中国组织工程研究

中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (34): 5437-5442.doi: 10.3969/j.issn.2095-4344.2017.34.005

• 组织工程骨及软骨材料 tissue-engineered bone and cartilage materials • 上一篇    下一篇

加载骨形态发生蛋白2的新型多组分组织工程骨支架材料

孙官文，王  剑，史  斌，贾  鹏
  

  1. 内蒙古自治区人民医院创伤骨科，内蒙古自治区呼和浩特市  010017
  • 收稿日期:2017-10-14 出版日期:2017-12-08 发布日期:2018-01-04
  • 通讯作者: 孙官文， 博士，主任医师，内蒙古自治区人民医院创伤骨科，内蒙古自治区呼和浩特市 010017
  • 作者简介:孙官文，男，1973年生，内蒙古自治区乌兰察布市察右前旗人，蒙古族，2015年武汉大学毕业，博士，主任医师，主要从事骨与软组织缺损的组织工程修复的相关研究。
  • 基金资助:
    内蒙古自治区自然科学基金项目(2015MS0383)

A novel tissue-engineered bone scaffold carrying bone morphogenetic protein 2

Sun Guan-wen, Wang Jian, Shi Bin, Jia Peng
  

  1. Department of Traumatic Orthopedics, Inner Mongolia People’s Hospital, Hohhot 010017, Inner Mongolia Autonomous Region, China
  • Received:2017-10-14 Online:2017-12-08 Published:2018-01-04
  • Contact: Sun Guan-wen, Department of Traumatic Orthopedics, Inner Mongolia People’s Hospital, Hohhot 010017, Inner Mongolia Autonomous Region, China
  • About author:Sun Guan-wen, M.D., Chief physician, Department of Traumatic Orthopedics, Inner Mongolia People’s Hospital, Hohhot 010017, Inner Mongolia Autonomous Region, China
  • Supported by:
    the Natural Science Foundation of Inner Mongolia Autonomous Region, No. 2015MS0383

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被引次数

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摘要：

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文题释义：
骨形态发生蛋白2：是转化生长因子β超家族成员之一，具有诱导未分化间充质干细胞向成软骨细胞和成骨细胞定向分化与增殖能力，促进成骨细胞分化成熟，参与骨和软骨生长发育及其重建过程，进而加速骨缺损修复，在体内以前体形式合成。
 
背景：实验证实在聚合物支架材料中加入羟基磷灰石，可具有良好的生物相容性。壳聚糖已与其他材料，如羟基磷灰石、透明质酸、藻酸盐和潜在的生长因子复合并应用于组织工程。同时大量研究证实骨形态发生蛋白2 (bone morphogenetic protein-2，BMP-2)能促进成骨细胞的生长和诱导体外和体内成骨。那么，是否能将上述4种材料成分结合构建一种新型的组织工程支架呢？
目的：制备新型加载BMP-2的聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid)，PLGA)、羟基磷灰石和不同浓度壳聚糖的组织工程骨支架材料，并观察其结构、亲水性、对成骨细胞的黏附作用以及壳聚糖的最佳修饰浓度。
方法：①利用固体-液相分离技术，制备包含PLGA、羟基磷灰石和BMP-2的组织工程骨支架材料，然后利用壳聚糖(0.25%、0.5%和1%)对支架材料进行改性修饰。作为对照，制备单纯PLGA/羟基磷灰石支架和PLGA /羟基磷灰石/BMP-2支架。扫描电镜下观察各支架的空间结构。检测各支架的亲水性，及PLGA/羟基磷灰石/ BMP-2/壳聚糖支架的BMP-2释放率；②取前成骨细胞悬液分别接种在各支架上，分别于细胞培养1，4 和7 d时，采用CCK-8法测定前成骨细胞在各支架上的黏附和增殖；细胞培养7 d时进行荧光素二乙酸酯示踪；细胞培养4，7和14 d时检测碱性磷酸酶活性。
结果与结论：①各支架在外观上均呈白色烧杯状，扫描电镜下材料呈多孔态，孔径约100 µm，孔隙间相互沟通；②支架材料的亲水性随着壳聚糖浓度增加而增强；③在第60天时，PLGA /羟基磷灰石/BMP-2支架，PLGA/羟基磷灰石/BMP-2/壳聚糖(0.25%，0.5%，1%)支架BMP-2累积释放分别为44%、34%、27%和26%，提示壳聚糖能有效抑制BMP-2的快速释放；④在第7天时，前成骨细胞在PLGA/羟基磷灰石/BMP-2/壳聚糖(0.25%)支架的活性最高(P < 0.05)。另外，PLGA/羟基磷灰石/BMP-2/壳聚糖(0.5%或1%)支架的细胞活性也高于无壳聚糖支架。经荧光素二乙酸酯染色，在共聚焦激光显微镜下观察，活细胞呈绿色荧光，均匀分布于支架上；⑤PLGA/羟基磷灰石/BMP-2/壳聚糖(0.25%)支架上的细胞中，碱性磷酸酶活性最高，其次是羟基磷灰石/BMP-2/壳聚糖(0.5%和1%)支架，PLGA/羟基磷灰石/BMP-2支架，在PLGA/羟基磷灰石支架上最低   (P < 0.05)；⑥结果提示，PLGA/羟基磷灰石/BMP-2/壳聚糖复合支架可以释放BMP-2，促进成骨细胞黏附、分化和增殖，优化了组织工程人工骨，且壳聚糖的最佳修饰浓度为0.25%。

关键词: 生物材料, 骨生物材料, 聚乳酸-羟基乙酸共聚物, 羟基磷灰石, 骨形态发生蛋白2, 壳聚糖, 支架, 骨组织工程, 前成骨细胞, 亲水性, 细胞黏附, 细胞增殖, 碱性磷酸酶, 细胞活性, 内蒙古自治区自然科学基金

Abstract:

BACKGROUND: It has been confirmed that a polymer scaffold with hydroxyapatite (HA) has good biocompatibility. Chitosan that is combined with other materials, such as HA, hyaluronic acid, alginate, and potential growth factors, can be applied in tissue engineering field. Meanwhile, numerous studies have confirmed that bone morphogenetic protein-2 (BMP-2) can promote the growth of osteoblasts and induce osteogenesis in vitro and in vivo. So, can we prepare a new tissue-engineered scaffold with these four kinds of materials?

OBJECTIVE: To prepare a novel BMP-2-loaded tissue-engineered bone scaffold using poly(lactic-co-glycolic acid) (PLGA), HA and different concentrations of chitosan, and to observe the scaffold structure, hydrophilicity, and adherence to osteoblasts as well as the optimal modification concentration of chitosan.

METHODS: (1) A tissue-engineered scaffold containing PLGA, HA and BMP-2 was prepared using the solid-liquid phase separation and modified by chitosan (0.25%, 0.5% and 1%). Additionally, PLGA/HA and PLGA/HA/BMP-2 scaffolds were prepared as controls. Scaffold structure was observed under a scanning electron microscope. The hydrophilicity of each scaffold and BMP-2 release of the PLGA/HA/BMP-2/chitosan scaffold were examined. (2) Pre-osteoblastic suspensions were seeded onto each scaffold. Cell adhesion and proliferation were detected using cell counting kit-8 at 1, 4, 7 days of cell culture. Fluorescein diacetate was used for a vital staining of cells at 7 days of cell culture. Alkaline phosphatase activity was detected at 4, 7 and 14 days of cell culture.

RESULTS AND CONCLUSION: (1) All the scaffolds were white beaker-shaped and had porous structure with a pore size of about 100 µm, and interconnected pores were observed under the scanning electron microscope. (2) The scaffold hydrophilicity was increased with the increasing concentration of chitosan. (3) BMP-2 cumulative release amount was 44% for the PLGA/HA/BMP-2, 34% for PLGA/HA/BMP-2/0.25% chitosan, 27% for PLGA/HA/BMP-2/0.5% chitosan, and 26% for PLGA/HA/BMP-2/1% chitosan, indicating that chitosan can effectively slow the release of BMP-2. (4) Cell viability of pre-osteoblasts seeded onto the PLGA/HA/BMP-2/0.25% chitosan scaffold was highest at 7 days of cell culture. Higher cell viability of pre-osteoblasts seeded onto the PLGA/HA/BMP-2/chitosan (0.5%, 1%) scaffolds was also observed compared with two control scaffolds. After fluorescein diacetate staining, living cells with green fluorescence were evenly distributed on the scaffolds under the confocal laser microscope. (5) The alkaline phosphatase activity in cells seeded onto different scaffolds was ranked as follows: the PLGA/HA/BMP-2/0.25% chitosan scaffold > the PLGA/HA/BMP-2/0.5% chitosan scaffold > the PLGA/HA/BMP-2/1% chitosan scaffold > the PLGA/HA/BMP-2 scaffold > the PLGA/HA scaffold (P < 0.05). Taken together, the PLGA/HA/BMP-2/chitosan scaffold is suitable to release bioactive BMP-2 for stimulating cell adhesion, differentiation and proliferation, which is designed to optimize the tissue-engineered bone scaffold in bone tissue engineering strategies. And moreover, the optimal modification concentration of chitosan is 0.25%. 

Key words: Hydroxyapatites, Bone Morphogenetic Proteins, Chitosan, Stents, Tissue Engineering

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