课题背景：以往的骨组织工程大多应用骨髓间充质干细胞，近年来因获取和培养扩增脂肪干细胞比骨髓间充质干细胞具有优势，所以本实验选择脂肪干细胞作为种子细胞,定向诱导其向骨细胞分化,并且体外诱导后移植到体内，试图观察多因素干预对骨缺损的效果。

应用要点：①通过全骨髓贴壁法分离纯化骨髓间充质干细胞，获取了较量多数量的细胞，并经过表皮生长因子和碱性成纤维细胞生长因子诱导后，细胞Nestin、NSE、GFAP表达阳性，表明在适当的条件下骨髓间充质干细胞可以诱导分化出神经干细胞。②为了解决神经干细胞在实验研究中的示踪问题，采用BrdU对神经干细胞进行体外和体内标记，1周后其标记率高达85%以上，说明BrdU标记和检测的准确性高、标记率高，是跟踪监测神经干细胞移植的理想标记物。

相关链接：对神经干细胞的研究近年来许多实验均致力于神经干细胞来源的问题，以期获得一个可靠的获取大量纯化神经干细胞的方法，其中最受关注的是骨髓间充质干细胞，骨髓间充质干细胞可以来源于自体,并且获取简单，从而克服了许多异体神经干细胞移植的弊端，为神经干细胞治疗带来新的希望。但是要将骨髓间充质干细胞真正应用于临床,仍然存在一些问题：①需要进一步检测从骨髓间充质干细胞分化而来的神经干细胞是否具有神经干细胞的一系列功能。②需要深入了解调控骨髓间充质干细胞向神经干细胞转化的分子机制等。

摘要
目的：探索兔自体脂肪干细胞、成骨诱导后的脂肪干细胞联合骨形态发生蛋白-2/纤维蛋白胶（bone morphogenetic protein-2/ Fibrin glue，BMP-2/ FG）经皮移植到骨缺损后体内成骨能力的差异。
方法：实验于2006-08/2007-11在北京军区总医院全军骨科研究所完成。①实验材料：清洁级日本大耳白兔，2.0~2.5 kg,雌雄不限，由北京维通利华实验动物技术有限公司提供，实验过程中对动物处置符合动物伦理学标准。②实验方法：制作新西兰大白兔桡骨中段15 mm骨缺损模型35侧，随机分为5组，每组7侧：未诱导细胞组、诱导细胞组、未诱导细胞+BMP-2/FG组、诱导细胞+ BMP-2/FG组、空白对照组。未诱导细胞组、诱导细胞组直接用生理盐水重悬未诱导脂肪干细胞和经成骨诱导的脂肪干细胞，未诱导细胞+BMP-2/FG、诱导细胞+ BMP-2/FG组分别用含20 g/L 骨形态发生蛋白-2、5 g/L纤维蛋白原的生理盐水重悬未诱导和经成骨诱导的脂肪干细胞。③实验评估: X射线片观察各组骨缺损修复情况，并进行生物力学检测。
结果：①12周时，未诱导细胞+BMP-2/FG组、诱导细胞+ BMP-2/FG组在骨缺损区新生骨的数量明显优于未诱导细胞组、诱导细胞组(P均 < 0.05)。②术后4周X射线显示，未诱导细胞+BMP-2/FG组、诱导细胞+ BMP-2/FG组阻射密度值高于其他组(P均 < 0.05)，未诱导细胞组与诱导细胞组无差别。术后8，12周时，诱导细胞组高于未诱导细胞组，未诱导细胞+BMP-2/FG、诱导细胞+ BMP-2/FG组均高于其他组，诱导细胞+ BMP-2/FG组高于未诱导细胞+BMP-2/FG组(P均 < 0.05)。③生物力学检测显示，未诱导细胞+BMP-2/FG组、诱导细胞+ BMP-2/FG组桡骨标本的四点弯曲断裂载荷均明显高于其他组，并且诱导细胞+ BMP-2/FG组高于未诱导细 胞+BMP-2/FG组(P均 < 0.05)。未诱导细胞组与诱导细胞组无差别。
结论: 成骨诱导脂肪干细胞及联合BMP-2/FG移植后8周和12周时具有更强的成骨能力，可作为种子细胞应用于骨组织工程。
关键词：脂肪干细胞；骨形态发生蛋白；细胞培养；细胞移植；经皮注射；纤维蛋白胶

马舟涌，李放，雷伟，陈晓斌，常玉立，苏强，余兆仲.兔自体脂肪干细胞成骨诱导后经皮注射修复骨缺损[J].中国组织工程研究与临床康复，2008，12(12):2245-2249 [www.zglckf.com/zglckf/ejournal/upfiles/08-12k/12k-2245(ps).pdf]

Percutaneous autologous adipose derived stem cell transplantation for bone defect after osteogenic induction in rabbits

Abstract

AIM：To explore in vivo osteogenic potential in size-critical bone defect after percutaneous autologous transplantation of culture-expanded rabbit autologous adipose derived stem cells (ADSCs) combined with or without those induced by osteogenic supplement (OS) and with or without bone morphogenetic protein-2/Fibrin glue (BMP-2/FG).

METHODS: Experiments were performed at the Institute of Department of Orthopaedics, General Hospital of Beijing Military Area Command of Chinese PLA from August 2006 to November 2007. ①Japanese big ear rabbits (2.0-2.5 kg), irrespective of gender, were provided by Vital River Lab Animal Technology Co., Ltd. (Beijing, China). Animal intervention met the animal ethical standards. ②The 15 mm bone defect experimental models at both radius of New Zealand rabbits were made (35 sides). Rabbit models were divided into 5 groups at random with 7 experimental models per group: Group A: ADSCs, Group B: ADSCs induced by OS, Group C: ADSCs non-induced by OS+BMP-2/FG, Group D: ADSCs induced by OS + BMP-2/FG, Group E: blank control. Rabbit models in the Group A and Group B were treated with saline. Rabbit models in the Group C and Group D received saline containing 20 g/L BMP-2/5 g/L FG. ③Osteogenesis at the defect area was observed by regular radiography and biomechanics examination was performed.

RESULTS: ①A large quantity of callus was found in the Group D and Group C while the others show little callus after cell transplantation at the 12th week (P <0.05). ②The value of X-ray analysis: 4 weeks after operation, the value of Group C and Group D was increased as compared with group A and Group B (P < 0.05). The value of Group A had no difference, as compared with that of Group B. Eight and twelve weeks after operation, the value of Group B was higher than Group A, while the value of Group C and Group D was increased as compared with Group A and Group B respectively. The value of Group D was higher than Group C (P < 0.05). ③The maximal load of Group C and Group D were better than the other groups, and the maximal load of Group D were higher than Group C (P < 0.05). The maximal load of Group A and Group B had no difference.

CONCLUSION: The osteo-induced ADSCs combined with BMP-2/FG after transplanting 8 and 12 weeks have excellent osteogenic potential in vivo. ADSCs as a seed cell is beneficial to bone tissue engineering.

Ma ZY, Li F, Lei W, Chen XB, Chang YL, Su Q, Yu ZZ.Percutaneous autologous adipose derived stem cell transplantation for bone defect after osteogenic induction in rabbits.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(12):2245-2249(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2245(ps).pdf]