MC 3T3-E1细胞在纳米羟基磷灰石/壳聚糖复合支架上的增殖和分化

Proliferation and Differentiation of MC 3T3-E1 Cells Cultured on Nanohydroxyapatite/chitosan Composite Scaffolds

摘要:

用原位合成纳米羟基磷灰石的方法制备多孔纳米羟基磷灰石/壳聚糖复合支架；在支架上接种MC 3T3-E1细胞，瑞氏染色检测细胞形态，MTT法检测其增殖情况；在诱导培养基中培养30d后，碱性磷酸酶染色比较其分化水平；定量检测细胞的碱性磷酸酶活性；RT-PCR检测成骨相关基因的表达情况。实验结果表明：MC 3T3-E1细胞在纳米级羟基磷灰石/壳聚糖复合支架上粘附铺展良好，其增殖率显著高于培养于纯壳聚糖支架上的细胞。碱性磷酸酶染色表明复合支架上的细胞有较高水平的碱性磷酸酶表达。进一步定量检测细胞的碱性磷酸酶活性，结果说明在复合支架上细胞比纯壳聚糖支架上培养的细胞碱性磷酸酶活性提高了约8倍。此外，骨分化相关特征基因骨桥蛋白OPN在复合支架上培养的细胞中的表达水平也明显高于纯壳聚糖上培养的细胞。分化成熟标志基因骨钙素OC在复合支架上培养的细胞中有表达，但是纯壳聚糖支架上培养的细胞中却未检测到。支架中纳米羟基磷灰石的加入不仅提高了前成骨细胞在复合支架上的增殖，而且还促进了它的分化。纳米羟基磷灰石/壳聚糖复合支架表现出良好的生物相容性和生物活性，是极具前景的骨组织工程支架材料。

Abstract:

Nanohydroxyapatite/chitosan composite scaffolds were fabricated and the proliferation and differentiation of preosteoblast MC 3T3-E1 on them were examined for the assessment of their biocompatibility. Nanohydroxyapatite was combined with chitosan in situ using a chemical method and a porous structure obtained was then lyophilized. Preosteoblast MC 3T3-E1 cells were inoculated into the porous composite scaffolds and chitosan scaffolds, respectively. The morphology of cells cultured on the scaffolds was examined after staining it with Wright's stain. Their proliferation was assessed using MTT assay. After being cultured in conditioned medium for 30 days, the cells' alkaline phosphatase activities on the scaffolds were studied in situ to compare their differentiation levelabout. Moreover, the alkaline phosphatase activities were assessed with a kit. The expression level of characteristic osteogenic gene was evaluated using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The results indicated that MC 3T3-E1 cells grown on the composite scaffolds showed a higher proliferation rate and spread better than that on chitosan scaffolds. The alkaline phosphatase stain results showed that the alkaline phosphatase activity of cells on composite scaffolds was significantly higher than that on the chitosan scaffolds. In addition, the quantitative examination of alkaline phosphatase activity indicated that the cells cultured on the composite scaffolds expressed an activity level about 8 times higher than that on chitosan scaffolds. Simultaneously, the osteogenic gene osteopontin (OPN) of cells cultured on composite scaffolds showed a higher expression level than that on chitosan scaffolds. Another osteogenic gene osteocalcin (OC) was expressed in cells cultured on composite scaffolds, whereas it was not detected in cells on chitosan scaffolds. The addition of nanohydroxyapatite in the scaffolds improved not only the proliferation but also the differentiation of preosteoblast cultured on them. The composite scaffolds showed good biocompatibility and bioactivity. These scaffolds would be promising in bone tissue engineering.