深低温保存组织工程化骨的实验研究  

Experimental study of cryopreservation for tissue-engineered bone

作　　者：尹宏宇[1] 崔磊[2,3] 刘广鹏[3] 曹谊林[1,2,3] 

机构地区：[1]中国医学科学院北京协和医学院整形外科医院研究中心,北京100041 [2]上海交通大学医学院附属第九人民医院整复外科,上海200011 [3]上海组织工程研究与开发中心,上海200234

出　　处：《中国美容医学》2009年第4期479-483,共5页Chinese Journal of Aesthetic Medicine

基　　金：北京市科技计划(H060920051230)

摘　　要：目的:深低温保存是组织工程化骨(TEB)实现大规模临床应用的必要保证。深低温保存方法分慢速冻存和玻璃化冻存。目前,玻璃化冻存是最有发展前景的冻存方法,并被认为更适用于TEB的深低温保存。因此,希望通过改进玻璃化液来提高TEB的玻璃化冻存效果。方法:通过检测第2(P2)代成骨诱导的犬骨髓基质干细胞(cBMSCs)在部分脱钙骨(pDBM)上的粘附率和生长曲线来获得适宜的细胞接种密度和体外培养时间,将在此基础上构建的TEB进行深低温保存。由TEB冻存后的细胞活性来选择一种理想的玻璃化液,并将其用于玻璃化冻存和慢速冻存TEB的对比实验研究。结果:P2代成骨诱导的cBMSCs在pDBM上适宜的细胞接种密度是50×106/ml,最佳的体外培养时间是8天。VS442作为玻璃化液用于TEB的深低温保存。尽管玻璃化冻存TEB的细胞存活率仅为46.2%±7.2%,低于慢速冻存的77.4%±5.1%,但经过体外培养11天后的细胞活性超过了后者,可基本恢复到冻存前的水平。结论:VS442是玻璃化冻存TEB理想的玻璃化液。尽管实验采用的慢速冻存方法可简单有效的保存TEB,但对于TEB而言,玻璃化冻存还是更具有发展潜力的深低温保存方法。Objective The large-scale clinical use of tissue-engineered bone （TEB） will require provisions for its mass availability and accessibility, which has cryopreservation be necessary to bank TEB. At present, vitrification preservation is the most promising methods of cryopreservation, which is thought as an attractive approach for the bank of TEB. The effect of vitrification method for TEB was wished to enhance by improving the vitrified solution. Methods The optimal seeding density and cultured time in vitro were selected by detecting the seeding efficacy and growth curve of osteoinduced canine bone marrow mesenchymal stem cells （cBMSCs） of passage 2 （P2） on partially demineralized bone matrix （pDBM）. According to the cellular viability of TEB composed of osteogenically induced cBMSCs at P2 and pDBM af- ter rewarming, the ideal cryoprotectant was chosen to cryopreserve TEB by vitrification in order to compare with the experimental result of slow freezing method. Results The optimal seeding density of osteo-induced cBMSCs of P2 on pDBM was 50×10^6/ml and the ideal culture time in vitro was eight days. A new vitrification solution named as VS442 was explored for vitrification preservation of TEB. The cell survival of vitreous cryopreservation and slow-freezing of TEB post-thaw with VS442 was 46.2%±7.2% and 77.4%±5.1%, respectively. However, the celt viability of cooling TEB by vitrification has exceeded that of slow-freezing approach and recovered to the level of pre-cryopreservation in day 11 post-rewarm. Conclusion Based on these results, it can be concluded that VS442 was an ideal vitreous cryoprotec- tant for vitrification preservation of TEB; though the slow-freezing method using in this study might simply and effectively cryopreserve TEB, vitrification preservation was still the more development potential of cryopreservation methods for TEB.

关 键 词：组织工程 深低温保存 玻璃化冻存 组织工程化骨 

分 类 号：Q813.1[生物学—生物工程]