【作者】 尹宏宇；

【导师】 曹谊林； 崔磊； 刘伟； 滕利；

【作者基本信息】 中国协和医科大学 ， 外科学， 2009， 博士

【摘要】 目的组织工程化骨(TEB)是目前骨缺损修复理想的骨移植材料之一,但常规的体外构建过程需要一段时间,无法满足临床上即刻骨缺损修复的要求。本研究针对这一问题进行了两种解决方案的体外实验研究。1有实验报道用骨髓单个核细胞(BMNCs)复合材料即刻构建的组织工程化骨进行体内骨缺损的即刻修复有一定的效果。但无体外实验对此方案的成骨效果进行论证,为此本实验通过在成骨诱导环境的体外检测来对此方案进一步加以验证。2常规构建的组织工程化骨若能获得理想的深低温保存效果则可作为实现即刻骨缺损修复的另一方案。玻璃化冻存是目前最有发展前景的冻存方法,因此本实验进行体外检测来验证玻璃化冻存TEB的可行性。方法1用1.063g/mL Percoll液分离Beagle犬骨髓单个核细胞(cBMNCs)。将猪股骨头制备成直径5毫米(mm)、厚度1mm的部分脱钙骨(pDBM)支架材料。通过cBMNCs在pDBM上粘附率的检测,获得理想的细胞接种密度。用此密度的cBMNCs分别经过诱导培养或常规培养的第二(P2)代成骨诱导或未诱导犬骨髓基质干细胞(cBMSCs)复合pDBM常规构建的TEB与该密度cBMNCs复合pDBM即刻构建的TEB共同进行成骨诱导培养,体外分析比较各自的细胞活性和成骨能力。2通过用不同组成和浓度的玻璃化液在不同的预处理条件下对P2代成骨诱导cBMSCs复合pDBM常规构建的TEB进行玻璃化冻存,选出适宜的玻璃化液和预处理条件。以VS55作对照,用新研制的玻璃化液分别玻璃化冻存TEB 7天或3个月,观察TEB经玻璃化冻存后的细胞活性和成骨能力。结果1采用Percoll密度梯度离心法可获得纯度较高的cBMNCs。在pDBM上接种cBMNCs适宜的密度是30×10~6/mL。通过P2代成骨诱导和未诱导cBMSCs复合pDBM常规构建TEB与cBMNCs复合pDBM即刻构建TEB的体外成骨培养检测中发现尽管即刻构建的TEB在相同的检测时间内细胞活性和成骨能力较低,但随着培养时间的延长可具有很大的提升空间。2用玻璃化液VS442在0℃/15分钟的预处理条件下玻璃化冻存由P2代成骨诱导cBMSCs复合pDBM常规构建的TEB可获得较好的冻存效果。在与VS55的比较中,用VS442进行TEB玻璃化冻存后的细胞活性和成骨能力明显强于VS55,将玻璃化冻存时间从7天延长至3个月时并未对细胞活性和成骨能力造成影响。结论1 1.063g/mL Percoll液能够较好的分离获取Beagle犬BMNCs。30×10~6/mL是cBMNCs复合pDBM即刻构建TEB适宜的细胞接种密度。通过在成骨诱导环境的体外检测证实了即刻构建的TEB是可以直接回植来满足体内即刻骨缺损修复的要求。2 VS442是玻璃化冻存常规构建TEB适宜的玻璃化液。在同VS55比较中可见VS442对保持TEB玻璃化冻存后的细胞活性和成骨能力方面效果显著,因此用VS442玻璃化冻存的TEB是可以满足临床上骨缺损的即刻修复需求。更多还原

【Abstract】 ObjectiveTissue-engineered bone(TEB) is one of ideal bone graft material for repairing bone defects.It will take some time to construct TEB by traditional method,which is unable to meet the instantly clinical requirements of repairing bone defects.This research has conducted two projects of in vitro experimental study in view of this question.1 Experiments have reported that the instantly constructed TEB with bone marrow mononuclear cells(BMNCs) and materials of scaffold had the certain effect of immediately repairing bone defects in vivo.However,there was not in vitro experiment to prove the osteogenic effect of this project,which was further verified by in vitro tests under the osteogenically inducted environment in our study.2 If the ideal cryopreservation effects of TEB constructed by traditional method can be obtained,it is possible to be used as another method to meet the immediately clinical requirements of repairing bone defects.At present,vitrification preservation is the most promising methods of cryopreservation.In this study,we examined the feasibility of cryopreservation of TEB by vitrification in vitro.Methods1 BMNCs of Beagle were separated by 1.063g/mL Percoll gradient solution. Prepared from porcine femoral head,Partially demineralized bone matrix (pDBM) scaffolds were molded into discs(5 mm in diameter and 1 mm in thickness).The ideal seeding density was obtained by detecting the seeding efficacy of canine bone marrow mononuclear cells(cBMNCs) on pDBM.After the cBMNCs of this density were cultured in osteogenic culture medium(OCM)or basic culture medium(BCM) respectively,conventionally constructed TEB with osteo-induced or non-induced cBMSCs at P2 and pDBM was compared with immediately constructed TEB composed of cBMNCs and pDBM under OCM together for analyzing respectively the cell activity and osteogenic capacity in vitro.2 Using the different composition and concentration of vitrification fluid,the freezing experiments on traditionally constructed TEB with osteo-induced cBMSCs at P2 and pDBM were performed under the different preconditioning so that the proper vitrification media and optimal pretreatments were chosen.Afterwards,TEB in VS442 was subjected to vitrification preservation for 7 days and 3 months,respectively.Cell viability,proliferation and osteogenic differentiation of cBMSCs in TEB after vitreous cryopreservation were examined with parallel comparisons made with those cryopreserved in VS55 vitreous cryoprotectant.Results1 With the density gradient centrifugation method,cBMNCs were successfully isolated,and 1.063g/mL Percoll medium could obtain more purified monocytes.The optimal seeding density of cBMNCs on pDBM was 30×10~6/mL.After analyzing the cell activity and osteogenic capacity of conventionally constructed TEB,which were composed of osteo-induced and non-induced cBMSCs at P2 and pDBM,and immediately constructed TEB with cBMNCs and pDBM under the same culture of osteogenic induction in vitro, it was found that despite that of immediately constructed TEB were the lowest, the potentiality of enhancement would be bigger following the culture time.2 In the experiments of vitrification preservation for TEB conventionally constructed with osteo-induced cBMSCs at P2 and pDBM,the effect of vitreous cryopreservation with VS442 under 0℃/15min of preconditioning was better.After cell viability,proliferation and osteogenic differentiation of cBMSCs in TEB after vitreous cryopreservation were examined with parallel comparisons made with those cryopreserved in VS55 vitreous cryoprotectant,post-rewarm cell viability and osteogenic capability of TEB in VS442 were significantly higher.Furthermore,we observed that extending the cryopreservation of TEB from 7 days to 3 months did not have a significant impact on its survival and osteogenic potential.Conclusions1 cBMNCs of Beagle were isolated through the 1.063g/mL Percoll density gradient centrifugation.The optimal seeding density of cBMNCs was 30×10~6/mL.In vitro examination under the osteogenically inducted environment confirmed that the directly replantation of instantly constructed TEB might satisfy the immediately clinical requirements of repairing bone defects.2 VS442 was an optimal vitreous cryoprotectant for vitrification preservation of TEB.As compared with VS55,VS442 was demonstrated to be more efficient in maintaining cellular viability and osteogenic function for vitreous cryopreservation of TEB.Consequently,TEB cryopreserved in VS442 by vitrification could satisfy the instantly clinical requirements of repairing bone defects.更多还原