组织工程化人工胸骨支架材料的制备及生物学评价

The comparison and biological evaluation of PCL/BP scaffold for tissue engineered artificial sternum

目的通过生物学评价探讨不同比例聚己内酯／天然骨粉（polycaprolactone／bonepowder，PCL／BP）作为组织工程化人工胸骨材料的安全性。方法将聚己内酯（PCL）与天然骨粉（BP）分别以8：2及6：4混合成备用材料，并利用材料的浸提液进行热源、溶血、急性全身毒性等生物学实验，同时将受试材料分别与骨髓基质干细胞进行培养，观察其接种后细胞的生长情况。结果受试材料均无急性全身毒性反应、溶血和热源反应。培养的骨髓基质干细胞经材料浸提液处理后形态良好，增殖旺盛。细胞增殖试验显示，受试材料组细胞生长无明显差别（P≥0．05）。结论PCL／BP复合物具有良好的生物相容性，不同比例的PCL／BP不影响其生物安全性。

Objective To prepare the composite of Polyeaprolactone（PCL） and Bone powder（BP） for bone repairing, and evaluate the biocompatibility of the material. Methods The materials were prepared in college of chemical engineering and materials science, Shanghai Jiaotong University , and the animal tests were fulfilled in Animal Experimental Center of the Second Military Medical University of PLA from June 2010 to March 2011. The PCL and BP were blended together to get the copolymer by haake （ Rheocord 900） torque rheometer. Then the extracts of the material in DMEM for 24 hours were obtained. Firstly biological safety detection of the materials was performed：（1）cytotoxicity test ： the extracts were mixed with DMEM to seed rabbit osteoblasts for 24 hours. The cell proliferation was detected by MTT method . The cell proliferation rate and toxicity grate were also calculated （ eytotoxicity of grade 0 or 1 was qualified）. （2）hemolysis test ： the extracts were mixed with fresh rabbit blood, hemolysis rate were calculated （ hemolysis rate less than 5% was qualified）. （3）pyrogenic test： the extracts were injected into the rabbit ear vein with the dose of 10 ml/kg. The anus temperature was measured per hour for three times. The elevator degree was calculated by subtracting the natural temperature from the highest one. Secondly, cellular adhesion test of the material composite was performed. The composite was casted on the cover glasses. The samples were put in 6-well tissue culture plates, totally 6 x 104 osteoblasts were seeded each well and cultured for 3 days. The cells were observed at 24 and 72 hours of culture, and photography was performed using inverted phase contrast microscope. （4）general toxicity test ： the extracts were injected into mouse vein with the dose of 50 ml/kg. The toxic symptoms were observed within 72 hours. Results The novel copolymer of PCL/BP was prepared. （1）Biological safety： the cell viability of the composite was 87.8% and cytotoxicity was grade 1. The hemolysis rate was 2.4% ,less than 5%. The maximal variety of body temperature in rabbits were 0.4~C ,which accorded with the national standard （ 〈0.6℃ ）. The general toxicity test showed that there were no adverse effects in the ani- mals, such as death, convulsion, paralysis, respiratory depression, diarrhea and decreasing weight. （2）Cell adhesion ： adhesive cells manifold obviously and assembled 24 hours later, inosculated gradually 3 days later, and the cells were in good fettle. Conclusion The composite of PCL/BP accords with cytotoxicity demand of the biological materials, presenting no toxicity, pyrogenicity or hemolysis. It possesses good biocompatibility and cellular adhesion.