成骨细胞、血管内皮细胞复合组织工程骨修复骨缺损的实验研究

Study on the treatment of bone defect with tissue engineering bone compounded by the osteoblast and the vascular endothelial cell

目的采用异种骨脱细胞基质（heterogenous bone acellular matrix，HBACM）复合细胞构建组织工程骨并植入动物体内，观察其对骨缺损的修复作用。方法培养与鉴定兔成骨细胞（osteoblast，0B）、血管内皮细胞（vascular endothelial cell，VEC）以及两种细胞的混合细胞。使用猪肋骨制备HBACM，并分别与0B、VEC以及两种细胞的混合细胞共同培养制备组织工程骨。27只新西兰大白兔随机分为三组，切除兔双前肢桡骨制成骨缺损动物模型，左侧植入HBACM与各组细胞复合培养的组织工程骨，右侧植入HBACM为对照。分别于术后3、6、12周取材，观察骨折愈合情况。结果大体形态观察和X线检查表明，组织工程骨修复骨缺损的速度和程度为：联合培养细胞组〉OB组〉VEC组〉单纯HBACM组。BrdU标记细胞示踪检测结果显示：6周时，HBACM上仍可检测到体外培养的标记细胞，并可见新生软骨形成。常规组织学检查可见12周时组织工程骨与正常骨之间的髓腔再通，骨折愈合。Ⅰ型胶原免疫组化研究发现三组均表达Ⅰ型胶原，VEC组略弱。组织切片图像分析示血管面积联合培养细胞组〉VEC组〉OB组，差异有统计学意义。结论联合培养细胞作为组织工程骨种子细胞可以缩短骨折愈合时间、促进骨缺损的修复。

Objective This study investigated delivery methods and the use of allograft cell therapy to enhance healing of a bone defect using beterogenic bone as a scaffold. Methods The osteoblasts （OB） and the vascular endothelial cells （VEC） were harvested from New Zealand white rabbits. The mononuclear cell fraction was isolated and culture expanded. Meanwhile, the OB and the VEC were co-cultured in vitro. Heterogenous bone acellular matrix （HBACM） was made from ribs of pig. The scaffold was coated with the OB （group OB）, coated with the VEC （group VEC） or loaded with both above cells （group MIX）. 27 radial defects were surgically made in 27 rabbits which were randomly divided into 3 groups. Left 1.5 cm defects in the radius were filled with these above bone graft substitutes. Right side acted as a control implanted with HBACM. To investigate bone healing, gross morphology, X-ray, the BrdU marked cells tracing, histological examination, type Ⅰ collagen immunohistocbemistry was performed 3, 6 and 12 weeks after surgery. Results Gross morphology and X-ray showed that there was more new bone in the MIX group compared to other groups. The BrdU-marked cell tracing revealed that the marked cells could still be tested in vivo after 6 weeks. The cartilaginous ossification was the major repairing of bone defect confirmed by histological section. Meanwhile type Ⅰ collagen were expressed in these groups. Histological image analysis identified the quantity of revascularization of tissue engineering bone was higher in the MIX group compared to other groups. Conclusion These observations suggest that the provision of OB and the VES is sufficient to enhance bone healing.