Kong Li-Jun, Ao Qiang, Xi Jing, Zhang Ling, Gong Yan-Dao, Zhao Nan-Ming, Zhang Xiu-Fang
Department of Biological Sciences and Biotechnology , State Key Lab of Biomembrane and Membrane Biotechnology, Tsinghua University, Beijing 100084, China.
Sheng Wu Gong Cheng Xue Bao. 2007 Mar;23(2):262-7.
with chitosan in situ using a chemical method and a porous structure obtained was then lyophilized. Preosteoblast MC 3T3-E1 the scaffolds was examined after staining it with Wright's stain. Their proliferation was assessed using MTZ assay. After being 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 333-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.
采用化学方法将纳米羟基磷灰石与壳聚糖原位复合，得到多孔结构后进行冻干处理。将前成骨细胞MC 3T3-E1接种到多孔复合支架和壳聚糖支架上。用瑞氏染色法对支架上培养的细胞进行染色后，观察其形态。采用MTT法评估细胞增殖情况。在条件培养基中培养30天后，原位研究支架上细胞的碱性磷酸酶活性，以比较其分化水平。此外，使用试剂盒评估碱性磷酸酶活性。采用逆转录-聚合酶链反应（RT-PCR）评估特征性成骨基因的表达水平。结果表明，在复合支架上生长的MC 3T3-E1细胞比在壳聚糖支架上具有更高的增殖率和更好的铺展性。碱性磷酸酶染色结果显示，复合支架上细胞的碱性磷酸酶活性明显高于壳聚糖支架上的细胞。此外，碱性磷酸酶活性的定量检测表明，在复合支架上培养的细胞表达的活性水平比在壳聚糖支架上高约8倍。同时，在复合支架上培养的细胞中成骨基因骨桥蛋白（OPN）的表达水平高于壳聚糖支架上的细胞。另一个成骨基因骨钙素（OC）在复合支架上培养的细胞中表达，而在壳聚糖支架上的细胞中未检测到。支架中添加纳米羟基磷灰石不仅提高了其上培养的前成骨细胞的增殖能力，还提高了其分化能力。复合支架具有良好的生物相容性和生物活性。这些支架在骨组织工程中具有广阔的应用前景。
