Abstract

BACKGROUND:The ideal biomaterial means absence of cytotoxic effect and immunological rejection, degradation at right moment, and a well histocompatibility. Whether bio-derived bone can be used in vivo for long time and exerts functions deserves to be studied.

OBJECTIVE: To investigate the local histocompatibility after bio-derived bone implanted into mouse and the effect on immunofunctions.

DESIGN: A randomized controlled animal experiment.

SETTING: Key Laboratory of Pathobiology, Ministry of Education, School of Basic Medical Sciences, Jilin University.

MATERIALS: This study was performed at the Key Laboratory of Pathobiology, Ministry of Science, School of Basic Medical Sciences, Jilin University from March to July in 2006. Eighteen BALB/C mice (weighing 20±2 g, half male and half female), one male Kunming mouse (weighing 20 g), and one female rabbit (weighing 2.5 kg) were included in the experiment. All the experimental animals were provided by Laboratory Animal Center, School of Basic Medical Science, Jilin University. The disposal of the experimental animals in the test process accorded with ethical guidelines for the use and care of animals. Porcine cancellous bone (iliac bone) was purchased from the market. Iscove's Modified Dulbecco's Medium (IMDM, Hycolone, USA), fetal bovine serum (FBS, Gibco Co., Ltd, USA), methyl thiazolyl tetrazolium （MTT, Sigma, USA）, and concanavalin A (ConA, Sigma Co., Ltd, USA) were used.

METHODS: Bio-derived bone was prepared from commercial porcine bone. ① Eighteen BALB/C mice were randomly divided into three groups with 6 mice in each group: a control group (simple local muscle injury without implantation), a bio-derived bone implantation group ( implanting bio-derived bone into the lower limb), and a xenogenic bone implantation group (femoral bone from Kunming mouse was implanted into the muscle of lower limb). ② Twenty-one days after operation, the implant material and surrounding tissue were obtained for gross observation and haematoxylin-eosin staining to investigate the histocompatibility of bio-derived bone. Mouse immunofunction was assessed by complement-mediated cytotoxicity test. Absorbance was determined with an automatic ELISA reader at 570 nm to assess the cytotoxicity.

MAIN OUTCOME MEASURES: ①Histocompatibility of implant surrounded tissue. ②Lymphocyte stimulation indices after induction of concanavalin A. ③ Cytotoxicity in each group after complement-dependent cytotoxicity test.

RESULTS: Eighteen BALB/C mice were included in the final analysis. ①Histocompatibility of implant surrounded tissue: In the bio-derived bone implantation group, 21 days after bio-derived bone implantation, there were no presentation of congestion, degeneration, necrosis and diapyesis around the implant in gross, plenty of fibrous connective tissue invaded into the pores of the bio-derived bone, encapsulation and forming the fibrous capsule. A great quantity of neutrophils and macrophages were not detected around the implant by haematoxylin-eosin staining. Bio-derived bone was encapsulated with fibrous tissue, and part of the biomaterial began to degrade, and being replaced with fibrous tissue. Regarding xenogenic bone implantation group, necrotic tissue was detected in the cross-section of the muscle in gross. A lot of neutrophils, macrophages and necrotic tissue were detected around the implant by haematoxylin-eosin staining. ②Lymphocyte stimulation indices: The stimulation index of xenogenic bone implantation group was significantly larger than that of control group (P < 0.05). There was no statistical difference between the bio-derived bone group and the control group (P > 0.05). ③Cytotoxicity: The cytotoxicity of xenogenic bone implantaion group was significantly larger than that of control group (P < 0.05). There was no significant difference in cytotoxicity between the bio-derived bone implantation group and the control group (P > 0.05).

CONCLUSION: The obtained bio-derived bone causes little immunoreactions, has no obvious cytotoxicity or inflammatory reactions, and possesses a good histocompatibility and bio-safety.

Key Laboratory of Pathobiology, Minis-try of Education, School of Basic Medical Sciences, Jilin University, Changchun 130021, Jilin Prov-ince, China

Niu Yun☆, Studying for doctorate, Key Laboratory of Patho-biology, Ministry of Education, School of Basic Medical Sci-ences, Jilin Univer-sity, Changchun 130021, Jilin Prov-ince, China niu_yun@163.com

Correspondence to: Li Yu-lin, Professor, Doctor's tutor, Key Laboratory of Patho-biology, Ministry of Education, School of Basic Medical Sci-ences, Jilin Univer-sity, Changchun 130021, Jilin Prov-ince, China ylli@mail.jlu.edu.cn

Supported by: the National 863 Major Program, No. 2004AA205020*; Doctoral Programs Foundation of Minis-try of Education of China, No. 20020183064*; the National Natural Science Foundation of China, No. 30700872*

Received: 2007-09-27 Accepted: 2007-10-22 (07-50-9-5309/YWY)

Niu Y, He X, Zhang LH, Gao T, Xu H, Li YL.Preparation of bio-derived bone and its histocompatibil-ity.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(7): 1385-1389(China)
[www.zglckf.com/zglckf/ejournal/upfiles/ 08-7/7k-1385
(ps).pdf]

摘要
背景：理想的支架材料必须对机体无毒性，无免疫排斥反应，并能适
时地降解，具有良好的生物相容性，生物衍生骨支架材料能否长期存留体内并发挥功能值得研究。
目的：观察生物衍生骨支架材料植入鼠体内后机体局部组织生物相容性及对免疫功能的影响。
设计：随机对照动物实验。
单位：吉林大学基础医学院病理生物学教育部重点实验室。
材料：实验于2006-03/2006-07在吉林大学基础医学院病理生物学教育部重点实验室完成，选用18只雄性BALB/C小鼠，体质量（202）g，雌雄各半；1只昆明小鼠，体质量20 g；1只雌性家兔，体质量2.5 kg，以上实验动物均由吉林大学基础医院实验动物部提供，实验过程中对动物的处置符合动物伦理学标准。猪松质骨（髂骨）为市售。实验用IMDM 培养基为美国Hycolone公司产品，FBS购自美国GIBCO公司，四甲基偶氮唑盐及ConA均为美国Sigma公司产品。
方法：采用猪髂骨制备生物衍生骨支架材料。①采用随机抽签法将BALB/C小鼠分成支架材料植入组、异种骨植入组及对照组，每组6只。支架植入组将支架植入到BALB/C小鼠的下肢肌肉内，异种骨植入组将昆明鼠骨植入到BALB/C小鼠下肢肌肉内，对照组仅将局部肌肉损伤。②术后21 d取材料植入部位及周围组织，大体及苏木精-伊红染色法进行观察材料与组织相容性；采用刀豆蛋白A诱导各组脾淋巴细胞转化，酶联免疫检测仪在λ570 nm波段检测并记录淋巴细胞刺激指数。以补体依赖性细胞毒实验评估各组小鼠免疫功能，即采用酶联免疫检测仪在λ570 nm波段检测并记录各组吸光度值，计算细胞杀伤率。
主要检测指标：①植入物周围组织相容性。②刀豆蛋白A诱导后淋巴细胞刺激指数。③补体依赖性细胞毒实验各组细胞杀伤率。
结果：18只BALB/C小鼠均进入结果分析。①植入物周围组织相容性：支架材料植入组植入21 d后，支架材料组肉眼可见，埋植物周围软组织均无明显充血、变性、坏死、化脓及积液等表现，大量纤维结缔组织长入材料的孔隙之中，包裹并形成纤维囊。苏木精-伊红染色结果显示埋植物周围未见大量中性粒细胞及多核巨细胞等炎细胞浸润。材料周围有大量纤维结缔组织增生，材料被纤维囊包裹。同时有部分材料已经开始降解，材料降解后的空间有纤维结缔组织增生。异种骨组肉眼见肌肉剖面有坏死组织，苏木精-伊红染色结果显示埋植物周围有大量的中性粒细胞和巨噬细胞浸润，并可见坏死组织。②淋巴细胞刺激指数：异种骨植入组淋巴细胞刺激指数高于对照组，差异有显著性意义（P < 0.05），支架材料组与对照组之间差异无统计学意义（P > 0.05）。③细胞杀伤率: 异种骨植入组的细胞杀伤率明显高于对照组，差异有显著性意义（P < 0.05），支架材料组的细胞杀伤率与对照组差异无统计学意义（P > 0.05）。
结论：本实验获得的生物衍生骨支架材料引起的免疫反应较弱，无明显细胞毒性，不产生明显的炎症反应，具有良好的生物相容性和生物安全性。
关键词：生物衍生骨支架；组织相容性；骨组织工程；组织构建

吉林大学基础医学院病理生物学教育部重点实验室，吉林省长春市 130021
牛 云☆，女，1979年生,河北省唐山市人，汉族，吉林大学在读博士，主要从事干细胞组织工程学研究。
通讯作者：李玉林，教授，博士生导师，吉林大学基础医学院病理生物学教育部重点实验室，吉林省长春市 130021
国家“八六三”重大专项(2004AA205020)*，教育部博士学科点专项科研基金（20020183064）*，国家自然科学基金（30700872）*


中图分类号: R318 文献标识码: A 文章编号: 1673-8225(2008)07-01385-05
牛云，何旭，张丽红，高婷，许华，李玉林.生物衍生骨支架材料制备及在体内的组织相容性[J].中国组织工程研究与临床康复，2008，12(7):1385-1389
[www.zglckf.com/zglckf/ejournal/upfiles/08-7/7k-1385(ps).pdf]
(Edited by Gorustovich A/Song LP/Wang L)