Zhang Yumin, Li Baoxing, Li Ji
Research Center of Tissue Engineering, Southern Medical University, Guangzhou Guangdong, 510515, P. R. China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2007 Feb;21(2):135-9.
To fabricate a novel porous bioactive composite biomaterial consisting of poly lactic acid (PLA)-bone matrix gelatin (BMG) by using the supercritical carbon dioxide fluid technique (SC-CO2) and to evaluate its osteoinductive activity.
The cortical bones selected from healthy adult donors were processed into BMG by the defatting, demineralizing, and deproteinizing processes. PLA and BMG were mixed at a volume radio of 3 : 1; then, the PLA-BMG mixed material and the pure PLA material were respectively placed in the supercritical carbon dioxide reaction kettles, and were respectively added by the NaCl particles 100-200 microm in diameter for the porosity of the materials so that the porous PLA-BMG composite material and the porous PLA composite material could be formed. The mouse osteoblast-like MC3T3-E1 cells were cultured in the dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum. Then, 20 microl of the MC3T3-E1 cell suspensions containing 2 X 10(6) cells /ml were delivered into the culturing plate (24 wells/plate) made of the different materials, which were co-cultured for 2 weeks. In the PLA-BMG group, 100 microg of the crushed PLA-BMG material was contained in each well; in the PLA group, 100 microg of the crushed PLA material was contained in each well; and in the DMEM group, only DMEM was contained, which served as the control group. There were 6 wells in each group. The quantitative analysis on the calcification area was performed by the staining of the alizarin red S. The co-cultured cells were harvested and lysated in 1 ml of 0. 2% Nonidet P-40 by the ultrasonic lysating technique. Then, the ALP activity and the Ca content were measured according to the illuminations of the reagent kits.
The porous PLA-BMG composite material showed a good homological porosity with a pore diameter of 50-150 microm and a good connectivity between the pores. The ALP activity, the Ca content, and the calcification area were significantly greater in the PLA-BMG group than in the PLA group and the control group (325.59 +/- 70.40 U/gprot, 3.51+/- 1.64 mmol/gprot, 42.98 +/- 4.44% vs. 63. 62 +/- 30.01 U/gprot, 1.04+/-0.21 mmol/gprot, 9.55+/-1.94%, and 2.40+/-1.47 U/gprot, 0.70+/-0.24 mmol/gprot, 0.86+/-0.41%; P<0.05). Meanwhile, there was a statistically significant difference between the PLA group and the control group in the ALP activity and the calcification area (P< 0.05).
The porous PLA-BMG composite material prepared by the use of SC-CO2 has a good osteoinductive activity and can be used as a promising bone biomaterial and a bone tissue engineered scaffold.
采用超临界二氧化碳流体技术（SC-CO2）制备一种新型的由聚乳酸（PLA）-骨基质明胶（BMG）组成的多孔生物活性复合生物材料，并评估其骨诱导活性。
选取健康成年供体的皮质骨，经脱脂、脱矿和脱蛋白处理制成BMG。将PLA与BMG按体积比3∶1混合；然后，分别将PLA-BMG混合材料和纯PLA材料置于超临界二氧化碳反应釜中，并分别加入直径为100 - 200微米的NaCl颗粒以形成材料的孔隙率，从而制成多孔PLA-BMG复合材料和多孔PLA复合材料。将小鼠成骨样MC3T3-E1细胞培养于添加10%胎牛血清的杜氏改良伊格尔培养基（DMEM）中。然后，将20微升含2×10(6)个细胞/毫升的MC3T3-E1细胞悬液接种到由不同材料制成的培养板（24孔/板）中，共培养2周。在PLA-BMG组中，每孔含100微克粉碎的PLA-BMG材料；在PLA组中，每孔含100微克粉碎的PLA材料；在DMEM组中，仅含DMEM，作为对照组。每组6孔。采用茜素红S染色对钙化面积进行定量分析。通过超声裂解技术将共培养的细胞收获并裂解于1毫升0.2%的Nonidet P-40中。然后，根据试剂盒说明测量碱性磷酸酶（ALP）活性和钙含量。
多孔PLA-BMG复合材料具有良好的同源孔隙率，孔径为50 - 150微米，孔间连通性良好。PLA-BMG组的ALP活性、钙含量和钙化面积显著高于PLA组和对照组（325.59±70.40 U/gprot，3.51±1.64 mmol/gprot，42.98±4.44% vs. 63.62±30.01 U/gprot，1.04±0.21 mmol/gprot，9.55±1.94%，以及2.40±1.47 U/gprot，0.70±0.24 mmol/gprot，0.86±0.41%；P<0.05）。同时，PLA组和对照组在ALP活性和钙化面积方面存在统计学显著差异（P<0.05）。
采用SC-CO2制备的多孔PLA-BMG复合材料具有良好的骨诱导活性，可作为一种有前景的骨生物材料和骨组织工程支架。
