玻璃化冻存组织工程骨修复兔骨缺损的实验研究  

Experimental study on vitrified frozen tissue engineering bone for repairing rabbit bone defects

作　　者：炕玉宾 韩昊天 崔磊 KANG Yu-bin;HAN Hao-tian;CUI Lei(Plastic Surgery Department,Beijing Shijitan Hospital,Capital Medical University,Beijing 100038,China)

机构地区：[1]首都医科大学附属北京世纪坛医院整形外科,北京100038

出　　处：《中国医药生物技术》2023年第4期311-318,共8页Chinese Medicinal Biotechnology

摘　　要：目的研究探讨日本大耳白兔脂肪干细胞与脱钙骨基质构筑的组织工程骨在长期玻璃化冻存后的活性和成骨能力,以及它们复苏后在兔桡骨骨缺损修复中的作用。方法分离脂肪干细胞,将其扩增至第3代后接种于猪松质骨来源的脱钙骨基质,经过1周的成骨诱导,将建立的组织工程骨置于装有玻璃化冻存液的2 ml冻存管内,置液氮环境中冻存。在冻存1周和12周时,取出组织工程骨,分别在复苏后的第1、4、7和11天,使用Hochest33258法检测细胞数量。复苏后的第1、4、7天,使用碱性磷酸酶(ALP)检验试剂盒评估ALP的活性,并使用RT-PCR反应来确认骨钙素(OCN)基因的表达量。建立16只日本大耳白兔的2 cm的桡骨中段缺损模型,随机分成4组:未经冰冻的组织工程骨组、空白对照组、经过12周冻存组织工程骨组和单独材料组。12周后进行X线观察、Micro-CT评价和HE染色组织学观察。结果经过Hochest33258测试,在冻存1周和12周之后,复苏后第1天的细胞数量分别达到了冻存前的74.4%和72.3%,第4~7天细胞数量达到最高峰,而第7~11天则进入了平台期。冻存1周或12周后,ALP表达量出现了轻微的下降,但随后又开始回升。冻存1周、12周复苏后第1天OCN表达量稍微降低,随后OCN表达量逐渐升高,第4~7天升高速率快。X线结果显示,冻存12周组与新鲜组织工程骨组都有连续性骨组织形成。Micro-CT结果显示,冻存组与新鲜组织工程骨组可见明显的骨质形成,连续性完整。冻存组与新鲜组织工程骨组在骨体积、骨小梁数量、骨密度上均无明显差异(P>0.05),但均明显高于对照组和单纯支架材料组(P<0.05)。HE染色结果显示,新鲜组织工程骨组和冻存12周组织工程骨组可见明显的连续的新生骨形成。结论长期冻存后的组织工程骨仍能保持足够的细胞数量,仍具备成骨能力和修复兔桡骨骨缺损的能力。Objective To investigate the viability and osteogenic ability of tissue-engineered bone constructed with adipose stem cells from Japanese big eared white rabbit and demineralized bone matrix after long-term vitrification and its role in the repair of radial bone defects in rabbits after re-warming.Methods Adipose-derived stem cells(ADSCs)were isolated and expanded to the third generation and seeded into demineralized bone matrix derived from porcine cancellous bone.After one week of osteogenic induction,the established tissue-engineered bone was placed in a 2 ml cryopreservation tube containing vitrification solution,and then frozen in liquid nitrogen environment.After cryopreservation for 1 and 12 weeks,the tissue-engineered bone was removed and the number of cells on it was examined in detail using the Hochest33258 method at 1,4,7,and 11 days after re-warming.At 1,4,and 7 days after re-warming,we assessed alkaline phosphatase(ALP)activity using an ALP assay kit and confirmed osteocalcin(OCN)gene expression using RT-PCR reactions.In this study,a 2 cm mid-radius defect was established in 16 Japanese white rabbits and they were randomly divided into four groups:unfrozen tissue-engineered bone group,blank control group,freeze-treated tissue-engineered bone group after 12 weeks,and material alone group.After 12 weeks,X-ray observation,Micro-CT evaluation and HE staining were performed.Results The results from Hochest 33258 test showed that after 1 week and 12 week of cryopreservation,the number of cells on the first day after cell resuscitation reached 74.4%and 72.3%of those before cryopreservation,respectively.In the following days,the growth trend of the number of cells gradually accelerated,reaching the peak on the 4th to 7th days,and entering a stable phase of proliferation on the 7th to 11th days.After 1 or 12 weeks,there was a slight decrease in ALP expression,but then it began to rise again.OCN expression decreased slightly on the first day after cryopreservation for 1 week and 12 weeks,and then gradually inc

关 键 词：组织工程骨 玻璃化冻存 骨缺损 脂肪间充质干细胞 

分 类 号：R68[医药卫生—骨科学] R318.08[医药卫生—外科学]