RESUMO
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.