去抗原异种松质骨作为骨组织工程载体的可行性

Feasibility of antigen-extracted xenogeneic cancellous bone as carrier in bone tissue engineering

目的:对自行研制的去抗原异种松质骨材料与骨髓基质细胞复合培养进行生物相容性进行评价,为骨组织工程中生物材料的选择提供实验依据。方法:实验于2004-05/2005-05在教育部省部共建新疆民族与地方高发病重点实验室完成。①取新生小牛股骨下端松质骨,锯成15mm×5mm×5mm大小骨块,经1∶1氯仿,HCl,H2O2和PBS+NaOH+NaN3混合液等物理化学处理,制成去抗原异种松质骨载体。②选用清洁级健康3月龄新西兰大白兔2只,抽取骨髓,通过密度梯度离心和贴壁筛选法分离纯化骨髓基质细胞,将传代至第3代细胞接种于24孔培养板内,实验分为2组:实验组每孔加入5mm×5mm×3mm去抗原异种松质骨材料一块。对照组单纯接种细胞,每组4孔。于4,8d后进行处理,用于观察及检测。③通过扫描电镜、MTT测试法、考马斯亮蓝和碱性磷酸酶活性检测法,观察去抗原异种松质骨材料对细胞生长、增殖及功能表达的影响。结果:①骨髓基质细胞生长情况:倒置相差显微镜观察,骨髓基质细胞能在去抗原异种松质骨材料上黏附、增殖,细胞形态良好。②骨髓基质细胞在去抗原异种松质骨材料生长结果:扫描电镜观察,骨髓基质细胞在去抗原异种松质骨材料生长良好。③实验组于接种后4和8d骨髓基质细胞增殖、蛋白质含量和碱性磷酸酶活性(A值)分别为0.50±0.03,0.78±0.10,0.44±0.02和0.73±0.14,1.21±0.07,0.59±0.03,与对照组(0.51±0.02,0.80±0.15,0.45±0.01;0.70±0.11,1.20±0.04,0.60±0.02)比较,差异不明显(P>0.05)。结论:去抗原异种松质骨对骨髓基质细胞体外增殖和蛋白质合成无不良影响,对骨髓基质细胞的生长增殖无明显的干扰作用,对骨髓基质细胞向成骨细胞转化无影响,即去抗原异种松质骨材料无细胞毒性;同时具有良好的细胞相容性,可用作骨组织工程的支架材料。

AIM： To evaluate the biocompatibility of antigen-extracted xenogeneic cancellous bone （AEXCB） loaded with bone marrow stromal cells （BMSCs）, so as to provide experimental basis for the choice of biomaterial in bone tissue engineering. METHODS： The experiment was carried out at the Key Laboratory of Xingjiang Endemic and Ethnic Diseases Shihezi University from May 2004 to May 2005. ①The new-born calf femroal distal cancellous bone was saw into 15 mm×5 mm×5 mm bone block, and subjected to physical and chemical treatment with 1：1 Chloroform, HCl, H2O2 and PBS＋NaOH＋NaN3 to prepare antigen-extracted xenogeneic cancellous bone carrier. ②Totally 2 clean degree 3-month-old New Zealand rabbits were selected and the marrow was extracted. Purified BMSCs were isolated with density-gradient centrifugation method and adherence culture method, and the third generation cells were seeded into the plates with 24 wells. There were two groups in the experiment： experiment group： One 5 mm×5 mm×3 mm AEXCB material was added into each well; control group was only seeded the cells with 4 wells in each group. The observation and detection were performed after 4 and 8 days. ③SEM, MTT assay and alkaline phosphatase （ALP） activity were carried out to observe the effect of AEXCB on cell growth, proliferation and functional expression. RESULTS： ① Growth. condition of BMSCs： BMSCs attached and proliferated well on AEXCB and good cell morphology could be observed by inverted phase contrast microscope. ②Growth results of BMSCs： BMSCs grew well on AEXCB observed by SEM. ③BMSCs of the experiment group were proliferated, and the cells protein content and ALP （,4 value） were 0.50±0.03, 0.78±0.10, 0.44±0.02; 0.73±0.14, 1.21±0.07, 0.59±0.03 after 4 and 8 days, which had no significant difference compared with the control group （0.51±0.02, 0.80±0.15, 0.45±0.01; 0.70±0.11, 1.20±0.04, 0.60±0.02, P 〉 0.05）. CONCLUSION： Antigen-extracted xenogeneic cancellous bone has no bad effect or intervention to the in vitro proliferation of BMSCs, and has no effect on the transformation of BMSCs into osteoblast, indicating that antigen-extracted xenogeneic cancellous bone is nontoxic for ceils; meanwhile, it has good cell compatibility, and can serve as a good scaffold material for bone tissue engineering.