成人骨髓源成骨细胞体内异位成骨的实验研究

Experimental study on the ectopia osteogenesised by adult human osteoblasts derived from bone marrow stromal cells in vivo

目的 观察成人骨髓源成骨细胞与珊瑚羟基磷灰石 (CHA)复合构建的组织工程化骨组织的体内异位成骨能力 ,探讨适宜的组织工程化骨组织的构建方式。 方法 抽取健康成人骨髓 ,采用全骨髓法培养 ,使成人骨髓基质干细胞 (hBMSCs)定向诱导分化为成骨细胞 ,然后种植于CHA上 ,复合培养 5d后植入裸鼠股部肌袋内 ,以未种植细胞的CHA作为对照。术后 4,8,12周取材作一般观察、X线摄片、组织学检查和源于成人的碱性磷酸酶 (ALP)及骨钙素 (OCN)的RT PCR检测。 结果 原代和传代培养的细胞具有活跃的增殖能力 ,成骨细胞与CHA复合生长良好。实验组术后 4、8、12周均有新骨形成 ,随着时间延长 ,新骨生成量增多 ;对照组则均无新骨形成。术后 4周实验组RT PCR检测源于人的ALP及OCN表达均为阳性 ,对照组阴性。 结论 成人骨髓源成骨细胞与CHA复合培养后构建的组织工程化骨组织 ,具有良好的异位成骨能力 ,可望应用于临床修复骨缺损。

Objective To explore the appropriate mode of fabricating tissue engineered bone, the ectopia osteogenesised of the tissue engineered bone fabricated by osteoblasts which derived from adult human bone marrow stromal cells(hBMSCs) co cultured with coral hydroxyapatite(CHA) was observed. Methods Bone marrow was aspirated from the healthy adult human and culture expanded in vitro by the method of total marrow. hBMSCs were induced to differentiate into osteoblasts. Then these differentiated osteoblasts were seeded onto CHA, co cultured for 5 days, the compounds with cells and CHA were implanted into musclar pouch of thigh of nude mouse and CHA alone was implanted as a control. The effectiveness of bone formation was assessed separately by means of gross observation, roentgenography, histology after implantation for 4,8,12 weeks. Human Alkaline phosphatase(ALP) and osteocalcin(OCN)of the compound were detected by RT PCR at the end of the 4th week. Results hBMSCs proliferated actively in primary and passage culture. These adult human osteoblasts derived from bone marrow could grow as well on CHA as normal cultured cells: New osteogenesis was detected at the end of the 4th, 8th and 12th week after implantation separately in experimental group. With time going by, the new bone formation increased. ALP and OCN derived from human by RT PCR were positively expressed, while no new bone formation was found in each of the control group. Conclusions The tissue engineered bone fabricated by osteoblasts which derived from hBMSCs cultured onto CHA have ablilities of ectopia osteogenesis in vivo. It is supposed to be a good way to repair clinical bone defect. [Key words] Tissue engineering; Bone marrow stromal cells; Cell culture; Osteoblast; Coral hydroxyapatite