原代人脂肪基质细胞体内成骨能力的检测

目的:检测原代人脂肪基质细胞(human adipose-derived stromal sells,hASCs)的体内成骨能力,为应用其构建组织工程化骨奠定基础。方法:酶消化法分离原代hASCs,细胞经成骨向和成脂肪向诱导后,碱性磷酸酶(al-kaline phosphatase,ALP)染色和茜素红矿化染色检测其成骨向分化的能力,油红O染色检测其成脂肪向分化的能力。体内实验使用12只裸鼠,于其背部一侧皮下植入原代hASCs+支架材料作为实验组,同一裸鼠的另一侧植入单纯支架材料作为对照组。植入4周和8周后取材,制成组织切片,进行HE染色和免疫组织化学染色观察。结果:300 mL脂肪组织中可以获得约6×107个原代hASCs。体外实验证实原代hASCs具有成骨向和成脂肪向分化的潜能。HE染色可见,植入4周和8周后植入物中有类骨组织形成,免疫组织化学染色显示骨钙素、ALP和抗人核抗体呈阳性表达。结论:原代人脂肪基质细胞与支架材料构建的复合物可以在裸鼠体内成骨。

Osteogenic capability of primary human adipose-derived stromal cells in vivo

Objective: To investigate the osteogenic capability of primary human adipose-derived stromal cells(hASCs) in vivo.Methods: hASCs were isolated from adipose tissue by the method of collagenase digestion.After 7 and 14 days of osteogenic induction,alkaline phosphatase(ALP) staining and Alizarin Red staining were performed to test the osteogenic potential of hASCs in vitro.After 14 days of adipogenic induction,the adipogenic potential of hASCs was assayed by Oil Red O staining.In the in vivo part,12 nude mice were used.Test group(scaffold with hASCs) and control group(scaffold only) were symmetrically implanted into the back of nude mice.After 4 weeks and 8 weeks of implantation,samples were collected.Histological and immunohistochemical staining were performed to investigate the osteoge-nic capability of hASCs.Results: Approximately 6×107 hASCs could be isolated from 300 mL adipose tissue.ALP,Alizarin Red and Oil Red O staining of hASCs showed positive results after specific inductions.These results demonstrated the osteogenic and adipogenic potentials of hASCs in vitro.Bone-like tissue could be observed in the test group at 4 weeks and 8 weeks after the implantation.Immunohistochemical staining showed that there were positive results of osteocalcin,ALP and anti-human nuclei in the bone-like tissue areas.Conclusion: A large number of primary hASCs can be isolated from human adipose tissue;hASCs combined with scaffold show osteogenic capability in vivo.