中国组织工程研究

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (10): 1477-1482.doi: 10.3969/j.issn.2095-4344.0704

• 组织工程骨及软骨材料 tissue-engineered bone and cartilage materials •    下一篇

淫羊藿苷诱导骨膜细胞增殖构建组织工程骨修复骨缺损

钟秀霞，罗美兰
  

  1. 深圳龙华区人民医院，广东省深圳市  518109
  • 收稿日期:2017-11-14 出版日期:2018-04-08 发布日期:2018-04-08
  • 通讯作者: 罗美兰，护师，深圳龙华区人民医院，广东省深圳市 518109
  • 作者简介:钟秀霞，女，1986年生，广东省五华县人，汉族，2015年广州中医大学毕业，药师，主要从事药剂研究。
  • 基金资助:
    江西省科技计划项目(20161BBG02315)

Tissue-engineered bone constructed by icariin-induced periosteal cells repairs bone defects

Zhong Xiu-xia, Luo Mei-lan
  

  1. the People’s Hospital of Longhua of Shenzhen, Shenzhen 518109, Guangdong Province, China
  • Received:2017-11-14 Online:2018-04-08 Published:2018-04-08
  • Contact: Luo Mei-lan, Nurse, the People’s Hospital of Longhua of Shenzhen, Shenzhen 518109, Guangdong Province, China
  • About author:Zhong Xiu-xia, Pharmacist, the People’s Hospital of Longhua of Shenzhen, Shenzhen 518109, Guangdong Province, China
  • Supported by:
     the Science and Technology Program of Jiangxi Province, No. 20161BBG02315

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文题释义：
淫羊藿苷：淫羊藿苷主要为柔毛淫羊藿、朝鲜淫羊藿、箭叶淫羊藿和巫山淫羊藿巫等植物的提取物，相对分子质量为676.65，为结晶状淡黄色粉末，有增加心脑血管血流、促进骨髓造血、增强免疫力等功能。
聚己内酯三维支架：是一种细胞培养的支架，主要成分是聚己内酯，具有标准的有利于细胞增殖的三维结构，其三维构造是由纵横交错的纤维构造而成，孔径一般是300 μm或500 μm。可制成不同三维形态，可置于细胞培养板中并进行三维细胞培养。
 
摘要
背景：淫羊藿苷有明确的诱导骨髓基质干细胞分化作用，但是对骨膜细胞诱导并用于动物实验报道较少。聚己内酯三维支架是一种新型的细胞培养三维支架，有可降解、孔径合适、组织相容性好的特点。
目的：探讨淫羊藿苷对骨膜细胞的诱导增殖作用，构建骨膜细胞和三维支架复合体组织工程骨并研究其对兔桡骨缺损模型的修复作用，为骨缺损的治疗提供一种新的方式。
方法：取兔下颌骨骨膜，组织块法获得原代骨膜细胞后，传代培养第3代并种植于24孔板。实验组分别用10-4，10-3和10-2 mol/L的淫羊藿苷加入到相应培养孔中；对照组仅加入等体积的PBS；阳性对照组加入10 μg/L碱性成纤维细胞生长因子。于第2，4，6和8天用MTT法检测不同组细胞吸光度值。将聚己内酯三维支架置于细胞培养板中，加入10-2 mol/L的淫羊藿苷及107 L-1的骨膜细胞联合培养，构建骨膜细胞三维支架复合体。建立兔骨缺损模型后，随机分为3组，每组各15只。实验组植入淫羊藿苷诱导形成的骨膜细胞三维支架复合体，聚己内酯组仅植入聚己内酯三维支架，对照组不植入任何材料。分别在植入后第2，4和8周取兔桡骨缺损处行苏木精-伊红染色观察骨愈合情况及成骨细胞的数量；于植入后4周拍摄各组兔桡骨X射线片。
结果与结论：①与对照组相比，不同浓度的淫羊藿苷组在不同的时间点均显著促进骨膜细胞增殖(P < 0. 05)，尤其是10-2 mol/L淫羊藿苷组(P < 0.05)，提示淫羊藿苷的最佳浓度为10-2 mol/L；②植入后4周，实验组骨细胞数量多，微小血管相对少；聚己内酯组可见较多的新生血管，少量编织样的新生骨和骨细胞；对照组见较多肉芽组织，骨细胞数量较少；植入后8周，对照组骨端可见纤维瘢痕组织，髓腔封闭，形成骨不连；聚己内酯组，骨缺损部分修复；实验组骨缺损部位完全被新生骨所替代，骨缺损修复。实验组成骨细胞数量显著多于其他2组(P < 0.05)；③植入后4周X射线情况：实验组骨缺损处基本愈合；聚己内酯组骨痂已经形成，骨断端部分相连；对照组骨缺损间隙仍清晰存在；④结果提示，不同浓度的淫羊藿苷均可促进骨膜细胞增殖，以10-2 mol/L最为显著。淫羊藿苷可诱导骨膜细胞在聚己内酯三维支架中形成细胞支架复合组织工程骨，可有效修复兔骨缺损。

关键词: 淫羊藿苷, 骨膜细胞, 聚己内酯, 组织构建, 碱性成纤维细胞生长因子, 吸光度值, 骨愈合, 聚己内酯, 骨缺损

Abstract:

BACKGROUND: Although icariin has a clear role in inducing differentiation of bone marrow stromal cells, no studies have reported on its use in proliferation of periosteal cells in animal experiments. Three-dimensional polycaprolactone scaffold is a new scaffold, and it has the characteristics of biodegradability, proper pore size, and good histocompatibility.
OBJECTIVE: To investigate the effect of icariin on the proliferation of periosteal cells, to construct periosteal cells and three-dimensional scaffold composite tissue-engineered bone and study its therapeutic effect on radical defects in rabbits, so as to provide a new way for the treatment of bone defects .
METHODS: The mandibular periosteum was obtained from the rabbit and the primary cells were obtained by tissue explant method. The passage 3 cells were planted on 24-well plates. In experimental group, icariin at 10-4, 10-3 and 10-2 mol/L were added into the corresponding cell culture wells; equal volume of PBS was added to control group; positive control group was treated with 10 μg/L basic fibroblast growth factor. The cell proliferation in each group was measured by MTT method on the 2nd, 4th, 6th and 8th days, respectively. The three-dimensional polycaprolactone scaffolds, 10-2 mol/L icariin and 107/L periosteum cells were placed in cell culture plates and cultured in vitro to construct the cell-scaffold complex. After the rabbit radical defect model was established, the model rabbits were randomized into three groups (n=15 per group), followed by implanted with scaffold complex (experimental group), polycaprolactone scaffold (polycaprolactone group) or nothing (control group). Pathological sections were taken at the 2nd, 4th and 8th weeks respectively for hematoxylin-eosin staining to observe the bone and count the number of osteoblasts. The X-ray films of the radius were obtained at the 4th week.
RESULTS AND CONCLUSION: (1) The proliferation of periosteal cells induced by different concentrations of icariin, especially 10-2 mol/L icariin, was better than that in the control group (P < 0.05), Therefore, the optimal concentration of icariin was 10-2 mol/L. (2) At 4 weeks after transplantation, in the experimental group, there were more osterocytes and less microvessels; in the polycaprolactone group, there were more new vessels, less woven newly born bone and osterocytes; in the control group, the defect region was filled with more granulation tissue and less osterocytes. At the 8th week, complete healing in the defect region was observed in the cell-scaffold complex group, while partial healing in the polycaprolactone scaffold group. In the control group, however, fibroblasts and scar tissues were visible in the defect region, with presence of closed medullary cavity and nonunion. The number of osteocytes in the experimental group was significantly higher than that in the other two groups (P < 0.05). X-ray examination at the 4th week showed that the defect region in the experimental group achieved healing; in the polycaprolactone group, the callus formed and defect region connected partly; and there was a clear gap in the defect region of the control group. Our findings indicate that icariin at different concentrations can promote the proliferation of periosteal cells and the concentration of 10-2 mol/L has the greatest role. Icariin can induce proliferation of periosteal cells in the three-dimensional polycaprolactone scaffold, which contributes to bone repair.

Key words: Phytoestrogens, Cell Proliferation, Tissue Scaffolds, Tissue Engineering

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