ABSTRACT
BACKGROUND:It is important to prepare a mal eable putty to repair irregular bone defects because the preformed bone substitute material cannot suit to bone defects. OBJECTIVE:To prepare mal eable demineralized bone matrix/hyaluronic acid putty and to screen out the optimal composite ratio, as wel as to evaluate its cellcompatibility. METHODS:Demineralized bone matrix was prepared from the cortical bone of healthy donors. The hyaluronic acid was dissolved at concentrations of 0.75%, 1.5%, 3%, 4.5%, and the viscosity of them was measured. Then 450 mg demineralized bone matrix was compounded with 1 mL hyaluronic acid solution to prepare the demineralized bone matrix/hyaluronic acid putty, and the optimal ratio of demineralized bone matrix/hyaluronic acid was screened by the disintegrated time of the putty at 37 ℃. L-929 mouse fibroblasts were cultured in the leaching liquor of demineralized bone matrix/hyaluronic acid, and the cytotoxicity was tested by cellcount kit-8 assay at days 1, 4 and 7. RESULTS AND CONCLUSION:With the increase of hyaluronic acid concentration, the viscosity of hyaluronic acid solution was increased;the demineralized bone matrix/hyaluronic acid putty was strengthened and the disintegrated time was prolonged gradual y. When the ratio of demineralized bone matrix/hyaluronic acid was 15/1, demineralized bone matrix/hyaluronic acid was molded easily, and the disintegrated time was 8 hours in 7 ℃ PBS. The results of cytotoxicity showed that cellproliferation rates were 93.72%, 101.65%and 97.68%at days 1, 4 and 7 respectively, while the cytotoxicity was in grade 0 or 1. These findings indicate that demineralized bone matrix/hyaluronic acid putty at a compound ratio of 15:1 can be molded easily and difficult to disintegrate, with low cytotoxicity.
ABSTRACT
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.