载辛伐他汀β-磷酸三钙支架复合脂肪干细胞修复兔颅骨缺损

上海口腔医学 ›› 2013, Vol. 22 ›› Issue (4): 361-367.

• 基础研究 • 下一篇

载辛伐他汀β-磷酸三钙支架复合脂肪干细胞修复兔颅骨缺损

徐恋祎¹，孙小娟²，张秀丽¹，金玉琴¹，吴玉琼¹，蒋欣泉¹

(1.上海交通大学医学院附属第九人民医院?口腔医学院口腔修复科，口腔生物工程/再生医学实验室，上海市口腔医学重点实验室，上海 200011；2.宁夏医科大学总医院口腔颌面外科，宁夏银川 750004)

收稿日期:2012-12-07 修回日期:2013-01-08 出版日期:2013-08-10 发布日期:2013-08-10
通讯作者: 蒋欣泉，Tel:021-23271132,Fax:021-63136856,E-mail: xinquanj@yahoo.cn
作者简介:徐恋祎(1985-),女,硕士,住院医师,E-mail:xulianyi.xly@gmail.com
基金资助:
国家自然科学基金(81170939)；国家重大科学研究计划“973”课题(2012CB933600)

Repair of calvarial defect using a tissue-engineered bone with simvastatin-loaded β-tricalcium phosphate scaffold and adipose derived stem cells in rabbits

XU Lian-yi¹，SUN Xiao-juan²，ZHANG Xiu-li¹，JIN Yu-qin¹， WU Yu-qiong¹, JIANG Xin-quan¹

1.Department of Prosthodontics, Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine; Oral Bioengineering Lab/Oral Tissue Engineering Lab, Shanghai Key Laboratory of Stomatology.Shanghai 200011;2. Department of Oral and Maxillofacial Surgery, General Hospital of Ningxia Medical University. Yinchuan 750004, Ningxia Province, China

Received:2012-12-07 Revised:2013-01-08 Online:2013-08-10 Published:2013-08-10
Supported by:
Supported by National Natural Science Foundation of China (81170939) and National Basic Research Program (973 Program) (2012CB933600).

摘要/Abstract

摘要： 目的：应用辛伐他汀(simvastatin)作用于脂肪干细胞，研究其对细胞生长、成骨、成血管分化的影响；利用β-磷酸三钙(β-tricalcium phosphate，β-TCP)三维支架材料负载辛伐他汀，与脂肪干细胞复合，构建组织工程骨，用于兔颅骨缺损模型的修复。方法：原代培养兔脂肪干细胞(rabbit adipose-derived stem cells, rASCs)，分别以含0、0.01、0.1和1 μmol/L辛伐他汀的培养液培养，计数法检测细胞数目；以0、0.05、0.1 μmol/L浓度辛伐他汀培养ASCs，1、7 d后，实时定量PCR检测成骨、成血管基因的表达(RUNX2、OPN、OCN、VEGF)； 7 d后行ALP染色；14 d后行茜素红染色。12只新西兰大白兔颅顶双侧8 mm缺损，分别以4组材料修复(A: β-TCP，B: β-TCP/Cell，C: β-TCP/Sim，D: β-TCP/Cell/Sim)，每组6例，植入8 周后取材，进行组织学观察。采用SPSS17.0软件包对数据进行统计学分析。结果：0.05 μmol/L辛伐他汀对脂肪干细胞RUNX2、OCN、OPN和VEGF等成骨、成血管基因的表达具有明显促进作用，碱性磷酸酶染色和von Kossa染色更为明显；植入体内8周后，β-TCP/Cell/Sim组材料的成骨面积显著大于其他3组。结论：0.05 μmol/L辛伐他汀在体外对ASCs具有明显的促成骨作用，载辛伐他汀β-TCP复合脂肪干细胞可促进兔颅骨缺损的修复。

关键词: 脂肪干细胞, 辛伐他汀, β-磷酸三钙, 组织工程骨, 兔

Abstract: PURPOSE: The osteogenic-angiogenic differentiation effects of simvastatin (Sim) were explored on adipose tissue-derived stem cells (ASCs). A tissue-engineered bone with simvastatin loaded β-tricalcium phosphate (β-TCP) scaffold and ASCs was constructed to repair the calvarial defect in rabbits. METHODS：ASCs were obtained from the groin of rabbits. After 14 days of osteogenic inducing culture, sufficient cells were expanded for the following experiments. Cell counting was conducted to ASCs in osteogenic inducing medium containing 0, 0.01, 0.1 and 1 μmol/L simvastatin. Concentrations of 0.05 and 0.1 μmol/L simvastatin were administrated to ASCs for real-time PCR of angiogenesis-osteogenesis related genes like RUNX2, OPN, OCN, and VEGF on day 1, 7. ALP staining was performed on day 7, Alizarin red staining for calcium deposits was carried out on day 14. Bilateral critical-sized defects were created on 12 New Zealand rabbits. Four groups of tissue-engineered bone were randomly allocated to them. Group A: β-tricalcium phosphate (β-TCP) (n=6); group B: β-TCP/Cell (n=6); group C: β-TCP/Sim (n=6); group D: β-TCP/Cell/Sim (n=6). Specimens were decalcified and stained by HE 8 weeks after operation. The data was statistically analyzed using SPSS 17.0 software package. RESULTS: The use of simvastatin with the concentration of 0.05 μmol/L enhanced the expression of angiogenic-osteogenic related genes like RUNX2, OPN, OCN, and VEGF. ALP activity and von Kossa were significantly stronger in osteogenic inducing medium containing 0.05 μmol/L simvastatin. The new bone formation area of β-TCP/Cell/Sim group at 8-week after implantation was significantly larger than the other groups. CONCLUSIONS: 0.05 μmol/L simvastatin enhances the angiogenic-osteogenic differentiation of ASCs. Simvastatin loaded β-TCP scaffold and ASCs successfully repair the calvarial defect in rabbits. These results indicate a promising future in application of simvastatin for bone regeneration.

Key words: Adipose-derived stem cells, Simvastatin, β-tricalcium phosphate, Tissue-engineered bone, Rabbit

中图分类号:

R318.08

徐恋祎, 孙小娟, 张秀丽, 金玉琴, 吴玉琼, 蒋欣泉. 载辛伐他汀β-磷酸三钙支架复合脂肪干细胞修复兔颅骨缺损[J]. 上海口腔医学, 2013, 22(4): 361-367.

XU Lian-yi, SUN Xiao-juan, ZHANG Xiu-li, JIN Yu-qin, WU Yu-qiong, JIANG Xin-quan. Repair of calvarial defect using a tissue-engineered bone with simvastatin-loaded β-tricalcium phosphate scaffold and adipose derived stem cells in rabbits[J]. Shanghai Journal of Stomatology, 2013, 22(4): 361-367.

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载辛伐他汀β-磷酸三钙支架复合脂肪干细胞修复兔颅骨缺损

Repair of calvarial defect using a tissue-engineered bone with simvastatin-loaded β-tricalcium phosphate scaffold and adipose derived stem cells in rabbits

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