双相接种法体外构建组织工程骨

Tissue-engineering bone fabricated with biphasic seeding in vitro

目的观察双相接种法构建组织工程骨的效果。方法取健康志愿者骨髓,密度梯度离心法分离骨髓间充质干细胞并进行体外扩增、诱导分化,然后应用双相接种技术与脱钙骨基质(DBM)复合构建组织工程骨,并与静置接种方法进行比较,计算接种效率、测定碱性磷酸酶活性,并用倒置显微镜、扫描电镜观察。结果与静置接种法相比,双相接种法不仅获得近100%的接种效率,而且在体外培养过程中,组织块中细胞的碱性磷酸酶活性均高于静置接种法,后者在第14天时碱性磷酸酶活性与前者第5天的水平相当。扫描电镜见双相接种法构建的组织块切面较平坦,孔隙较小,细胞包埋在胶原中,而静置接种法构建的组织块可见细胞,细胞外基质较少。结论双相接种法是一种高效的组织工程骨构建方法,有利于提高接种效率和促进组织工程骨的体外成熟。

Objective To observe the effect of fabricating tissue-engineering bone using biphasic seeding method. Methods Bone marrow was obtained from healthy adult volunteers, and then mesenchymal stem cells （MSCs） were isolated using density gradient centrifugation and cultured in vitro. After induced with 50 mg/L ascorbic acid, 10 mmol/L sodium β-glycerophosphate and 10^-8 moL/L dexamethasone for 5 d, MSCs were seeded to demineralyed bone matrix （DBM） using biphasic seeding as experimental group and using normal seeding as a control group. The adhersive rates were calculated respectively. At different time points, alkaline phosphatase （ALP） activity was detected and the structures were observed by invert microscope or scanning electron microscope （SEM）. Results MSCs-DBM complex fabricated with biphasic seeding had higher adhersive rates and ALP activity than the control group （ P 〈 0. 05 ）. The ALP activity of the experimental group at day 5 after being fabricated was equal to that of the control group at day 14. Conclusion Biphasic seeding was an effective method in fabricating tissue-engineering bone. It could improve the efficiency of seeding and be benefitial to the MSCs-DBM complex maturation in vitro.