Abstract
BACKGROUND:To repair bone defect, histocompatibility, growing characteristics, biodegradation and repairing mechanism of nanometer need to be further studied in clinic.

OBJECTIVE: To observe the growing characteristics and histocompatibility of nano-hydroxyapatite (Nano-HA) for repairing jaw defect of rabbits.

DESIGN: Randomized grouping animal study.

SETTING: Beijing Jishuitan Hospital and Stomatology College of Jiamusi University.

MATERIALS: A total of 24 New Zealand rabbits, either gender, weighing 2.5-3.5 kg, were provided by Animal Experimental Center of Jiamusi University. The animal experiment had got confirmed consent from local ethic committee. Nano-HA was provided by Material Engineering College of Jiamusi University and dealt with routine hyperthermia/hypertension sterilization. In addition, hydroxyapatite was provided by Wuhan Industry University, and the diameter was 1.0-2.0 μm.

METHODS: The experiment was carried out in the Experimental Animal Center of Jiamusi University from November 2001 to May 2006. All rabbits were randomly divided into experimental group and control group with 12 in each group. Bone defect in the diameter of 1.0 cm was produced on body of mandible. Nano-HA was used to repair the bone defect of rabbits in the experimental group, while hydroxyapatite was used to repair the bone defect of rabbits in the control group. At 1, 4, 8 and 12 weeks after operation, all rabbits were sacrificed. In addition, medical imaging analysis system was used to analyze generative quantity of tissue in the two groups; meanwhile, histological quality and quantity were also analyzed so as to observe histocompatibility and newborn osteogenesis.

MAIN OUTCOME MEASURES: Histocompatibility and newborn osteogenesis.

RESULTS: With the time passing by, the amount of repairing materials was decreased because of the combination with newborn tissue into bone in bone defect-repaired region in the experimental group. When it was closed to normal bone, the amount was stable. However, bony callus was not able to grow in materials in the control group. Results of correlation analysis demonstrated that materials were negatively straight-line correlation with newborn bone (r = －0.912 0, P < 0.01). During the repairing procedure of bone defect, newborn bone was closely correlative with Nano-HA; while, with the increase of newborn bone, the amount of repairing materials was decreased because of the combination with newborn tissue into bone.

CONCLUSION: Nano-HA can combine with newborn bone tissue so as to rapidly generate bone, while it has an excellent biocompatibility.

INTRODUCTION

Since 2001, nano-hydroxyapatite (Nano-HA) is used to repair bone defect. Based on histocompatibility, growing characteristics, biodegradation and repairing mechanism, the effect of Nano-HA is studied as compared with hydroxyapatite so as to investigate the feasibility of Nano-HA in clinic. This study was designed to observe and analyze the growing characteristics and histocompatibility of Nano-HA for repairing jaw defect of rabbits based on histology.

MATERIALS AND METHODS

Materials
The experiment was carried out in the Experimental Animal Center (Provincial Laboratory) of Jiamusi University from November 2001 to May 2006. A total of 24 New Zealand rabbits, either gender, weighing 2.5－3.5 kg, were provided by Animal Experimental Center of Jiamusi University (certification: P01105002). All rabbits were randomly divided into experimental group and control group with 12 in each group. The animal experiment had got confirmed consent from local ethic committee. Nano-HA was provided by Material Engineering College of Jiamusi University and dealt with routine hyperthermia/hypertension sterilization. In addition, hydroxyapatite was provided by Wuhan Industry University, and the diameter was 1-2 μm.

Methods
Model establishment:The experimental animals were intramuscularly anesthetized with 0.3 mL/kg sumianxin parenteral solution (Veterinary Institute of Changchun Military Command University) inferior jaw region, fixed on operation table and sterilized with 0.5% iodophor. Jaw was cut and separated till surface of bone, and then bone defect in the size of 1.0 cm×1.0 cm was established at axletooth postzone of inferior maxilla near to angle of jaw and ascending branch by using Volvere GX miniature engine (NSK NAKANISHI INC, Japan). Bone defect was repaired with Nano-HA and hydroxyapatite in the experimental group and the control group, respectively. After operation, rabbits were given antibiotic for 5 days.
At 1, 4, 8 and 12 weeks after operation, rabbits in the experimental group and the control group were sacrificed with 3 rabbits once. Compound tissue blocks
of bone samples selected from lower jaw were fixed in 100 g/L neutral formaldehyde for over 24 hours for decalcification; and then, they were cut into tissue sections routinely and stained with HE staining. Olympus BH-2 microscope was used to obtain image which was input into computer.
Histological analysis: ①Qualitative analysis: Histological changes and bone generation were observed under optic microscope with different amplifying powers.②Quantitative analysis: Microscope (×4 times) and computer monitor were used for quantitative analysis. In the experimental group, 2-4 parallel images were selected from defect region of section. In addition, domain division was used to draw domain area of newborn bone and rest repairing materials, while computer imaging analysis system was used to calculate area percentage of various components in measuring region and perform statistical analysis.
Statistical analysis:SAS software was used by the fifth author in this study. Domain division was used to calculate percentage of newborn bone and rest materials accounting for measuring region at different time points (4, 8 and 12 weeks). Correlation coefficient was calculated, while correlation regression analysis was performed because of close correlation of generation of newborn bone with application of repairing materials.

RESULTS

Quantitative analysis of the experimental animals
All 24 rabbits were involved in the final analysis. Experimental animals did not have fracture, and bone defect and lower alveolar nerve were not observed. Trauma was healed well after operation.

Qualitative analysis of histology
Bone defect in the experimental group was observed in the first week. Fiber bony callus was formed around materials, while newborn bone fiber internally grew towards materials. When materials were cut open, there were a lot of newborn fiber bony callus; however, inflammatory cells, macrophages and osteoclasts were not observed (Figure 1).

In the fourth week, a lot of bone matrixes were observed in the experimental group (Figure 2a); bone matrix went around osteoblasts, and areola was found between them. This suggested that Golgi's body was plentiful and was young form of osteocytes (Figure 2b). High-time microscope showed that bone matrix was average; bone trabecula grew in materials; the growth of bone trabecula in materials was well; osteoblast grew around materials (Figure 2c). This suggested that materials had a great compatibility to bone tissue. Fiber bony callus started to calcify into bone, and calcification line was transparent (Figure 2d). Fiber bony callus was observed in the control group; terminal line between bony callus and materials was clear; bony callus was able to internally grow in materials (Figure 3).

In the eighth week, there were a lot of newborn bone trabecula around materials in the experimental group, and bone trabecula arrayed in a good order (Figure 4a). Meanwhile, newborn bone marrow tissue was observed around materials, and a lot of bone marrow tissues were observed in specific regions (Figure 4b). This suggested that bone rebuild reached a certain degree.

Newborn tissue in the control group did not internally grow in materials. While, the newborn tissue in the particular domain bordered with materials was scar tissue (Figure 5a). High-time microscope indicated that collagen fiber was not even and shaped as streak, while nucleus shaped as fusiform (Figure 5b).
 

In the 12th week, mature bone was observed in the experimental group, bone trabecula arrayed in a good order, and there was still a few of materials. High-time microscope demonstrated that mature bone and bone lacuna were observed around materials, while osteocytes were also observed (Figure 6a). Harvard's system was formed, and both newborn bone and medullary cavity of bone were mature (Figure 6b). Fiber bone callus bordered with materials in the control group was gradually mature and started ossification. In addition, osteoblasts were observed, new band of bone was observed in some regions, and newborn bone was gradually mature (Figure 7).

Quantitative analysis of histology (Table 1)

Results of correlation analysis demonstrated that materials were negatively straight-line correlation with newborn bone (r = -0.912 0, P < 0.01). Based on correlation regression analysis scatter diagram and regression line analysis of percentage between newborn bone and materials accounting for measuring area, most points were located in 95% confidence intervals. This proved that, during the repairing procedure of bone defect, newborn bone was closely correlative with Nano-HA; while, with the increase of newborn bone, the amount of repairing materials was decreased because of the combination with newborn tissue into bone.

DISCUSSION

Nano-HA had many particular characteristics for repairing jaw defect as compared with pervious plerosis of bone defect.
In the first week, optic-histological microscope showed that there were not inflammatory cells and inflammatory reaction in the experimental group. This suggested that materials after transplanting into jaw had light stimulation, slight reaction which rapidly disappeared, and an excellent biocompatibility to jaw[1-3]. Reaction between inflammation and peripheral tissue of graft could be described by using model of Williams[4]. The principles of this model were detailed as follows: Materials which transplanted into tissue needed some operative measures. However, operation still caused wound healing without transplantation; therefore, response of tissue to materials was regarded as the changes and elongation of wound healing. Response of tissue to ecdemic stimulation was the same, but reactive level and dynamic characteristics were different. Nano-HA was used to repair bone defect in this study. In the first week, inflammatory infiltration was not observed under histological-sectional microscope. This suggested that transplanted Nano-HA had slight or even no stimulation on organism, while organism had slight or even no protective response to Nano-HA. These results were coincident with repairing materials and normal component of bone; meanwhile, those were also correlation with Nano-HA[5,6]. Nano-HA, which was regarded as a component of bone mineral, had a high activity due to small particles, high superficial energy and increase of superficial atom[7]. Therefore, Nano-HA was easy to combine with peripherally newborn bone tissues so as to maintain the equilibration between inflammation and repairing tissue as soon as possible and start plerosis of bone defect. On the other hand, histological sections predicted that Nano-HA observed in in vitro culture did not have effect on activity of normal cells, but had inhibitive effect on growth of cancer cells[8]; therefore, Nano-HA could inhibit onset of inflammatory reaction.
The results in this study demonstrated that Nano-HA had a good biocompatibility to jaw. However, the concept of biocompatibility was still controversial[9]. Recent concept of biocompatibility pointed to the ability for materials to induce reasonable host response in a specific application. Thus, interaction on each side was paid more and more attention, especially development of tissue reaction was the most important. Host reaction of graft could control functional manifestation after implantation. In addition, host reaction was affected by material characteristics, especially chemical stability in vivo. Histological-sectional observation indicated that there were no side effects of Nano-HA on repairing bone defect, and generation of bone was fast. This suggested that the materials had a suitable response to jaw, and materials rapidly combined with host and played functions. The results proved that Nano-HA had a good biocompatibility to jaw, while those were proved in many researches during recent years[10-13].
This study also demonstrated that Nano-HA had a good biodegradation in vivo. Surface of nanometer-sized hydroxyapatite was not smooth and formed a lot of roug atomic steps. So its superficial area increased, and superficial key state and electronic state were different from internal part of granules. Incompletely superficial atomic coordinate might cause increase of superficially active sites so as to easily absorb with protein[7,14]. Newborn osteoblasts secreted extracellular matrix at interface. Especially, collagen might absorb and apply Nano-HA so as to rapidly generate bone[15]. This conclusion was as the same as establishing compound materials by using Nano-HA to repair bone defect[16-19]; meanwhile, it had a good biocompatibility. Degradation of Nano-HA for repairing jaw defect was regarded as a component of newborn bone for absorption and application during bone formation but not reacted with bone tissue and outputted metabolites. The results of tissue quantity of histological sections indicated that degradation of materials was suitable for formation of newborn bone. With the formation of newborn bone, materials were degraded and applied gradually. Materials were negatively straight-line correlation with newborn bone. In the 12th week, HE staining histological-sectional optic microscope observed that transplanted Nano-HA was generally applied with the maturity of bone. A few rest of materials were only observed under high-time microscope, and this suggested that bone was formed rapidly.

REFERENCES

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纳米羟基磷灰石修复兔颌骨缺损的
组织学分析★

富建明1，苗 波2，贾刘合1， 姚海涛3，祝丽玲4，吕奎龙5，梁 楠6，滕立群7
1北京积水潭医院口腔科，北京市 100035；佳木斯大学，2口腔医院颌面外科，3病理教研室，4公卫教研室，5材料工程学院，黑龙江省佳木斯市 154002； 6上海市同仁医院口腔科，上海市 200050；7大连口腔医院儿童牙病科，辽宁省大连市 116021
富建明★，男，1965年生，辽宁省康平县人，蒙古族，1989年毕业于佳木斯医学院口腔系，硕士，副教授，副主任医师，主要从事口腔颌面外科的临床、教学和科研。
通讯作者：苗 波，主任医师，佳木斯大学口腔医院颌面外科， 黑龙江省佳木斯市 154002
摘要
背景：纳米材料修复骨缺损的临床应用组织相容性、生长特性、生物降解性及修复机制需进一步研究。
目的：观察纳米羟基磷灰石修复颌骨缺损模型兔的生长特性及生物相容性。
设计：随机分组动物实验。
单位：北京积水潭医院，佳木斯大学口腔医学院。
材料： 选用24只新西兰白兔，雌雄不拘, 体质量2.5~3.5 kg,由佳木斯大学动物实验中心提供。实验处置过程符合动物伦理标准。Nano-HA由佳木斯大学材料工程学院提供，常规高温高压消毒备用。普通HA购自武汉工业大学，粒径为1.0~2.0 μm。
方法：实验于2001-11/2006-05在佳木斯大学实验动物中心完成。摸球法将实验兔随机分为实验组和对照组，每组12只。各组实验兔在下颌骨体部造成直径1.5 cm的骨缺损，实验组以纳米羟基磷灰石修复，对照组以普通羟基磷灰石修复，于术后1，4，8，12周分别麻醉后处死，用医学图像分析系统分析各组分的组织生成量，并进行组织学定性和定量分析，观察材料的组织相容性及新生骨生成情况。
主要观察指标：材料的组织相容性及新生骨生成情况。
结果：实验组骨缺损修复区随时间增长修复材料被利用与新生组织结合成骨而不断减少，直至与正常骨接近而趋于稳定，对照组骨痂不能长入材料内。相关分析结果表明材料与新生骨之间呈直线负相关（r =-0.912 0，P < 0.01）。骨缺损的修复过程中新生骨与纳米羟基磷灰石之间相互关系密切，且随着新生骨不断产生、增多，修复材料被利用与新生组织结合成骨而不断减少。
结论：纳米羟基磷灰石可与新生骨组织结合且成骨较快，有良好的生物相容性。
关键词：纳米羟基磷灰石；骨缺损；生物相容性；组织学；图像分析
中图分类号: R318.08 文献标识码: A 文章编号: 1673-8225(2008)01-00157-04
富建明，苗波，贾刘合， 姚海涛，祝丽玲，吕奎龙，梁楠， 滕立群.纳米羟基磷灰石修复兔颌骨缺损的组织学分析[J].中国组织工程研究与临床康复，2008，12(1):157-160
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(Edited by Zhan DS/Ji H/Wang L)