多孔聚乳酸／骨基质明胶生物活性复合材料骨诱导活性实验

背景: 目前复合材料已成为骨移植材料研究的热点, 多孔聚乳酸 /骨基质明胶复合活性材料的应用有待研究。目的: 采用超临界二氧化碳合成法制备多孔聚乳酸 / 骨基质明胶复合活性材料, 体外评价其骨形成能力。设计: 观察对比实验。单位: 南方医科大学组织工程研究中心, 中国辐射防护研究院生物材料制药技术研究所。材料: 小鼠 MC3T3-E1 成骨前体细胞从日本 RIKEN 细胞库获得, 聚乳酸由暨南大学提供。碱性磷酸酶检测试剂为南京建成生物工程研究所产品。小鼠 MC3T3-E1 成骨前体细胞在含 100 g/L 胎牛血清 ( 四季青) 、100 mg/L 青霉素和 100 U/L 链霉素的 DMEM 培养基(Gibco, 美国) 培养, 用 0.25%胰酶(Gibco, 美国) 传代。方法: 实验于 2005- 10/2006- 07 在南方医科大学组织工程研究中心广东省重点实验室完成。采用超临界二氧化碳法制备多孔聚乳酸 / 骨基质明胶复合材料, 并通过大体及扫描电镜观察复合情况。在体外将聚乳酸、聚乳酸 / 骨基质明胶与 MC3T3-E1 小鼠成骨前体细胞共培养, 分别为聚乳酸组及聚乳酸 / 骨基质明胶组, 正常培养基作为对照组, 数码相机拍摄每组培养孔图像, 图像处理与分析软件测定被染色面积占培养孔面积百分比, 即为钙化结节面积百分比。采用细胞超声裂解法检测各组细胞内钙含量及碱性磷酸酶活性。主要观察指标: ①聚乳酸 / 骨基质明胶大体及扫描电镜观察结果; ②钙化面积的定量测定。③钙含量及碱性磷酸酶活性测定。结果: ①聚乳酸 / 骨基质明胶大体及扫描电镜观察结果: 复合材料骨基质明胶与聚乳酸混合均匀, 材料孔隙分布均匀, 孔隙大小在 50～150 μm之间, 孔洞联通性好。在这些大孔隙的壁上, 仍有大量 5～10 μm 的孔隙。②钙化结节面积测定结果: 聚乳酸 / 骨基质明胶复合材料组、聚乳酸组、空白对照组钙化结节形成面积百分比分别为 (42.98±4.44)%,(9.55±1.94)%, (0.86±0.41)%, 组间比较差异均有统计学意义 (P <0.01) 。③钙含量及碱性磷酸酶测定结果: 聚乳酸 / 骨基质明胶复合材料组、聚乳酸组和空白对照组的碱性磷酸酶活性分别为 (5 427.58±1 173.57), (1 060.54±500.27), (40.01±24.50) nkat/g, 组间比较均有统计学意义(P < 0.05～0.01) ; 聚乳酸 / 骨基质明胶复合材料组钙含量为(3.51±1.64)mmol/g, 高于聚乳酸组和空白对照组[(1.04±0.21) , (0.70±0.24)mmol/g, P < 0.01]。结论: 采用超临界二氧化碳合成法制备的聚乳酸 /骨基质明胶多孔复合支架材料具有良好的骨诱导活性, 有可能作为一种有前景的骨植入材料及骨组织工程支架材料。

Osteoinductive activity of porous poly (lactic-acid)/bone matrix gelatin bioactive composite materials

BACKGROUND:The need for bone graft and its substitutes has dramatically increased during the past decade. Either bio-derived graft materials or synthetic materials cannot satisfy this need. Study of composite bone graft has become a focused field.OBJECTIVE: To develop a novel porous poly (lactic-acid) (PLA)/bone matrix gelatin (BMG) bioactive composite material by means of supercritical carbon dioxide fluid technique (SC-CO2) and evaluate the bone forming potential in vitro.DESIGN: A comparative study and observation.SETTING: Research Center of Tissue Engineering, Southern Medical University; Institute of Biomaterials and Pharmaceutical Technique, China Institute for Radiation Protection.MATERTALS: Mouse osteoblast-like MC3T3-E1 cells were obtained from RIKEN Cell Bank in Japan. PLA was supplied by the Institute of Bio-technique of Jinan University. Alkaline phosphatase (ALP) kit was the product of Nanjing Jiancheng Bio-engineering Institute. MC3T3-E1 cells were cultured in Dulbecco's modified eagle medium (DMEM) (Gibco Laboratories, USA) supplemented with 10% fetal bovine serum (FBS, Si Ji Qing, China), 100 mg/L penicillin and 100 U/L streptomycin in a 37℃ humidified atmosphere. The cells were passaged with 0.25% trypsin (Gibco Laboratories, USA).METHODS: The experiment was carried out in the Research Center of Tissue Engineering, Southern Medical University from October 2005 to July 2006. The porous PLA/BMG composite biomaterials and PLA were prepared with SC-CO2 technique, and then macroscopic and microscopic observations were performed. The MC3T3-E1 cells were co-cultured with PLA and PLA/BMG in vitro, and DMEM was taken as the blank control group. Each well was pictured with digital camera.The percentage of the stained area, standing for the calcification deposition, was determined with an image processing and analysis software. The ALP activity and calcium content were determined with the method of ultrasonic cell lysis.MArN OUTCOME MEASURES: ① Macroscopic and microscopic observation of PLA/BMG; ③ Quantitative measurement of the calcification area; ② ALP activity and calcium content.RESULTS: ① Macroscopic and microscopic observation of PLA/BMG: The PLA/BMG showed good homological porosity with the size of 50-150 μm and connectivity: There were many holes with the size of 5-10 μm in the PLA/BMG walls. The PLA and BMG were mixed uniformly. ② Calcification areas: The percents of calcification area were significantly different among the PLA/BMG group, PLA group and blank control group [(42.98±4.44)%, (9.55±1.94)%, (0.86±0.41)%, P ＜ 0.01]. ③ Results of calcium content and ALP activity: The ALP activities were significantly different among the PLA/BMG group, PLA group and blank control group [(5 427.58±1173.57), (1 060.54±500.27),(40.01±24.50) nkat/g, P ＜ 0.05-0.01]; The calcium content in the PLA/BMG group was higher than those in the PLA group and blank control group [(3.51±1.64), (1.04±0.21), 0.70±0.24] mmol/g, P ＜ 0.01].CONCLUSTON: The PLA/BMG prepared by means of SC-CO2 has a good osteoinductive activity and it is worth studying further as bone biomaterial and bone tissue engineered scaffold.