不同预湿方法对构建组织工程骨膜细胞黏附率的影响★

王军胜1，王栓科2，高恩建1，卢则陈1

课题背景：用骨膜修复骨缺损，已被大量实验所证实。但其供源有限，且由于抗原性问题，只能自体应用而不能用于异体，限制了临床大量应用。因此，用组织工程学的原理和方法构建人工骨膜，将为骨缺损的治疗提供一种全新的思路和方法。

应用要点：①为了便于骨髓间充质干细胞与小肠黏膜下层黏附，需要在复合后不加培养液的情况下静置4~6 h。在这段时间里，经胎牛血清预湿的小肠黏膜下层，细胞的黏附率明显增加，而且细胞更易伸展。②重悬细胞的培养液过多，细胞易于弥散至小肠黏膜下层以远，过少，又易引起细胞缺水和酸中毒。用恰到好处的培养液重悬细胞，无疑可提高接种效率。③材料的含水量要适当，在预湿2 h后，将培养板翻转，置孵箱5 h以上，使多余的液体积聚于翻转培养板的底面，既保证了适当的含水量，又避免了传统做法用吸水纸吸水的烦琐和增加污染危险。

同行评价：作者应用胎牛血清、磷酸盐缓冲液对的猪小肠黏膜下层组织预湿处理后复合细胞, 发现用胎牛血清预湿小肠黏膜下层,有利于提高接种效率,实验设计简单合理，数据分析可靠，对应用小肠黏膜下层组织构建组织工程组织有很好的帮助。�

摘要
目的：将经成骨诱导的兔骨髓来源的间充质干细胞与猪小肠黏膜下层复合，构建的组织工程骨膜,与天然骨膜在形态结构和功能上非常类似。实验着重观察不同预湿方法对构建的组织工程骨膜细胞黏附率的影响，为有效构建组织工程化骨膜奠定理论基础。
方法：实验于2005-09/2007-03在兰州大学附属第二医院骨科研究所完成。①实验材料：20 d的新西兰纯种兔2只。②实验过程及分组：取健康新西兰幼兔骨髓,密度梯度离心法分离骨髓间充质干细胞并进行体外扩增、诱导分化,然后与经过胎牛血清（实验组）、磷酸盐缓冲液（对照组）预湿处理的猪小肠黏膜下层复合构建组织工程骨膜，体外培养2周。③实验评估：计算贴附率、测定碱性磷酸酶活性,并用倒置显微镜、扫描电镜观察生长、分裂、增殖情况。
结果：①细胞贴附率：接种后5 h实验组细胞贴附率高于对照组[（57.34±2.23）%，（39.16±5.22）%，P < 0.01]；12 h实验组细胞贴附率高于对照组[（72.45±1.15）%，（54.47±3.46）%，P < 0.01]。②倒置显微镜观察：实验组比对照组更易黏附、伸展。③扫描电镜观察：复合培养第3天，实验组可见材料表面的细胞已完全伸展，附着在材料表面；对照组细胞大多呈不规则形，未完全伸展。培养7 d以后两组细胞差异不大。④碱性磷酸酶活性测定：复合培养第5，10，14天，实验组的碱性磷酸酶活性均明显高于对照组，差异有非常显著性（P < 0.01）。
结论：用胎牛血清预湿小肠黏膜下层,有利于提高接种效率,从而能更有效的构建组织工程骨膜。
关键词：组织工程骨膜；间质干细胞；小肠黏膜下层；组织构建

王军胜，王栓科，高恩建，卢则陈. 不同预湿方法对构建组织工程骨膜细胞黏附率的影响[J].中国组织工程研究与临床康复，2008，12(15):2806-2810 [www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2806(ps).pdf]

1淮安市第二人民医院骨科，江苏省淮安市 223002； 2 兰州大学第二附属医院骨科，甘肃省兰州市 730000

王军胜★，男，1974年生，甘肃省通渭县人，汉族，2007年兰州大学毕业，硕士，主治医师，主要从事骨组织工程研究。
nick121@sina.com

中图分类号:R318
文献标识码:A
文章编号:1673-8225
(2008)15-02806-05

收稿日期：2007-09-13 修回日期：2008-11-26 (07-50-9-5021/W·Y)

Effect of different wetting methods on cellular adhesion during tissue-engineered periosteum fabrication

Abstract

AIM：Tissue engineered periosteum fabricated by porcine small intestinal submucosa (SIS) combined with rabbit bone marrow-derived mesenchymal stem cells (BMSCs), are similar to the natural periosteum in the morphology, structure and function. The study was designed to explore the cell attachment in process of constructing tissue-engineered periosteum by different wetting methods to determine the most effective method of fabricating tissue-engineered periosteum.
METHODS: The experiment was carried out in the Institute of Orthopedics, the Second Affiliated Hospital of Lanzhou University from September 2005 to March 2007.①Two New Zealand purebred rabbits, 20 days old, were used in this study.②Bone marrow was obtained from healthy rabbits, and BMSCs were isolated by using density gradient centrifugation for culture in vitro. After induction, BMSCs were seeded to SIS wetted with fetal bovine serum (experimental group) and pretreated with phosphate buffered saline (control group), to construct the tissue-engineered periosteum that was cultured for two weeks in vitro. ③The adhesion rates were calculated, and alkaline phosphatase (ALP) activity was detected after seeding. The growth, dissociation and proliferation of the cells were observed by inverted microscope and scanning electron microscope.
RESULTS: ①At 5 hours and 12 hours after seeding, the adhesion rates were higher in the experimental group than in the control group [(57.34±2.23)%, (39.16±5.22)%, P < 0.01; (72.45±1.15)%, (54.47±3.46)%, P < 0.01].②The inverted microscope confirmed that cells in the experimental group were easier adhesive than ones in the control group.③Scanning electron microscope results showed that, cells in the experimental group spread completely and adhered on the material surface; most of the cells in the control group were irregular on the third day of seeding. No obvious differences were found after 7 days.④ALP activity of the BMSC-SIS complex in the experimental group were higher significantly than that in the control group (P < 0.01).
CONCLUSION: The SIS pretreated with fetal bovine serum can improve the seeding efficiency and facilitate the fabrication of tissue-engineered periosteum.

Wang JS, Wang SK, Gao EJ, Lu ZC. Effect of different wetting methods on cellular adhesion during tissue-engineered periosteum fabrication.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(15):2806-2810(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2806(ps).pdf]

1Department of Orthopedics, Second People's Hospital, Huaian 223002, Jiangsu Province, China; 2Department of Orthopedics, Second Affiliated Hospital of Lanzhou University, Lanzhou 730000, Gansu Province, China

Wang Jun-sheng★, Master, Attending physician, Department of Orthopedics, Second People's Hospital, Huaian 223002, Jiangsu Province, China
nick121@sina.com

Received: 2007-09-13
Accepted: 2007-11-26