活性维生素D促组织工程骨血管化

目的探讨以1,25-(OH)2D3为活性因子,与人骨髓间质成骨细胞(human marrow stromal osteoblast,hMSO)、脐静脉血管内皮细胞(human umbilical vein endothelial cell,hUVEC)、珊瑚羟基磷灰石人工骨(coral hydroxyapatite,CHA)联合构建组织工程骨的异位成骨能力和体内快速血管化能力。方法体外分离、培养hMSO和hUVEC。hMSO以5×105/ml,hUVEC以2.5×105/ml按2∶1比例接种至经1,25-(OH)2D3处理过的CHA上,为实验组;相同比例hMSO和hUVEC接种于单纯CHA作为对照组。体外培养3d后,18只裸鼠背部皮下其中6只双侧均植入实验组组织工程骨1块,6只双侧均植入对照组组织工程骨1块,余6只植入实验组及对照组组织工程骨每侧1块。术后4、8、12周后取材,行大体观察、常规组织学、扫描电镜观察,并行植入物成骨的定量判断和新生血管定量分析。结果组织学观察可见,4周时,各组原始类骨组织均长入支架材料内部,实验组有大量不成熟骨组织伴随众多微血管出现;8周,骨组织成熟与微血管相伴出现;12周,各组均有成熟骨组织出现,实验组有典型骨单位。对照组在各时间点微血管量均少于实验组。扫描电镜观察:4周,实验组大量细胞外基质将细胞包埋,材料表面可见大量微血管并有部分穿入材料内部;对照组虽然细胞外基质也较丰富,但微血管量少;8周,实验组部分材料表面出现了束状条索样的类骨质结构,细胞外基质丰富,可见大量微血管相伴出现;对照组微血管量少,骨组织成熟度亦差。植入物成骨的定量判断,8周实验组为3.10±0.52,对照组为2.30±0.59;12周实验组为4.63±0.55,对照组为3.53±0.62;两时间点两组比较差异均有统计学意义(P<0.05)。新生血管定量分析,4周实验组为28.74%±7.81%,对照组为19.52%±4.57%;8周实验组为24.66%±7.38%,对照组为17.84%±5.22%;两时间点两组比较差异均有统计学意义(P<0.05)。结论1,25-(OH)2D3作为活性因子通过加强hMSO与hUVEC之间的相互作用,增强了构建的组织工程骨的异位成骨能力和快速血管化能力。

A RESEARCH ON ECTOPIC OSTEOGENESIS AND VASCULARIZATION OF TISSUE ENGINEERED BONE PROMOTED BY 1,25-(OH)2D3

OBJECTIVE: To study the ectopic osteogenesis and vascularization of the tissue engineered bone promoted by an artificial bone composite that consists of coral hydroxyapatite (CHA), 1,25-(OH)2D3, human marrow stromal osteoblast (hMSO), and human umbilical vein endothelial cell (hUVEC). METHODS: After the isolation and the culture in vitro, hMSO and hUVEC were obtained. Then, hMSO (5 x 10(5)/ml) and hUVEC (2.5 x 10(5)/ml) were seeded at a ratio of 2 : 1 onto the CHA scaffolds coated with 1,25-(OH)2D3 (the experimental group) or onto the CHA scaffolds without 1,25-(OH)2D3 (the control group). The scaffolds were cultured in vitro for 3 days, and then the scaffolds were implanted into the pockets that had been made on the backs of 18 nude mice. Then, 6 of the mice were implanted with one experimental engineered bone bilaterally; another 6 mice were implanted with one control engineered bone bilaterally; the remaining 6 mice were implanted with one experimental engineered bone and one control engineered bone on each side. At 4, 8 and 12 weeks after operation, the retrieved scaffolds and cells were examined by the nake eye and histology as well as by the scanning electron microscopy. The quantitative assessment of the newly-formed bone and the quantitative analysis of the newly-formed blood vessels were performed. RESULTS: The evaluations by the histology revealed that at 4 weeks the original bone tissues grew into the scaffolds in all the groups, but significantly more newly-formed bone tissues and newly-formed blood vessels were found in the experimental group. At 12 weeks the newly-formed bone tissues were found in all the groups, but there was a typical bone unit found in the experimental group. There was a significantly smaller amount of capillary vessels in the control group than in the experimental group at all the time points. The evaluations by the scanning electron microscopy revealed that at 4 weeks in the experimental group there were great amounts of extracelluar matrix that embedded the cells, and plenty of capillary vessels were found on the surface of the implanted bone materials and some of them grew into the materials; however, in the control group there was a smaller amount of capillary vessels although much extracelluar matrix was still found there. At 8 weeks sarciniform osteoids were found on some of the implanted materials, with much extracelluar matrix and many newly-formed capillary vessels in the experimental group; however, in the control group there were fewer capillary vessels and lower degrees of the bone maturity. The quantitative assessment of the newly-formed bone showed that the new-formed bones were 3.1 +/- 0.52 in the experimental group but 2.30 +/- 0.59 in the control group at 8 weeks (P < 0.05), and 4.63 +/- 0.55 vs. 3.53 +/- 0.62 at 12 weeks. There was a significant difference at these two time points between the two groups (P < 0.05). The quantitative analysis of the newly-formed blood vessels showed that the vascular areas were 28.74% +/- 7.81% in the experimental group but 19.52% +/- 4.57% in the control group at 4 weeks (P < 0.05), and 24.66% +/- 7.38% vs. 17.84% +/- 5.22% at 12 weeks. There was a significant difference at these two time points between the two groups (P < 0.05). CONCLUSION: 1,25-(OH)2D3 as an active factor can increase the interaction between hMSO and hUVEC, and thus promote the ectopic osteogenesis and vascularization in the tissue engineered bone.