimal concentration of FK506 to promote ALP activity was 2.0 μg/mL; the results of alizarin red staining showed that when the concentration of BIO was 0.1 μmol/L, the cells showed stronger ability of calcium nodule formation. The scaffolds slowly and stably degraded until the 35th day. FK506 and BIO were released sequentially, and the molecules were basically released within 14 days. ALP staining and alizarin red staining results showed that the experimental group with internal BIO and external FK506 showed stronger osteogenic differentiation ability compared with other groups. RT-qPCR results showed that scaffold with internal BIO and external FK506 significantly promoted the expressions of Runx2 and OCN in MC3T3-E1 cell line. Conclusion: Chitosan/sodium alginate was used as the scaffold to carry WNT signaling activator BIO inside and BMP signaling activator FK506 outside, and the sequential release of the drugs could promote the osteogenic differentiation of MC3T3-E1 cells in vitro, which opened a new approach for the treatment of bone defects and the promotion of bone regeneration clinically.

Key words: chitosan, sodium alginate, hydrogel, FK506, 6-bromoindirubin-3-oxim, BMP/WNT signaling pathway, osteogenic differentiation

摘要：

目的: 通过组织工程策略构建顺序释放骨形态发生蛋白（bone morphogenetic protein, BMP）和WNT信号通路激活剂的双层复合材料，并研究其在体外对成骨细胞分化的作用。方法: 用CCK-8法、碱性磷酸酶（alkaline phosphatase, ALP）染色法和茜素红染色法分别检测BMP信号通路激活剂他克莫司（tacrolimus, FK506）和WNT信号通路激活剂6-溴靛玉红-3-肟（6-bromoindirubin-3-oxim, BIO）对前成骨细胞系MC3T3-E1增殖活性和成骨向分化的影响，筛选出最合适的药物浓度；制备壳聚糖/海藻酸钠支架材料，采用称量法测定材料的降解曲线，制备内层壳聚糖载BIO、外层海藻酸钠载FK506的支架材料，采用紫外分光光度计测定药物的释放；制备内外单载不同药物的支架材料，用ALP染色法和茜素红染色法检测载药复合材料对前成骨细胞系MC3T3-E1成骨向分化的作用，应用实时定量聚合酶链式反应（real-time quantitative polymerase chain reaction，RT-qPCR）法检测载药支架材料对前成骨细胞系MC3T3-E1成骨相关基因表达的影响。结果: 在适宜范围内，FK506处理的细胞早期ALP活性较BIO处理的细胞更强，且FK506促进ALP活性最适合的质量浓度为2.0 μg/mL；茜素红染色结果表明，BIO在0.1 μmol/L时，细胞表现出更强的钙结节形成能力。支架材料缓慢稳定降解至第35天，期间FK506和BIO顺序释放，药物在14 d内基本释放完毕。应用载药材料浸提液ALP染色的支架材料和茜素红染色结果显示，内载BIO外载FK506的实验组相比较于其他组表现出更强的成骨向分化能力； RT-qPCR结果显示，内载BIO外载FK506的支架材料能够显著促进成骨相关基因Runx2和OCN的表达。结论: 以壳聚糖/海藻酸钠为支架材料，内层壳聚糖载WNT信号通路激活剂BIO，外层海藻酸钠载BMP信号通路激活剂FK506，两者顺序释放，即先激活BMP信号通路，后激活WNT信号通路，能够在体外促进MC3T3-E1细胞的成骨向分化，为临床治疗骨缺损，促进骨再生提供新思路。

关键词: 壳聚糖, 海藻酸钠, 水凝胶, 他克莫司, 6-溴靛玉红-3-肟, BMP/WNT信号通路, 成骨向分化

CLC Number: 

• R782