海藻酸钠凝胶复合异种骨构建组织工程骨及体内成骨

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(1×10-8mol/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(xenograftbone,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 dA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%4±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516%±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.

CARRIER COMBINATION OF TISSUE ENGINEERED BONE BY SODIUM ALGINATE AND XENOGRAFT BONE AND BONE FORMATION IN VIVO

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 effective carrier. METHODS: BMSCs were extracted from 2-week-old New Zealand rabbits and the BMSCs were induced by rhBMP-2 (1 x 10(-8)mol/L). The second generation of the induced BMSCs was combined with 1% (V/W) A by final concentration of 1 x 10(5)/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 final concentration of 1 x 10(5)/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 final concentration of 1 x 10(10)/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 final concentration was 1 x 10(10)/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 fission were found. Under the microscope, normal proliferation, many forming processes, larger nucleus, clear nucleolus and more nuclear fission 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 significant difference 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 significant difference 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 significant difference 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 efficient bone formation in vivo.