1，25-（OH）2VitD32促进组织工程骨的血管化和骨化

目的：观察以1，25．（OH）2VitD3为活性因子，与人骨髓间质成骨细胞、脐静脉血管内皮细胞、珊瑚羟基磷灰石人工骨联合构建组织工程骨时，1，25-（OHhVitD3在三维支架上对成骨细胞的作用及构建组织工程骨的体内快速血管化能力和异位成骨能力。方法：（1）实验材料及对象：实验于2005—09／2006—03在苏州大学细胞与基因教研室完成。取SPF级6—8周龄BALB／cnu雄性裸鼠18只，体质量27～32g，骨髓来源于健康成人（志愿者）、新生儿脐带取材时经产妇同意。（2）实验方法及评估：①体外实验：体外分离、培养人骨髓间质成骨细胞和脐静脉内皮细胞。人骨髓间质成骨细胞以5×10^8 L^-1，脐静脉内皮细胞以2．5×10^8 L^-1按2：1比例接种至经1，25-（0H）2VitD3处理过的珊瑚羟基磷灰石人工骨上，作为实验组；相同比例接种于单纯珊瑚羟基磷灰石人工骨上为对照组。体外培养3d后，检测骨钙素含量及碱性磷酸酶活性并始终行光学显微镜观察。②动物实验：18只裸鼠随机摸球法分为两组，9只植入实验组组织工程骨每侧1块，另9只植入对照组组织工程骨每侧1块。术后4，8，12周后取材，行常规组织学、扫描电镜观察，并行植入物新生血管定量分析和成骨的定量判断。结果：纳入裸鼠18只，均进入结果分析。①倒置显微镜下珊瑚羟基磷灰石人工骨周边人骨髓间质成骨细胞、脐静脉内皮细胞均良好生长。组织工程骨构建3d后，骨钙素含量和碱性磷酸酶活性实验组明显高于对照组（P〈0．05）。②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。8周时，骨组织成熟与微血管相伴出现。12周时，各组均有成熟骨组织出现。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周时，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

1, 25-（OH）2VitD3 promotes osteogenesis and vascularization of tissue-engineered bone

AIM： To study the osteogenesis and vascularization of artificial bone composite of coral hydroxyapatite, 1,25-（OH）2VitD3, human marrow stromal osteoblast and umbilical vein endothelial cells. METHODS： ①Experiments were performed at the Department of Cell and Gene of Soochow University from September 2005 to March 2006. Eighteen BALB/cnu male SPF nude mice aged 6-8 weeks weighting 27-32 g were selected. Bone marrow was collected from healthy voluntary adults. Umbilical cords in neonates were collected with the agreement of the parturiens.②Human marrow stromal osteoblast and umbilical vein endothelial cells were harvested in vitro. Human marrow stromal osteoblast （5×10^8 L^-1） and umbilical vein endothelial cells （2.5×10^8 L^-1） were incubated at the ratio of 2 ： 1 in coral hydroxyapatite artificial bone that was coated with 1, 25-（OH）2VitD3 as an experimental group. Those were incubated in only coral hydroxyapatite artificial bone as a control group. Three days later, osteocalcin levels and alkaline phosphatase activities were determined under a light microscopy. Eighteen nude mice were randomly assigned into two groups. Nine in the experimental group were implanted with a tissne-engineered bone at one side. Nine in the control group were implanted with a tissne-engineered bone at one side. 4, 8 and 12 weeks after the surgery, the retrieved scaffolds and cells were examined histologically and by a scanning electron microscopy, and the vascular area and the new-formed bone area were measured quantitatively.
RESULTS： Eighteen nude mice were involved in the result analysis. ①Human marrow stromal osteoblast and umbilical vein endothelial cells grew well around coral hydroxyapatite artificial bone under a invert microscope. Three days later, osteocalcin levels and alkaline phosphatase activities were higher in the experimental group than the control group （P 〈 0.05）. ②Histological observation showed that in the fourth week; Primitive bone tissue grew into the stent. In the eigh