RESUMO
OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.