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.

INTRODUCTION

Application of biomaterials has long been a very important issue in tissue engineering, which directly determine the success of this technology. Until now, biocompatibility remains the central theme for biomaterials applications in medicine [1-3]. Biocompatibility determines how long the biomaterial will exist in host and whether it will perform biological function after transplantation. The ideal biomaterial means absence of cytotoxic effect and immunological rejection, degradation at right moment, and a well histocompatibility. Research on biocompatibility of biomaterials includes experiment in vitro and in vivo, i.e. implantation of biomaterial.
In this study, we not only developed bio-derived bone, but also evaluated its histocompatibility by implanting it into muscles of mice, thus providing experimental evidence and material base for the selection of biomaterial in tissue engineering.

MATERIALS AND METHODS

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
Preparation of bio-derived bone
Commercial porcine bone was rinsed with 37 ℃ physiological saline. The soft tissue, periosteum and dense bone were removed. The bone was dissected at the size of 1 cm3. After cleaned with 37 ℃ physiological saline, the bone was incubated in hydrogen peroxide(30%volume fraction) for 3 days in the constant temperature cabinet, and hydrogen peroxide was renewed every 24 hours. The bone was washed with 37 ℃ distilled water, and then immersed into chloroform-methanol (3:1 in volume) for 4 hours. After washed again with 37 ℃distilled water, the bone was immersed in 0.6 mol/L HCl for 72 hours at room temperature. After washed with distilled water repeatedly, the bone was vacuum dehydrated for 4 hours. Next, it was placed in liquid nitrogen for 24 hours. After dried, it was irradiated with 60Co at the dose of 35Gy and sterilized for use.

In vivo implantation experiment
Eighteen BALB/C mice were randomly divided into three groups with 6 mice in each group: a control group (simple injury without implantation, dissecting muscle at one side of lower limb, and then suturing), a bio-derived bone implantation group (dissecting bio-derived bone into 0.2 cm× 0.2 cm × 0.3 cm parts, and implanting them into the muscle of one side of lower limb), and a xenogenic bone implantation group (one piece of femoral bone was collected from Kunming mouse, rinsed for several times with PBS, allowing it to soak in PBS overnight, then implanted into the muscle of one side of lower limb. Twenty-one days after operation, the implant material and surrounding tissue were obtained for haematoxylin-eosin staining.

Experiment of ConA-induced splenic lymphocyte transformation
Twenty-one days after in vivo implantation, blood serum was prepared by collecting the blood from mice's eyeballs. The spleens from individual mice were aseptically taken and homogenized by adding 1.5 mL serum-free IMDM for preparing cell suspension. Cells (1×1010 L-1) were inoculated to a 96-well flat-bottom plate at 3×104 per well. ConA (10 mg/L) was added to two wells of each group, and two wells of each group supplied with IMDM medium were taken as control. After incubation in a 5% CO2 atmosphere at 37 ℃ for 48 hours, 10 mL MTT (5 μg/L) was added to each well, then incubation was performed for another 5 hours, to dissolve formazan crystals. 100 mL dimethyl sulphoxide (DMSO) was added into each well. Absorbance was determined with an automatic ELISA reader at 570 nm and used to indicate T cell proliferation. Data are reported as the following:

Where, SI refers to lymphocyte stimulation indices. It detects the reaction of lymphocytes to mitogen stimulation

Analysis of complement-dependent cytotoxicity
Blood serum was collected from rabbit through cephalic artery, keeping the sample at 4 ℃ overnight for the preparation of complement. The serum was diluted with IMDM medium at the ratio of 1:4 and 1:8, respectively. The cell suspension (1×1010 L-1) above-mentioned was regulated to 8×108 L-1. Then 50 μL cell suspension and 50 μL corresponding mouse serum was respectively added to a 96-well plate, and cells supplied with 50 μL IMDM medium were taken as control group. After incubation in a 5% CO2 atmosphere at 37 ℃ for 45 minutes, rabbit serum prepared into different concentration (1∶5, 1∶10) with IMDM was added to corresponding wells. Subsequently, all wells were incubated for 1 hour in the CO2 incubator, and 20 μL MTT(5 μg/L) was added to each well. Four hours after incubation, 150 μL DMSO was added into each well. Absorbance was determined with an automatic ELISA reader at 570 nm. Data are reported as the following:
Cytotoxicity =(1-absorbance of experimental group/ absorbance of control group) ×100%
Here, SI is the lymphocyte stimulation indices. It detects the reaction of lymphocyte to mitogen stimulation.

Statistical analysis
Statistical analysis was perfomed by the first author with SPSS 10.0 software, and all data were expressed as Mean± SD. t test was used for comparison between each two groups.

RESULTS

Quantitative analysis of experimental animals
Eighteen BALB/C mice were included in the final analysis.

Structure of bio-derived bone
With an anatomical microscope, we found that bio-derived bone comprised a lot of pores, the diameter of which was about 300-500 μm, and the pores connected with each other (Figure 1).

Experimental results of in vivo implantation
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 (Figure 2). 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 (Figure 3).

Results of ConA induced splenic lymphocyte transformation (Table 1)
Results indicated that 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).

Results of the complement dependent cytotoxicity test (Table 2)

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).

DISCUSSION

Tissue engineering scaffold can provide three-dimensional space for seeded cells, and can induce the regeneration of the tissue from the recipients themselves post transplant. The ideal scaffold should be biocompatible, have structural integrity, and act as a temporary framework for the cells until the newly formed bone is generated [4]. In addition, the ideal scaffold should have a proper balance between mechanical properties, a porous architecture, and degradability while remaining osteoconductive[5-10]. A satisfactory biocompatibility is a fundamental component in the design of tissue engineering scaffolds, hence the necessarity of evaluation for biocompatibility of scaffold before a long term of implantation. The present tissue engineering bone is usually composed of artificial materials, e.g. poly-acidum aceticum, polylactic acid, hydroxyapatite ceramic and so on. However, the poor biocompatibility, the derangement of spatial structure and the absence of osteoinductive factor greatly limit its application. Bone from the same race is seconday to the autogenous bone as a substituent for the transplantation, which not only possesses the ability of bone conduction, but also some extent osteoinductive capacity [11-12]. However, the fresh bone has a strong antigenicity, and it derives mainly from the surface of bone marrow-derived cells, then the periosteum and soft tissue, which is easily to initiate the cell mediated immunoreaction [13]. We prepared bio-derived bone through freezing, deproteinization, decalcification, defatting and elimination of cells and antigens. Besides containing some osteoinductive factors, the bio-derived scaffold we have developed also has a low antigenicity and a good biocompatibility, as its essential component is collagen [14]. Above all, this scaffold maintaines the spongy structure of natural bone, which has an interconnecting porous structure, and high porosity [15-17]. To further evaluate its bio-safety, we implanted the bio-derived scaffold into the mice, and examined the inflammatory reaction and immunoreaction.
The experiment of implantation in vivo is indispensable to measure the biocompatibility of biomaterial. We can detect all the effects of biomaterial on the recipient through this experiment, e.g. inflammatory stimulation and immunoreaction. Our present study showed that there were no red swelling in the region around the bio-implant, and the wound healed well. It was easy to discern the scaffold group with our naked eyes. There was no obvious congestion, degeneration, necrosis, pus, or effusion. A great quantity of fibrous tissue had grown into the fenestra and had begun to form a fibrous capsule. Hematoxylin and eosin staining demonstrated no obvious infiltration of inflammatory cells such as neutrophils or multinucleated giant cells around the implant. A large quantity of fibrous connective tissue was seen around the scaffold, and the scaffold was encapsulated by fibrous tissue. At the same time, some parts of the scaffold had degraded and the resulting spaces were filled with the fibrous connective tissues. In contrast, the necrotic tissue could be detected in the injured muscle of the positive control group. Hematoxylin and eosin staining revealed obvious infiltration of neutrophils and macrophages along with necrotic tissues. The above results indicated that the bio-derived bone possessed a satisfactory biocompatibility and biodegradability, and could be used as an ideal scaffold material.
Test of ConA activated lymphocyte transformation is one good way to measure the lymphocyte immunity function in vitro. Small lymphocytes can be transformed into lymphocytoblasts with the stimulation of unspecific stimulant, e.g. PHA and ConA, and specific antigen. The rate of transformation reflects the level of cell-mediated immunity. Our results showed that the lymphocyte stimulation index of the Kunming mouse group was significantly higher than that of the control group. However, there was no significant difference between the bio-derived bone implantation group and the control group. It indicated that the bio-derived bone did not have a strong immunogenicity, and had no effect on the level of cell-mediated immunity.
To determine whether the recipient was stimulated to produce the specific antibody, we conducted the complement dependent cytotoxicity test. The results demonstrated that the cytotoxicity of the xenogenic bone implantation group was larger than that of the control group. There was significant difference in the cytotoxicity between the two groups. However, there was no significant difference between the bio-derived bone implantation group and the control group.
In conclusion, the bio-derived scaffold we have developed has a low antigenicity and a good biocompatibility, without obvious cytotoxicity and immunoreaction, which can be a promising scaffold for bone tissue engineering, and may play a very important role in the healing of bone defection. However, the plasticity of bio-derived bone is still low, while in the full-scale operation, it is difficult to control the quality, and the function will not be in-synchronism with its structural change. Therefore, further study is still needed about the preparation and quality control of the bio-derived bone. With the development of biochemical technology, immunology, tissue engineering, a great progress will be made in the study of bio-derived bone, and bio-derived bone will play more important role in the healing of the bone defection [18-20].

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生物衍生骨支架材料制备及在体内的
组织相容性***☆

牛 云，何 旭，张丽红，高 婷，许 华，李玉林
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）*
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
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)