冻干组织工程骨与组织工程骨成骨活性比较研究

EXPERIMENTAL COMPARATIVE STUDY ON OSTEOGENIC ACTIVITY BETWEEN FREEZE-DRIED TISSUE ENGINEERED BONE AND TISSUE ENGINEERED BONE

目的目前组织工程骨(tissue engineered bone,TEB)缺乏有效可行的储存、运输方法。评估TEB经过冻干处理后其成骨活性变化及其异位成骨能力,探索新的TEB储存和运输策略。方法取志愿者捐献的骨髓和骨组织分别培养hBMSCs和制备脱钙骨基质(decalcifi ed bone matrix,DBM),取第3代hBMSCs与DBM复合构建TEB,分别于体外孵育3、5、7、9、12、15d经过低温干燥后得到冻干组织工程骨(freeze-dried tissue engineered bone,FTEB),并将体外培养不同时间点的TEB和FTEB行扫描电镜观察。Western blot法检测TEB和FTEB内成骨相关蛋白BMP-2、TGF-β1、IGF-1的变化。将FTEB、TEB和DBM分别移植于30只6周龄BALB/C裸鼠皮下进行异位成骨实验(n=10),并行X线片评分、CT值检测以及HE染色观察。结果扫描电镜观察示,TEB中种子细胞保持正常形态,随着培养时间的延长分泌细胞外基质逐渐增加;FTEB内的种子细胞脱水皱缩而亡,细胞外基质位于疏松的DBM支架材料中。Western blot检测示,TEB和FTEB内BMP-2、TGF-β1和IGF-1表达均为阳性,除体外培养15d TEB和FTEB的TGF-β1蛋白积分吸光度(IA)比值比较差异有统计学意义(P<0.05)外,其余各时间点各蛋白含量比较差异均无统计学意义(P>0.05)。移植术后4周各种移植物未见明显钙化;术后8、12周,TEB和FTEB移植裸鼠皮下可以实现较好的异位成骨。通过X线片评分、CT值比较移植物钙化程度,TEB和FTEB差异无统计学意义(P>0.05),而DBM未见明显钙化。HE染色示TEB和FTEB出现钙化,DBM降解吸收。结论 FTEB与TEB具有相似的成骨活性,为TEB的储存和运输提供了一种新的策略和方法 。

Objective Tissue engineered bone（TEB） lacks of an effective and feasible method of storage and transportation.To evaluate the activity of osteogenesis and capability of ectopic osteogenesis for TEB after freeze-dried treatment in vitro and in vivo and to explore a new method of preserving and transporting TEB.Methods Human bone marrow mesenchymal stem cells（hBMSCs） and decalcified bone matrix（DBM） were harvested from bone marrow and bone tissue of the healthy donators.TEB was fabricated with the 3rd passage hBMSCs and DBM,and they were frozen and dried at extremely low temperatures after 3,5,7,9,12,and 15 days of culture in vitro to obtain freeze-dried tissue engineered bone（FTEB）.TEB and FTEB were observed by gross view and scanning electron microscope（SEM）.Western blot was used to detect the changes of relative osteogenic cytokines,including bone morphogenetic protein 2（BMP-2）,transforming growth factor β1（TGF-β1）,and insulin-like growth factor 1（IGF-1） between TEB and FTEB.The ectopic osteogenesis was evaluated by the methods of X-ray,CT score,and HE staining after TEB and FTEB were transplanted into hypodermatic space in athymic mouse.Results SEM showed that the cells had normal shape in TEB,and secretion of extracellular matrix increased with culture time;in FTEB,seeding cells were killed by the freeze-dried process,and considerable extracellular matrix were formed in the pore of DBM scaffold.The osteogenic cytokines（BMP-2,TGF-β1,and IGF-1） in TEB were not decreased after freeze-dried procedure,showing no significant difference between TEB and FTEB（P 0.05） except TGF-β1 15 days after culture（P 0.05）.The ectopic osteogenesis was observed in TEB and FTEB groups 8 and 12 weeks after transplantation,there was no significant difference in the calcified level of grafts between TEB and FTEB groups by the analysis of X-ray and CT score.On the contrary,there was no ectopic osteogenesis in group DBM 12 weeks after operation.HE staining showed that DBM scaffold degraded and disappeared 12 weeks after operation.Conclusion The osteogenic activity of TEB and FTEB is similar,which provides a new strategy to preserve and transport TEB.