【摘要】 目的采用海藻酸钠凝胶(sodium alginate,A)复合异种骨的方法,构建骨组织工程载体,观察载体中细胞的生物性能及体内成骨能力,为构建效率更高的骨组织工程载体提供实验依据。方法取2只2周龄新西兰兔的骨髓,以rhBMP-2(1×10-8 mol/L)诱导培养BMSCs。取诱导后第2代BMSCs接种于1%(V/W)A中,培养4 d HE染色观察凝胶中细胞形态。将第2代BMSCs分为单纯DMEM凝胶组和含1%A的DMEM凝胶组,培养7 d后行BMP-2免疫组织化学染色观察。第2代BMSCs与2%(V/W)A的DMEM凝胶混合,负压下复合去抗原牛松质骨(xenograft bone,X),4 d后扫描电镜观察细胞生长情况。取24只裸鼠,随机分为2组(n=12),于两侧股部肌袋中分别植入BMSCs-A-X复合体作为实验组,BMSCs-X复合体作为对照组。于术后2、4周后组织学观察复合体成骨情况,图像分析系统分析各组成骨或软骨的面积百分比。结果HE染色观察,培养4 d A中BMSCs细胞形态饱满,细胞悬浮于凝胶中,可见细胞分裂和核分裂相。单纯DMEM凝胶组和含1%A的DMEM凝胶组免疫组织化学观察,细胞分裂增殖正常,伸出多种形态的突起,胞核大,核仁清晰。单纯DMEM凝胶组BMP-2表达阳性率为44.10%±3.02%;含1%A的DMEM凝胶组为42.40%±4.83%,差异无统计学意义(P>0.05)。扫描电镜观察:A均匀复合于X微孔中,不同平面均有细胞生长。植入裸鼠体内2周后,实验组及对照组中均有软骨和骨组织形成;实验组软骨面积百分比为7.31%±0.32%,骨为5.26%±0.24%;对照组软骨为2.31%±0.21%,骨为2.16%±0.22%;两组差异有统计学意义(P<0.05)。术后4周,两组软骨和新生骨小梁及骨髓组织较2周时增多;实验组软骨面积百分比为9.31%±0.31%,骨为7.26%±0.26%;对照组软骨为3.31%±0.26%,骨为2.26%±0.28%;两组差异有统计学意义(P<0.05)。术后2、4周同组间比较差异有统计学意义(P<0.05)。结论以A-X构建骨组织工程载体,符合组织工程载体的超结构原理,最大限度地承载细胞,生物性能好,对BMSCs增殖和成骨表型及相关的生物性能无不良影响,在体内成骨效率较高。更多还原

【Abstract】 Objective To produce a new bone tissue engineered carrier through combination of xenograft bone(X) and sodium alginate(A) and to investigate the biological character of the cells in the carrier and the ability of bone-forming in vivo,so as to provide experimental evidence for a more e ective carrier.Methods BMSCs were extracted from 2-week-old New Zealand rabbits and the BMSCs were induced by rhBMP-2(1 × 10-8mol/L).The second generation of the induced BMSCs was combined with 1%(V/W) A by nal concentration of 1 × 105/mL.After 4-day culture,cells in gel were investigated by HE staining.The second generation of the induced BMSCs was divided into the DMEM gel group and the DMEM containing 1% A group.They were seeded into 48 well-cultivated cell clusters by nal concentration of 1 × 105/mL.Seven days later,the BMP-2 expressions of BMSCs in A and in commonly-cultivated cells were compared.The second generation of the induced BMSCs was mixed with 2% A DMEM at a nal concentration of 1 × 1010/mL.Then it was compounded with the no antigen X under negative pressure.After 4 days,cells growth was observed under SEM.Twenty-four nude mice were randomly divided into 2 groups(n=12).The compound of BMSCs-A-X(experimental group) and BMSCs-X(control group) with BMSCs whose nal concentration was 1 × 1010/mL was implanted in muscles of nude mice.Bone formation of the compound was histologically evaluated by Image Analysis System 2 and 4 weeks after the operation,respectively.Results Cells suspended in A and grew plump.Cell division and nuclear ssion were found.Under the microscope,normal proliferation,many forming processes,larger nucleus,clear nucleolus and more nuclear ssion could be seen.BMP-2 expression in the DMEM gel group was 44.10% ± 3.02% and in the DMEM containing 1% A group was 42.40% ± 4.83%.There was no statistically signi cant di erence between the two groups(P > 0.05).A was compounded evenly in the micropore of X and cells suspended in A 3-dimensionally with matrix secretion.At 2 weeks after the implantation,according to Image Analysis System,the compound of BMSCs-A-X was 5.26% ± 0.24% of the total area and the cartilage-like tissue was 7.31% ± 0.32% in the experimental group;the compound of BMSCs-X was 2.16% ± 0.22% of the total area and the cartilage-like tissue was 2.31% ± 0.21% in the control group.There was statistically signi cant di erence between the two groups(P < 0.05).At 4 weeks after the operation,the compound of BMSCs-A-X was 7.26% ± 0.26% of the total area and the cartilage-like tissue was 9.31% ± 0.31% in the experimental group;the compound of BMSCs-X was 2.26% ± 0.28% of the total area and the cartilage-like tissue was 3.31% ± 0.26% in the control group.There was statistically signi cant di erence between the two groups(P < 0.05).Conclusion The new carrier compounding A and no antigen X conforms to the superstructural principle of tissue engineering,with maximum cells load.BMSCs behave well in the compound carrier with e cient bone formation in vivo.更多还原