1，25-(OH)2VitD3促进组织工程骨的血管化和骨化☆
李 涛1，臧洪敏1，唐天驷2
课题背景：骨发育和骨折愈合是一个极其复杂的过程，血管化和骨形成是其中两个最基本环节，而且血管化的出现是早于骨形成的。这一复杂过程包含了数种细胞的迁移、增殖、分化、活化和这些细胞间的相互作用。本实验观察以1，25-(OH)2VitD3作为活性因子通过加强人骨髓间质成骨细胞、脐静脉内皮细胞间相互作用而加速血管化，进而促进组织工程骨的骨化。
应用要点：实验应用1，25-(OH)2 VitD3这一具有多重作用机制的细胞活性因子作用于成骨样细胞和血管内皮细胞两种种子细胞，分别通过1，25-(OH)2 VitD3对两种细胞单独的作用、对两种细胞间相互作用的加强、对各种生物活性因子作用的调节而发挥促进新生血管形成和骨形成的作用。减少了单一因子大剂量应用所带来的缺点。
偏倚或不足：作者因较长时间从事临床工作，对部分先进的仪器设备、检测方法了解不够，致使有部分可行的方法没有充分利用。如：更完善的免疫组织化学、免疫荧光技术，使珊瑚羟基磷灰石人工骨支架、成骨细胞、血管内皮细胞能以不同的颜色，显示其二维、三维情况下的共存情况及细胞功能；作为生物活性因子，1，25-(OH)2VitD3随细胞代谢的浓度变化情况；进一步研究该组织工程骨应用于临床的可行性。�
摘要
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-(OH)2VitD3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27~32 g，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5×108 L-1，脐静脉内皮细胞以2.5×108 L-1 按2∶1比例接种至经1，25-(OH)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3 d 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3 d 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周时，实验组部分材料表面出现了束状条索样的类骨质结构，可见大量微血管相伴出现。对照组微血管量少。新生血管定量分析显示，4周及8周时两组比较，差异显著 (P < 0.05)；植入物成骨的定量判断，8周及12周时两组比较差异均有显著性意义(P < 0.05)。
结论：1，25-(OH)2VitD3作为活性因子通过加强人骨髓间质成骨细胞与脐静脉内皮细胞之间的相互作用，增强了构建的组织
工程骨的快速血管化能力和异位成骨能力。
关键词：组织工程骨；人骨髓间质成骨细胞；脐静脉血管内皮细胞；异位成骨；血管化
李涛，臧洪敏，唐天驷.1，25-(OH)2VitD3促进组织工程骨的血管化和骨化[J].中国组织工程研究与临床康复，2008，12(11):2001-2005 [www.zglckf.com/zglckf/ejournal/upfiles/08-11/11k-2001(ps).pdf]
1淄博市中心医院骨科，山东省淄博市 255036；2苏州大学第一附属医院骨科，江苏省苏州市 215006
李 涛☆，男，1970年生，山东省淄博市人，汉族，2005年苏州大学毕业，博士，副主任医师，主要从事脊柱外科及组织工程的研究。
litaozhongguo@
163.com
中图分类号:R318
文献标识码:A
文章编号:1673-8225
(2008)11-02001-05
收稿日期：2007-08-08
修回日期：2007-11-02
(07-50-8-4289/WL·Q)
1，25-(OH)2VitD3 promotes osteogenesis and vascularization of tissue-engineered bone
Abstract
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 (5×108 L-1) and umbilical vein endothelial cells (2.5×108 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 tissue-engineered bone at one side. Nine in the control group were implanted with a tissue-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 eighth week, mature bone and conspicuous microvessel were found. In the twelfth week, mature bone appeared in either group. The amount of microvessel was less in the control group than the experimental group. Scanning electron microscope showed that a mass of extracellular matrix coated the cells in the experimental group, and a large quantity of microvessel was seen in the surface of the materials and some grew into the material in the fourth week; Extracellular matrix was rich in the control group, but the microvessel count was less. In the eighth week, fascicular cord-like osteoid was found in partial materials, with a large number of extracellular matrix and microvessel in the experimental group. Microvessel count and mature bone was less in the control group. Quantitative analysis of neovascularization showed that there were significant differences between the two groups in the fourth and eighth weeks (P < 0.05). There were significant differences in the implant osteogenesis between the two groups in the twelfth and eighth weeks (P < 0.05).
CONCLUSION: The 1，25-(OH)2VitD3 can improve the ability of ossification and vascularization of tissue-engineered bone by strengthening the association of human marrow stromal osteoblast and umbilical vein endothelial cells.
Li T, Zang HM, Tang TS.1，25-(OH)2VitD3 promotes osteogenesis and vascularization of tissue-engineered bone.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(11):2001-2005(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-11/11k-2001(ps).pdf]
1Department of Orthopaedics, Zibo Central Hospital, Zibo 255036, Shandong Province, China; 2Department of Orthopaedics, First Affiliated Hospital, Soochow University, Suzhou 215006, Jiangsu Province, China
Li Tao☆, Doctor, Associate chief physician, Department of Orthopaedics, Zibo Central Hospital, Zibo 255036, Shandong Province, China
litaozhongguo@163.com
Received: 2007-08-08
Accepted: 2007-11-02
Advertisement |
 |