正常人颌骨组织冻存后活性评估及组织库的建立

中国口腔颌面外科杂志 ›› 2017, Vol. 15 ›› Issue (4): 314-319.

正常人颌骨组织冻存后活性评估及组织库的建立

华洪飞¹, 张文杰², 张伟杰¹, 杨驰¹, 张志愿³, 王绍义¹

1．上海交通大学医学院附属第九人民医院口腔医学院口腔外科,
2．口腔修复科, 3．口腔颌面-头颈肿瘤科,上海市口腔医学重点实验室,上海 200011

出版日期:2017-08-10 发布日期:2017-08-14
通讯作者: 王绍义,E-mail:wangshaoyi163@aliyun.com
作者简介:华洪飞（1991-）,男,硕士,E-mail: huahongfei213@sina.com
基金资助:
国家自然科学基金（81271114）

Evaluation of viability of cryopreserved human mandible and establishment of experimental bone bank

HUA Hong-fei¹, ZHANG Wen-jie², ZHANG Wei-jie¹, YANG-Chi¹, ZHANG Zhi-yuan³, WANG Shao-yi¹

1.Department of Oral Surgery,
2.Department of Prosthodontics,
3.Department of Oromaxillofacial Head and Neck Oncology, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine； Shanghai Key Laboratory of Stomatology. Shanghai 200011, China

Online:2017-08-10 Published:2017-08-14

摘要/Abstract

摘要： 目的体外实验评估正常人颌骨冻存后的生物活性变化,初步建立颌骨组织库,探讨骨组织活性冻存后用于骨缺损修复的可能性。方法选择15~80岁的捐献者(于上海交通大学医学院附属第九人民医院口腔外科行阻生牙拔除术等牙槽手术的患者),术中无菌条件下用超声骨刀取骨。骨块处理后,置于含10%二甲基亚砜（dimethyl Sulphoxide,DMSO）的冻存保护液中,无菌独立包装,行慢速梯度降温至-80℃。登记必要信息后,保存入-80℃深低温冰箱,建立骨组织库。通过检测不同冻存时限（0、1、6、12个月）骨块来源的成骨系细胞爬出时间及集落形成率、形态变化、增殖活力、碱性磷酸酶合成能力、体外矿化能力、成骨相关基因表达能力、凋亡情况、染色体变异情况、细胞衰老情况等,研究低温冻存对骨组织生物活性的影响。使用SPSS17.0软件包对数据进行统计学分析。结果不同冻存时间骨块组织块培养法均能观察到细胞集落形成,形态无明显变化;增殖活力、碱性磷酸酶合成能力、体外矿化能力、成骨相关基因表达能力、染色体变异情况、细胞衰老等与新鲜对照组比较无显著差异（P>0.05）。结论低温冻存对人颌骨组织的体外生物学功能影响甚微,冻存颌骨组织库用于自体骨缺损修复或组织工程骨构建具有可行性。

关键词: 低温冻存, 自体骨, 骨组织样本库, 骨组织工程

Abstract: PURPOSE：To establish an experimental bony tissue bank and evaluate the viability of cryopreserved human mandible, with an attempt to explore the possibilities of applying cryopreserved bone (with cryoprotectant) for reconstruction of bone defects. METHODS: Bony tissue specimens were harvested from the donors aged from 15 to 80 years old who had impacted wisdom teeth necessitating extraction in the Department of Oral Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. Piezoelectric osteotomy was applied to acquire and pretreat the specimens, which were then packed individually with cryoprotectant and programmed cryopreserved in -80℃ refrigerator by slow-rate freezing. After registration of the necessary information, experimental bony tissue bank was set up. The cells migrated from the fragments which were subjected to a prescribed freezing period of 0, 1, 6, or 12 months were observed under inverted microscopy. Cell viability was evaluated by WST-8, and real-time PCR was used to detect the mRNA expression of Runx2, ALP and OCN. Alkaline phophatase and alizarin red staining were utilized to detect the osteogenic capacity in vitro. Cell senescence was evaluated by a histochemical staining with a Senescence Detection Kit and karyotyping was performed; cell apoptosis was also evaluated. Statistical analysis was performed using SPSS 17.0 software package. RESULTS: Cells could migrate from different fragments, proliferated well with no significant difference in cell senescence, cell apoptosis and osteogenic capacity in vitro (P>0.05). CONCLUSION: Cells derived from cryopreserved bone fragments could maintain good proliferation activity and osteogenic phenotype. It is feasible to utilize the cryopreserved bone to reconstruct bone defects.

Key words: Cryopreseration, Autogenous bone, Bony tissue bank, Bone tissue engineering

中图分类号:

R782.2

华洪飞, 张文杰, 张伟杰, 杨驰, 张志愿, 王绍义. 正常人颌骨组织冻存后活性评估及组织库的建立[J]. 中国口腔颌面外科杂志, 2017, 15(4): 314-319.

HUA Hong-fei, ZHANG Wen-jie, ZHANG Wei-jie, YANG-Chi, ZHANG Zhi-yuan, WANG Shao-yi. Evaluation of viability of cryopreserved human mandible and establishment of experimental bone bank[J]. China J Oral Maxillofac Surg, 2017, 15(4): 314-319.

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