双相接种技术构建组织工程骨的异位成骨观察

Ectopic bone formation of tissue engineering bone constructed with diphasic seeding technique

目的评价双相接种技术体外构建的组织工程骨在体内的成骨潜力和这种体外构建策略的可行性。方法8只山羊抽取自体骨髓体外扩增、定向成骨诱导,再分别采用静置接种法和双相接种法将羊骨髓间充质干细胞(m esen-chym al stem cells,MSCs)与脱钙骨基质(dem ineralized bone m atrix,DBM)复合,复合培养5 d;将这两种方法构建的组织工程骨分别植入羊的背部肌袋内,同时植入单纯DBM作为阴性对照;术后2、8周取标本,进行大体观察、组织学染色检查和免疫组织化学检测。结果双相接种技术体外复合MSCs与DBM构建组织工程骨,细胞在支架上体外生长情况优于普通方法,植入山羊肌肉内术后2周观察到异位成骨,并随时间延长成骨量增加,其组织学半定量评分术后2周为(2.46±0.32)、8周为(4.13±0.42),明显优于普通方法的术后2周(1.96±0.21)、8周(3.66±0.37)(P<0.01)。植入后的组织工程骨材料边缘均可检测到B rdU标记阳性的细胞。单纯DBM植入未见成骨表现,亦无B rdU标记阳性的细胞。结论双相接种技术有利于组织工程骨在体内的成骨,是一种值得推广的组织工程骨体外构建技术。

Objective To explore the potential of ectopia osteogeny by tissue engineering bone fabricated by diphasic seeding technique and its feasibility. Methods Mesenchymal stem cells （MSCs） were drawn from 8 goat bone marrow, amplified and induced to differentiated into osteoblasts, then seeded onto demineralized bone matrix （DBM） by normal seeding technique and diphasic seeding technique respectively, and co-cultured for 5 d in vitro. The two kinds of tissue engineering bone and DBM as negative control were implanted into musclar pouch in goat back separately. At week 2 and 8 postoperatively, the effectiveness of bone formation was assessed by means of gross, histological and immunohistologial observations. Results MSCs grew better in DBM network fabricated by diphasic seeding technique than by normal seeding technique. In 2 weeks after implantation new osteogenesis was detected in MSCs-DBM groups and increased in a time-dependent manner. The tissue score of osteogenesis in diphasic seeding group was （2.46±0.32） in 2 weeks and （4.13 ±0.42） in 8 weeks, which was higher than in normal seeding group [ （ 1.96±0.21 ） in 2 weeks, （3.66±0.37） at 8 weeks] （P 〈 0. 01 ）. The cells marked by BrdU were observed in tissue engineering bone, while none was detected in DBM alone group in which there was no new bone formation. Conclusion Diphasic seeding technique is more favorable than normal seeding method, which can facilitated the bone formation by tissue engineering bone in rive.