中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (6): 917-923.doi: 10.3969/j.issn.2095-4344.1547

• 纳米生物材料 nanobiomaterials • 上一篇    下一篇

载纳米银磷酸三钙的体外细胞毒性及兔体内毒性

程 坚1，2，张 军1，3，管 捷1，曾俊凯1，赵 鑫1，谢幼专1   

  1. 1上海交通大学医学院附属第九人民医院骨科，上海市内植物重点实验室，上海市 200011；2余姚市人民医院外五科，浙江省余姚市 315400；3上海青浦区中心医院骨科，上海市 200011
  • 收稿日期:2018-10-27 出版日期:2019-02-28 发布日期:2019-02-28
  • 通讯作者: 谢幼专，主任医师，上海交通大学医学院附属第九人民医院骨科，上海市内植物重点实验室，上海市 200011
  • 作者简介:程坚，男，1979年生，浙江省余姚市人，汉族，上海交通大学医学院在读硕士，副主任医师，主要从事创伤、脊柱研究。
  • 基金资助:

    国家自然科学基金(81071453)，项目负责人：谢幼专；上海市科委基金(1441901700)，项目负责人：谢幼专

Silver nanoparticle-doped tricalcium phosphate: in vitro and in vivo toxicity in rabbits

Cheng Jian1, 2, Zhang Jun1, 3, Guan Jie1, Zeng Junkai1, Zhao Xin1, Xie Youzhuan1   

  1. 1Department of Orthopedics, Ninth People’s Hospital of Shanghai Jiaotong University Medical School, Shangahi 200011, China; 2Fifth Department of Surgery, Yuyao People’s Hospital, Yuyao 315400, Zhejiang Province, China; 3Department of Orthopedics, Qingpu Central Hospital of Shanghai, Shanghai 200011, China
  • Received:2018-10-27 Online:2019-02-28 Published:2019-02-28
  • Contact: Xie Youzhuan, Chief physician, Department of Orthopedics, Ninth People’s Hospital of Shanghai Jiaotong University Medical School, Shangahi 200011, China
  • About author:Cheng Jian, Master candidate, Associate chief physician, Department of Orthopedics, Ninth People’s Hospital of Shanghai Jiaotong University Medical School, Shangahi 200011, China; Fifth Department of Surgery, Yuyao People’s Hospital, Yuyao 315400, Zhejiang Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81071453 (to XYZ); the Foundation of Shanghai Science and Technology Committee, No. 1441901700 (to XYZ)

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摘要：

文章快速阅读：

 

文题释义：
载纳米银磷酸三钙：β-磷酸三钙是一种自固化型骨水泥，具有与骨质无机质相似的化学组成，是一种能够被生物降解的生物材料，组织相容性好，可被组织逐渐降解并能被骨组织逐渐长入其中微孔中，还提供一定的机械力学稳定性；研究将质量分数10%的具备抗菌作用的纳米银颗粒融解于β-磷酸三钙中，形成含质量分数10%的载纳米银磷酸三钙。
CCK-8试剂：CCK-8试剂中含有WST-8[化学名：2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2，4-二磺酸苯)-2H-四唑单钠盐，它在电子载体1-甲氧基-5-甲基吩嗪硫酸二甲酯作用下被细胞线粒体中的脱氢酶还原为具有高度水溶性的黄色甲臜产物，生成的甲臜物数量与活细胞数量呈正比。用酶联免疫检测仪在450 nm波长处测定其光吸收值，可间接反映活细胞数量。该方法已被广泛用于一些生物活性因子的活性检测、大规模抗肿瘤药物筛选、细胞增殖实验、细胞毒性实验及药敏实验等。
 
 
背景：课题组前期研究发现，负载5%纳米银颗粒β-磷酸三钙材料具有抗菌性和良好的生物相容性。
目的：10%纳米银颗粒β-磷酸三钙材料的体内外毒性。
方法：①体外细胞毒性：分别采用含质量分数10%纳米银颗粒β-磷酸三钙材料浸提液、多孔β-磷酸三钙材料浸提液、正常组织培养液培养SD大鼠(上海西普尔-必凯实验动物有限公司提供)骨髓间充质干细胞，培养1，3，5，7 d，CCK-8法检测细胞增殖；②动物体内毒性：取20只新西兰大白兔(上海市甲干生物科技有限公司提供)，在左侧股骨外髁制作直径6 mm、深度13 mm的骨缺损，随机分2组，实验组缺损处植入含质量分数10%纳米银颗粒的β-磷酸三钙材料，对照组植入β-磷酸三钙材料。植入后12周，采用电感耦合等离子体质谱法检测血液和各组织器官内银离子浓度；苏木精-伊红染色切片观察各组织器官病理状态；Van Gieson染色切片观察材料植入区组织生长情况。

结果与结论：①β-磷酸三钙浸提液组、正常组织培养液组、含纳米银颗β-磷酸三钙材料浸提液组培养不同时间点的细胞增殖比较无差异(P < 0.05)；②实验组和对照组兔血液、心、肝、脾、肺、肾各脏器中的银离子浓度均小于0.1×10-6，小于WHO规定的人体内银离子的最高浓度0.5×10-6；③实验组与对照组兔心、肝、脾、肺、肾组织切片均未见明显病理性改变；④实验组与对照组材料轮廓可分辨，材料周围可见被染成红色的新骨组织，材料内部多孔结构内可见大量红染的新骨组织，两组材料多孔结构内的新骨组织分布未见明显差异；⑤结果表明，含质量分数10%纳米银颗粒的β-磷酸三钙材料无明显细胞毒性与动物体内毒性，具有良好的生物相容性。

ORCID: 0000-0003-2101-7605(程坚)

 

关键词: 纳米银颗粒, β-磷酸三钙, 生物毒性, 骨缺损, 载纳米银磷酸三钙

Abstract:

BACKGROUND: Previous studies by the research team found that 5% silver nanoparticle-doped β-tricalcium phosphate material has antibacterial properties and good biocompatibility.

OBJECTIVE: To study the biotoxicity of porous β-tricalcium phosphate containing 10% mass fraction of silver nanoparticles (10% SNPs-TCP) in vitro and in vivo.
METHODS: (1) In vitro cytotoxicity: Bone marrow mesenchymal stem cells from Sprague-Dawley rats (provided by Shanghai Xipuer-Beikai Animal Co., Ltd., China) were cultured with the extract containing 10% SNPs-TCP, porous TCP extract and normal tissue culture medium for 1, 3, 5, 7 days. Cell counting kit-8 method was used to detect cell proliferation. (2) In vivo toxicity: Twenty New Zealand white rabbits (provided by Shanghai Jiagan Biotechnology Co., Ltd., China) were used to make bone defects of the left femoral condyle (6 mm in diameter and 13 mm in depth), which were randomly divided into two groups. The experimental group was implanted with 10% SNPs-TCP, and the control group was implanted with single TCP. Twelve weeks after implantation, inductively coupled plasma mass spectrometry was used to detect the concentration of silver ions in the blood and tissues. Hematoxylin-eosin staining sections were made to observe the pathological changes of various tissues and organs. Van Gieson staining sections were used to observe the tissue growth in the implantation area.

RESULTS AND CONCLUSION: (1) There was no difference in cell proliferation between the TCP group, normal culture group and 10% SNPs-TCP group at different time points (P < 0.05). (2) The concentration of silver ions in the blood, heart, liver, spleen, lung and kidney of the experimental and control groups was less than 0.1×10-6, which was less than the highest concentration of silver ions (0.5×10-6)in the human body specified by the WHO. (3) There were no obvious pathological changes in the heart, liver, spleen, lung and kidney sections of the experimental and control groups. (4) The contours of the implantation materials in the experimental and control groups could be distinguished. New bone tissues stained with red were around the materials. A large amount of red-stained new bone tissues were visible inside the porous structure of the materials. There was no significant difference in the distribution of new bone tissue in the porous structure of the two groups of materials. To conclude, the 10% SNPs-TCP has no obvious cytotoxicity and toxicity in animals, and has good biocompatibility.

Key words: Silver, Calcium Phosphates, Toxicity Tests, Tissue Engineering

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