抗菌性骨缺损修复材料的理化与生物学性能实验研究
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摘要
最为理想的生物材料就是机体自身的组织,天然生物材料经过亿万年的演变进化,形成具有结构复杂精巧、效能奇妙多彩的功能原理和作用机制。新鲜自体骨是由无机材料和有机材料巧妙地结合在一起形成的复合体,其中无机材料的大部分是羟基磷灰石结晶,具有较高的骨诱导性,无免疫排斥反应,但是会增加手术创伤,并且病人还会出现局部疼痛、感觉异常或过敏等并发症,骨的来源也十分有限。近年来,利用组织工程化骨修复骨缺损,不仅提供了一种修复骨缺损的方法,而且也提供了一种全新思路,骨组织工程学的研究为治疗骨缺损带来了新的希望。目前,体外构建的组织工程化人工骨虽然还停留在实验室及动物实验阶段,但随着细胞生物学、工程学、免疫学、材料学等相关学科的发展,骨组织工程学的研究也将会取得突破性进展,在工业化生产和临床应用上有着极其广阔的应用前景,它将会成为21世纪极具发展潜力的高新技术产业,蕴藏着巨大的经济与社会效益。作为人类骨组织的主要无机成分,羟基磷灰石生物陶瓷价廉易得,具有良好的生物相容性,可降解又具备骨引导、骨融合性,尤其是含孔的珊瑚羟基磷灰石可提供很大的表面积/体积比,能保证细胞的增殖、分化和高代谢活动,所以特别适合用于骨组织工程。随着现代工业化机械化程度的提高,由于创伤、感染、肿瘤、事故、战争、地震等所致的大量污染性骨缺损发生率大幅度升高。对其传统治疗方法,即先清创、待伤口关闭、无感染迹象3-6个月后、再行二期植骨修复；而一期植骨常因发生较高的感染率而被视为禁忌,且临床上细菌耐药性的问题日益严重,植骨材料加载抗生素的作用越来越局限化,对抗菌剂提出了更高的要求。为解决传统治疗方法所带来的增加治疗时间、难度、患者的痛苦和经济负担等一系列问题,改变传统观念,近年来,国内外学者在抗菌骨移植材料的基础实验和临床实践方面进行了大量的研究,取得了一定的进展。为此,我们研制一种抗菌性骨支架修复材料,并经以下系列实验研究证明：这一抗菌性骨修复材料具有良好的生物相容性,可在局部缓释银离子,有效地抑制污染性骨缺损清创术后局部残留的细菌。
实验分四个部分进行观察研究：
课题研究第一部分：载银珊瑚羟基磷灰石人工骨的制备及性能表征。
目的：制备载银珊瑚羟基磷灰石人工骨并观察其理化性能表征。
方法：实验于2008-05/08在清华大学材料科学与工程系实验室完成。实验材料：在加工好的珊瑚羟基磷灰石(Coral Hydroxyapatite, CHA)颗粒与相应浓度硝酸银(Silver nitrate, AgNO3)溶液浸泡一定时间后置入冻干机冻干制得载银珊瑚羟基磷灰石(Silver-loaded Coral Hydroxyapatite, SLCHA或Ag+-CHA)。应用X射线衍射法对Ag+-CHA、CHA和纯珊瑚样品晶体的物相、结构和位相进行分析；应用FT-IR光谱法对样品的材料组成进行分析；应用扫描电镜观察材料的显微形貌和孔径；利用压缩试验及三点弯曲试验评价骨修复支架材料的机械性能。应用SPSS13.0软件对机械性能测试结果行单向方差分析及LSD法两两比较(a=0.05)。
结果：①X射线衍射法对Ag+-CHA、CHA和纯珊瑚三种材料晶体进行物相分析、结构分析和位相分析,图形显示Ag+-CHA样品的衍射峰与CHA的图谱基本相符,而且XRD图中无其他相的衍射峰,说明银离子与CHA发生反应后产物比较单一,无其他衍生物生成,仍然保持原有的晶形结构；同时晶面位于(211),(300)的峰值较CHA低,说明银离子可能与此晶面位置的HA发生反应。②FT-IR光谱法图中显示三者具有一定的相似性,1456 cm-1和1413cm-1是材料中C032-基团的特征峰位,与纯珊瑚基本一致,说明Ag+-CHA和CHA材料本身大部分为CaCO3成分。1031 cm-1、604 cm-1和563cm-1为HA的特征峰,即显示出P04-3离子根的振动峰,说明Ag+-CHA和CHA中已存在一部分HA。这些HA特征波峰变宽,对FT-IR光谱进行分析,Ag+-CHA的曲线的变化可能与Ag+与CHA的Ca2+之间存在离子键变换、Ag+的离子半径大于Ca2+有关。③扫描电镜、能谱分析及背散射电镜可见载银珊瑚羟基磷灰石样品仍维持着珊瑚本身的三维多孔结构,载银材料表面存在有钙、碳、银、磷和氧等多种元素,且载银材料表面沉淀物的银含量与制备过程中硝酸银的浓度成正比关系；④采用压缩试验法分析这三种材料间的压缩强度无显著性差异(F=3.147,P=0.196)；三种材料间的压缩模量无显著性差异(F=0.827,P=0.482)；及采用三点弯曲试验法分析三种材料间的三点弯曲折断功无显著性差异(F=2.543,P=0.159)。
结论：珊瑚羟基磷灰石经载银后未改变其理化性能,依然维持着三维多孔框架结构；制备原理主要为钙银离子之间的置换反应,其次为银离子的溶出-沉积反应,并且随着制备过程中硝酸银溶液浓度的递增,Ag+-CHA的载银量也随之递增。
课题研究第二部分：载银珊瑚羟基磷灰石人工骨的载银量及体外缓释实验研究
目的：研制载银珊瑚羟基磷灰石抗菌性人工骨支架材料,通过对其自身载银量及体外缓释实验完善对该抗菌性磷灰石类复合人工骨的认识。
方法：应用电感耦合等离子体发射光谱(inductively coupled plasma optical emission spectrometry, ICP-OES)测定载银材料中银的平均含量,并在模拟体液(Stimulated body fluid, SBF, pH=7.25)中通过体外释放试验测定银离子释放浓度与时间的变化曲线,探讨材料结构与释放银的关联性。实验数据应用SPSS13.0统计软件处理,用相关回归分析和曲线拟合载银量与AgNO3浓度关系曲线,建立回归方程。
结果：根据实验结果绘制的载银量-AgNO3浓度关系曲线。经统计学分析拟合出载银量-AgNO3浓度曲线方程为：y=3E+007x2+126863.3x+12.845(R SquareChange=0.997),对方程进行回归检验,F=959.625,P=-0.000,有统计学意义。载银羟基磷灰石颗粒3个试样中银离子均能从颗粒向模拟体液中扩散,具有明显缓释作用。第1个24小时释放量最大,达到885.27±122.49μg/L,低于文献中报道的银离子最低抑菌浓度(MIC) 1.25μg/mL.,达到减毒增效的效果。以后逐渐减少,7-14天维持一平台期释放,其后以较低浓度维持,第28天仍有79.80±6.69μg/L的银离子释放量。
结论：载银珊瑚羟基磷灰石之所以能保持长时间的银离子释放,与羟基磷灰石本身的空间晶格结构关系密切,这种可逆的离子置换过程使其释放性能更显著,能更有效地避免银离子突释,保持长时间的持续释放,有效地减少金属银的使用量,达到减毒增效的作用。
课题研究第三部分：载银珊瑚羟基磷灰石人工骨的细胞相容性研究
目的：研制载银珊瑚羟基磷灰石抗菌性人工骨支架材料,观察其在体外与小鼠胚胎成骨细胞株(MC3T3-E1)的细胞相容性。
方法：实验于2009-06/09在广州军区广州总医院实验科完成。采用体外细胞培养技术,将MC3T3-E1细胞种植于10-3、8×10-5、10-5mol/L Ag+-CHA, CHA及纯珊瑚支架材料上。实验评估：①MTT法测定MC3T3-E1细胞接种在不同支架材料上生长1,3,5d后的吸光光度值,绘制细胞在不同骨材料上的生长曲线,评价材料上细胞的增殖情况；②碱性磷酸酶(ALP)活性法测定细胞接种在不同支架材料上2,4,6d的吸光光度值,计算培养液中碱性磷酸酶值；③倒置相差显微镜下直接观察培养板上MC3T3-E1细胞的生长形貌；④应用激光共聚焦显微镜(Confocal laser scanning microscopes, CLSM)直接观察支架材料上MC3T3-E1细胞的生长形貌；⑤应用扫描电镜(SEM)观察细胞在材料上生长的微观形貌。应用SPSS 13.0软件对各框架材料上的细胞生长情况结果行析因方差分析及LSD法两两比较,(α=0.05)。
结果：①成骨细胞在各组材料上的增殖能力有显著性差异(F=76.457,P=0.000)。CHA组与8×10-5mol/L组比较有显著性差异(P=0.019),而与10-5mol/L组及纯珊瑚组两两比较均无显著性差异(P值分别为0.177、0.428),成骨细胞在CHA组上的活性最高,增殖能力最强,增长速度最快,其次为纯珊瑚组、10-5mol/L组及8×10-5mol/L组；而细胞在10-3mol/L组上的增殖能力最低,材料上细胞数目最少,与其他组比较均有显著性差异(P值分别为0.000、0.000、0.000、0.000、0.000),各组与对照组相比较均有显著性差异(P值分别为0.000、0.000、0.000、0.000、0.000),表明8×10-5、10-5mol/L Ag+-CHA及其他非载银材料适合用于培养成骨细胞,对细胞无明显毒性,可诱导细胞迅速增长,而10-3mol/L Ag+-CHA不适合成骨细胞在其上生长。②各组之间比较有显著性差异(F=15.582,P=0.000)；10-3mol/L组与其他各组之间比较有显著性差异(P值分别为0.000、0.000、0.000、0.000、0.000),说明细胞在此组材料上生长最慢,分泌ALP最少；8×10-5mol/L组与对照组比较无显著性差异(P值为0.292),而8×10-5mol/L组与10-5mol/L组及CHA组两两比较无显著性差异(P值分别为0.340、0.068),说明随着材料载银量的减少,上述载银材料对细胞已无明显毒性作用；10-5mol/L组、CHA组、纯珊瑚组三组之间比较无显著性差异(P值分别为0.366、0.292、0.880),而与对照组比较有显著性差异(P值分别为0.049、0.006、0.004),说明三组材料适合细胞在其表面粘附生长,同时材料的三维多孔空间结构拓展了细胞粘附的表面积,有利于细胞快速增殖,其结果与MTT试验结果一致。③MC3T3-E1细胞与不同框架材料联合培养1d时的生长形貌：10-3mol/L Ag+-CHA培养板上细胞未贴壁生长,为透明的圆球形,呈游离状态,未能很好舒展,且有些皱缩,生长活力也不旺盛；细胞与8×10-5mol/L组、10-5mol/L组、CHA组及纯珊瑚组联合培养的生长情况要明显好于10-3mol/L组,细胞相互融合成片,多呈长梭形,细胞紧密排列成束,聚集生长的趋势更明显,这些结果与MTT及ALP结果相一致。④CLSM图像显示三组材料上的细胞数量及形貌无明显差别。⑤SEM图像显示材料上细胞呈梭形,较扁平,表面有细小绒毛与皱褶,细胞在与材料接触部位伸出伪足,形成鸭蹼状粘附并包绕材料,表面可见其分泌的骨基质。
结论：通过MTT、ALP活性、倒置相差显微镜、激光共聚焦及扫描电镜全方位多角度观察了细胞在天然框架材料上的反应。细胞增殖、生长良好,表明材料具有良好的细胞相容性。MTT、ALP活性的定量结果表明低载银量珊瑚羟基磷灰石材料与非载银骨替代材料的生物相容性基本一致。
课题研究第四部分：载银珊瑚羟基磷灰石人工骨修复兔桡骨污染性骨缺损的实验研究
目的：通过体内动物实验检测该抗菌性人工骨植入动物体内的组织相容性、骨传导性和材料的生物降解等组织自愈合生物学性能。
方法：新西兰大白兔36只,随机分为4组,每组9只。选用新西兰大白兔制作兔桡骨15mm节段性污染性骨与骨膜缺损模型,将36只大白兔称重,按体重大小顺序编号,按随机数目表法随机分为A-D组。A组植入载银珊瑚羟基磷灰石材料；B组植入珊瑚羟基磷灰石材料；C组植入原位自体骨材料；D组无任何植入,做为对照组。分别于术后2,6,10周分三次处死动物取材,每次每组3只,通过大体观察、影像学检查、组织学检查观察比较各组骨缺损修复情况,应用SPSS13.0软件对X线观察结果行重复测量方差分析及LSD法两两比较,α=0.05,比较上述各组对大段骨缺损的修复作用。
结果：不同组材料对骨缺损修复的大体观察结果可见2w时A、B两组移植物表面有肉芽组织和部分半透明骨质覆盖,与宿主骨断端间有部分骨痂连接,移植物无明显移位；C组移植物与宿主骨有部分骨痂连接,移植物无活动；D组骨缺损区填充以肌纤维组织。6w时A、B两组移植物表面有薄层骨质覆盖,与宿主骨断端间有大量骨痂连接,较稳固；C组移植物与宿主骨皮质连续,但不均匀,移植物无明显移位；D组缺损区仍填充以肌纤维组织。10w时A、B两组移植物表面有明显连续骨质覆盖,移植物稳固；C组移植物与宿主骨皮质连续成一体；D组缺损区仍填充以肌纤维组织。X线检查结果可见不同时间点各组之间比较有显著性差异(F=11.537,P=0.000)；自体骨成骨情况最好,与其他各组之间比较有显著性差异(P值分别为0.004、0.010、0.000),说明自体骨组织在大段骨缺损修复时仍然是最好的材料；Ag+-CHA组与CHA组两组比较无显著性差异(P值分别为0.524),说明珊瑚羟基磷灰石经一定量载银后其成骨作用未受明显影响；对照组与其他各组之间比较有显著性差异(P=0.000、0.000、0.000),说明污染性兔桡骨大段骨缺损后其成骨作用明显受到抑制,其模型制作是成功的。
结论：根据仿生的思路和以往对骨修复框架材料的研究,我们制备了一种新型的抗菌性框架材料用于污染性骨缺损修复。此材料成分和结构均与天然骨无机成分有相似性,多孔结构与松质骨类似。通过体内动物实验,证明了此材料具有良好的生物相容性和骨传导性,是有效的骨修复框架材料,可以达到与自体骨材料相同的效果,有希望成为污染性骨缺损修复的优选材料,具有广阔的市场前景。
综上所述,抗菌性载银珊瑚羟基磷灰石骨替代材料的制备过程简单,抗菌剂银离子的缓释性能较为理想,在污染性大段骨缺损处有良好的骨传导性及成骨作用,可以预想该抗菌人工骨材料是一种治疗污染性骨缺损的良好骨修复材料。
The most ideal biological material is the personal bone tissue from oneself. As the evolution of natural biological materials for billions of years, biological materials are formed by a structure-complicated and sophisticated, wonderful principles and mechanism. As a complex, fresh autogenous bone is ingeniously combined with the inorganic and organic materials together. Hydroxyapatite mostly in inorganic materials are of highly osteoinductive, non-immunological rejection response. But autogenuous bone graft will increase the surgical trauma, and lead to local pain, paresthesia, or allergies and other complications in patients, and the source of bone is very limited. In recent years, the use of tissue-engineering bone to repair bone defects, not only has provided a method of repairing bone defects, but also has provided a new idea. New hope has been brought for the treatment of bone defects because of bone tissue engineering research. At present, artificial bones constructed by tissue engineering in vitro still remain in the laboratory and animal-experimental segments, but with the development of the related subjects, such as cell biology, engineering, immunology, material science, the research of bone tissue engineering will make a breakthrough progress and there is an extremely broad application prospects in industrial production and clinical applications. It will become a great potential for the development of high-tech industries in the 21st century, and offer enormous economic and social benefits. As the main inorganic component of human bone tissue, hydroxyapatite bioceramic is cheap, and has good biocompatibility, biodegradability, bone conductivity, and bone fusion. Particularly the porous coral hydroxyapatite can provide a lot of surface area/volume ratio, can ensure cell proliferation, differentiation, and high metabolic activity. Then it is particularly suitable for bone tissue engineering. With the increased mechanization of modern industrialization, a large number of significantly infectious bone defects increase caused by trauma, infection, cancer, accidents, wars, earthquakes. The traditional methods of treatment is that there is a debridement at first until the wound closed, and no signs of infection for 3 to 6 months, then there is a bone repairment secondly. The higher infective rate of bone graft in one-stage often occurs, and it has been regarded as taboos. And the clinical bacterial resistance to antibiotics is a growing problem, the role of fighting against microbial bone in antibiotics-loaded graft material is taken more and more limit, and microbial agent has been put forward higher requirements. To solve the traditional treatment methods to bring a range of issues, such as increasing treatment time, the difficulty, the patient's suffering and economic burden, traditional attitudes have been changed in recent years. Domestic and foreign scholars has made some progress for the anti-infective bone graft materials in basic experiments and clinical practice. To the end, we are cooperating with the Department of Materials Science and Engineering in Tsinghua University to develop an anti-infective bone scaffold materials, and a series of experimental research have shown that:The antibacterial bone repairment material has good biocompatibility, and can release slowly silver ions to effectively inhibit local residual bacteria in infective bone defect after debridement, as follow.
This experiment divides into four parts:
Section one:Development and Characterization of Silver-loaded coralline hydroxyapatite (Ag+-CHA) artificial bone.
Objective:To develop Ag+-CHA artificial bone and study the physical and chemical characterization.
Methods:The experiment was accomplished in Tsinghua University, Laboratory of Materials Science and Engineering from May to August in 2008. Experimental material:Ag+-CHA was prepared by immersing coralline hydroxyapatite (Coral Hydroxyapatite, CHA) particles into the some concentration of silver nitrate (Silver nitrate, AgNO3) solution, and frozen in freezed dryer after a certain time. X-ray diffraction method is applied on the Ag+-CHA, CHA and pure coral to investigate their crystal phase, structure and phase analysis; FT-IR spectroscopy is applied to analyze the composition of the sample material; SEM micrograph is applied to materials morphology and the aperture; Compression tests and three-point bending test are used to evaluate mechanical properties of bone repair scaffold. The experimental data is analyzed with One-Way ANOVA and LSD on mechanical properties by the software SPSS13.0.
Results:①X-ray diffraction results revealed the characteristic peaks of Ag+-CHA, CHA, and pure coral, and there was no secondary phases, indicating that there was no other derivatives generated in the reaction between silver ions and the CHA, remaining the original crystalline structure; the same crystal plane phase at (211), (300) peak were lower than the CHA's. It showed that silver ions might react with the location of the HA crystal surface.②FT-IR spectroscopy are shown that they have a certain similarity,1456 cm-1 and 1413cm-1 are the material characteristic peaks of CO32-group, the same as pure coral. It showed that the most component of Ag+-CHA and CHA was CaCO3. The characteristic peaks of HA are 1031 cm-1,604 cm-1 and 563cm-1, shows that HA exists in the part of the Ag+-CHA and CHA already. The broadened HA features peaks on the FT-IR spectrum may be related to the transformation between silver ion of Ag+-CHA and the calcium ion of CHA, because of Ag+ ionic radius is larger than the Ca2+.③The SEM and BSE images showed that the morphology of the SLCHAs depended on the content of Ag+, and the silver ions were uniformly distributed on the surface of SLCHAs. EDS results demonstrated that the silver content of the SLCHAs decreased along with the decrease of the concentration of silver nitrate.;④compression test method was used to analyze the compressive strength among the three kinds of materials, and there was no significant difference (F=3.147, P=0.196), the P values were 0.323,0.695,0.403 compared among them; The compression modulus among three kinds of material was also no significant difference (F=0.827, P=0.482), the P values were 0.491,0.604,0.247 compared among them; and the broken power in the three-point bending test was no significant difference (F=2.543, P=0.159), the P values were 0.080,0.731,0.131 compared.
Conclusion:The physical and chemical properties of coralline hydroxyapatite still was maintained after loading silver ion and a three-dimensional porous framework has not been changed; The principle of Preparation is mainly the replacement reaction between silver ions and calcium, followed by the dissolution of silver ions-deposition. And with the increase of the concentration of silver nitrate solution, the content of silver in Ag+-CHA increased.
Section two:Study on the content of silver in Ag+-CHA and releasing character in vitro.
Objective:To develop antimicrobial Ag+-CHA artificial bone scaffold material, and the knowledge of Ag+-CHA is taken by silver content and releasing experiment in vitro.
Methods:The total silver content in the SLCHAs and the average concentration in the releasing test were determined by inductively coupled plasma emission spectroscopy (ICP-OES), calculated the content of silver loading refer to the standard curve of realationship between the content of Ag and the concentration of AgN03. Simulated body fluid (SBF, pH=7.25) was used in the releasing test in vitro, and we try to reveal the relationship between the materials' strucure and the silver releasing. The experimental data is analyzed with Linear Regression and Curve Fit by the SPSS13.0.
Results:The equation of Linear Regression:y=3E+007 x2+126863.3x+ 12.845 (R Square Change=0.997). According to Linear regression test, F=959.625, P=0.000, there is statistical significance. The Ag+-CHA has a well silver delivery capability, and the samples each are soaked into the liqiud of SBF, the titer of silver releasing after the 24 hours is 885.27±122.49μg/L at its peak, lower than that reported in the literature minimum inhibitory concentration of silver ions (MIC) 1.25μg/mL. Then gradually reduced,7 to 14 days to release the maintenance of a plateau, followed by a slow releasing in the following days a lower concentration. After 28 days, there are 79.80±6.69μg/L of silver ion release.
Conclusion:The Ag+-CHA has been able to remain a long time the release of silver ions closely related to the 3D porous structure itself. The reversible ion exchange process shows more visiblely releasing performance and more effectively avoid the sudden release of silver ions to maintain the sustained release for a long time, effectively reducing the use of metallic silver to achieve its effect.
Section three:Study on cytocompatibility of Silver-loaded coralline hydroxya-patite artificial bone
Objective:To develop antimicrobial Ag+-CHA artificial bone scaffold material and study on the cytocompatibility of mouse embryonic osteoblasts line (MC3T3-E1) in vitro.
Methods:The experiment was accomplished in Department of Orthopaedics, Guangzhou General Hospital of Guangzhou Military Command from June to September in 2009. Using cell culture technique in vitro, MC3T3-E1 cells were seeded on composites 10-3,8×10-5,10-5 mol/L Ag+-CHA, CHA and pure coral scaffold materials. Cell growth and reciprocity were monitored using MTT assay, ALP activity measurement, Inverted phase contrast microscope, Confocal laser scanning microscopes (CLSM), and SEM. The experimental data is analyzed with Univariate ANOVA on proliferation of osteoblasts by the SPSS 13.0.
Results:①The results of MTT assay can be seen that significant differences-exist among the different groups (F=76.457, P=0.000). Proliferation of MC3T3-E1 cells is not significantly different from that on CHA, pure coral, and the lower silver concentrations (P=0.177、0.428) at days 1,3,5, but is significantly higher on the control group and 170μg/ml Ag+/CHA group (P=0.000、0.000、0.000、0.000、0.000). Compared to blank control group, the proliferation of the cells cultured in each scaffold increased with the increasing of culture time besides the CHA soaked with 170μg/ml Ag+ ions;②The results of ALP can be seen significant differences exist among the different groups(F=15.582, P=0.000). The group 10-3mol/L has more significant difference than other groups(P=0.000,0.000,0.000,0.000,0.004), indicating that cells have been growing slowest in this group, and ALP has been secreting at least; 8×10-5mol/L group,10-5mol/L group have no significant difference compared with the control group (P=0.340、0.068); CHA group showed no significant difference compared with pure coral group(P=0.292), and no significant difference exist in 8×10-5mol/L group and 10-5mol/L group(P=0.366、0.292、0.880), but there was significant difference between the control group (P=0.049、0.006、0.004, respectively), indicating that 8×10-5mol/L group,10-5mol/L group had no significant toxicity on cells, suitable for cell growth and adhesion on its surface. Meanwhile, the spatial structure of 3D porous material expands the surface area of cell adhesion, and is bebefit for the rapid proliferation of cell. The results are consistent with the MTT test.③Surface topography of MC3T3-E1 cells seeding for 1 days:The transparent and round cells seeding on 10-3mol/L Ag+-CHA were free, folded and unadhered, presenting shrinkage and low proliferation; The osteoblasts were proliferated much better on the 8×10-5mol/L group,10-5mol/L group than that on the surface 10-3mol/L. The confluent cells were shaped in fusiform and arranged tightly in fasciculation or finger print, with the narrow interval and gathering growth.④CLSM image shows the morphology of the 8×10-5mol/L group is the same as the other two groups.⑤SEM image shows material cells were spindle-shaped than the flat surface of the tiny villi and folds, the cell contact area with the material protruding pseudopods formed duck webbed adhesive and wrapping materials, the surface shows that their secretion of bone matrix.
Conclusion:The MTT, ALP activity, inverted phase contrast microscopy, laser confocal and scanning electron microscopy observation of the full range of multi-angle cells in response to the natural framework of the material. The cell proliferation, growth and well, indicates that material has good cytocompatibility. The results of MTT and ALP activity quantitatively show that the low-silver content loaded in Ag+-CHA and non-loaded materials are basically the same in the biocompatibility.
Section four:Study on the repairment with Ag+-CHA bone graft in infected radial bone defects in rabbits
Objective:To investigate the therapeutic effect of the Ag+-CHA artificial bone on a large number of bone defects.
Methods:36 New Zealand white rabbits were selected, and randomly assigned to four groups with nine rabbits in each group. A rabbit model of infected and defect radial bone segment with 15mm. The animals all were followed by implant of Ag+-CHA in group A, CHA in group B, situ autogenous bone in group C and those in group D were left without anything grafting, as a control group. Respectively, specimens were harvestd after 2,6,10 weeks after the above procedures and were then subjected to gross observation, radiographic examination, histological observation to compare their therapeutic effect on bone tissue repairment. The experimental data is analyzed with Repeated-Measures ANOVA and LSD on the results of radiographic examination by the SPSS 13.0.
Results:The different groups of materials for repairing_bone defects:2w general observations can be seen when the A, B groups the surface of granulation tissue graft and some translucent bone coverage, with the host bone callus stump some connection between the graft-free apparent shift; C group graft and host bone callus, some connected to graft inactivity; D group to muscle bone defect filled with fibrous tissue.6w, when A, B groups the surface of thin bone graft covered stump with the host bone callus between the large number of connections, a more solid; C group of cortical bone graft and host continuous, but uneven, no significant graft shift; D group in order to muscle defect was still filled with fibrous tissue. 10w, when A, B two consecutive graft bone covering the surface of a clear, stable graft; C group graft and host bone cortex into one row; D group in order to muscle defect was still filled with fibrous tissue. X-ray results can be seen among the groups at different time points, there was a significant difference (F=11.537, P=0.000); Autogenous bone was the best than the other groups compared with the significant difference (P=0.004,0.010, 0.000), indicating that autologous bone tissue in large segmental bone defect repair is still the best materials; Ag+-CHA group and the CHA group showed no significant difference(P=0.524), indicating that lower silver content of Ag+-CHA was the same as non-loaded silver; Control group had significant differences compared with other groups (P=0.000,0.000,0.000), and the role of bone repairment was obviously inhibited owing to the infection of large segment of rabbit radial bone the bone defect, and the animal model is successful.
Conclusion:Based on the idea of bionics and previous study on scaffold material for bone repair, we prepared a new type of antimicrobial scaffold materials for infected bone defects. The composition and structure of bone material are similar to natural bone inorganic ingredients and porous structure of cancellous bone. The animal experiments in vivo proved that this material has good biocompatibility and bone conductivity, and is an effective bone repair-scaffold materials, can be achieved the same effect as non-silver materials for bone repairment, and there is hope to become the preferred antiinfected bone repair material and has broad market prospects.
In summary, the preparation process of anti-bacterial Ag+-CHA bone substitute materials is simple, and antimicrobial properties of silver ions is achieved more desirablely by slow-release. There are good bone conductivity and osteogenic effect in the large infective bone defect, it is conceivable that the anti-bacterial artificial bone material may have a widespread application prospect in treating infective bone defect to prevent infection as a good-assisted treatment.
实验分四个部分进行观察研究：
课题研究第一部分：载银珊瑚羟基磷灰石人工骨的制备及性能表征。
目的：制备载银珊瑚羟基磷灰石人工骨并观察其理化性能表征。
方法：实验于2008-05/08在清华大学材料科学与工程系实验室完成。实验材料：在加工好的珊瑚羟基磷灰石(Coral Hydroxyapatite, CHA)颗粒与相应浓度硝酸银(Silver nitrate, AgNO3)溶液浸泡一定时间后置入冻干机冻干制得载银珊瑚羟基磷灰石(Silver-loaded Coral Hydroxyapatite, SLCHA或Ag+-CHA)。应用X射线衍射法对Ag+-CHA、CHA和纯珊瑚样品晶体的物相、结构和位相进行分析；应用FT-IR光谱法对样品的材料组成进行分析；应用扫描电镜观察材料的显微形貌和孔径；利用压缩试验及三点弯曲试验评价骨修复支架材料的机械性能。应用SPSS13.0软件对机械性能测试结果行单向方差分析及LSD法两两比较(a=0.05)。
结果：①X射线衍射法对Ag+-CHA、CHA和纯珊瑚三种材料晶体进行物相分析、结构分析和位相分析,图形显示Ag+-CHA样品的衍射峰与CHA的图谱基本相符,而且XRD图中无其他相的衍射峰,说明银离子与CHA发生反应后产物比较单一,无其他衍生物生成,仍然保持原有的晶形结构；同时晶面位于(211),(300)的峰值较CHA低,说明银离子可能与此晶面位置的HA发生反应。②FT-IR光谱法图中显示三者具有一定的相似性,1456 cm-1和1413cm-1是材料中C032-基团的特征峰位,与纯珊瑚基本一致,说明Ag+-CHA和CHA材料本身大部分为CaCO3成分。1031 cm-1、604 cm-1和563cm-1为HA的特征峰,即显示出P04-3离子根的振动峰,说明Ag+-CHA和CHA中已存在一部分HA。这些HA特征波峰变宽,对FT-IR光谱进行分析,Ag+-CHA的曲线的变化可能与Ag+与CHA的Ca2+之间存在离子键变换、Ag+的离子半径大于Ca2+有关。③扫描电镜、能谱分析及背散射电镜可见载银珊瑚羟基磷灰石样品仍维持着珊瑚本身的三维多孔结构,载银材料表面存在有钙、碳、银、磷和氧等多种元素,且载银材料表面沉淀物的银含量与制备过程中硝酸银的浓度成正比关系；④采用压缩试验法分析这三种材料间的压缩强度无显著性差异(F=3.147,P=0.196)；三种材料间的压缩模量无显著性差异(F=0.827,P=0.482)；及采用三点弯曲试验法分析三种材料间的三点弯曲折断功无显著性差异(F=2.543,P=0.159)。
结论：珊瑚羟基磷灰石经载银后未改变其理化性能,依然维持着三维多孔框架结构；制备原理主要为钙银离子之间的置换反应,其次为银离子的溶出-沉积反应,并且随着制备过程中硝酸银溶液浓度的递增,Ag+-CHA的载银量也随之递增。
课题研究第二部分：载银珊瑚羟基磷灰石人工骨的载银量及体外缓释实验研究
目的：研制载银珊瑚羟基磷灰石抗菌性人工骨支架材料,通过对其自身载银量及体外缓释实验完善对该抗菌性磷灰石类复合人工骨的认识。
方法：应用电感耦合等离子体发射光谱(inductively coupled plasma optical emission spectrometry, ICP-OES)测定载银材料中银的平均含量,并在模拟体液(Stimulated body fluid, SBF, pH=7.25)中通过体外释放试验测定银离子释放浓度与时间的变化曲线,探讨材料结构与释放银的关联性。实验数据应用SPSS13.0统计软件处理,用相关回归分析和曲线拟合载银量与AgNO3浓度关系曲线,建立回归方程。
结果：根据实验结果绘制的载银量-AgNO3浓度关系曲线。经统计学分析拟合出载银量-AgNO3浓度曲线方程为：y=3E+007x2+126863.3x+12.845(R SquareChange=0.997),对方程进行回归检验,F=959.625,P=-0.000,有统计学意义。载银羟基磷灰石颗粒3个试样中银离子均能从颗粒向模拟体液中扩散,具有明显缓释作用。第1个24小时释放量最大,达到885.27±122.49μg/L,低于文献中报道的银离子最低抑菌浓度(MIC) 1.25μg/mL.,达到减毒增效的效果。以后逐渐减少,7-14天维持一平台期释放,其后以较低浓度维持,第28天仍有79.80±6.69μg/L的银离子释放量。
结论：载银珊瑚羟基磷灰石之所以能保持长时间的银离子释放,与羟基磷灰石本身的空间晶格结构关系密切,这种可逆的离子置换过程使其释放性能更显著,能更有效地避免银离子突释,保持长时间的持续释放,有效地减少金属银的使用量,达到减毒增效的作用。
课题研究第三部分：载银珊瑚羟基磷灰石人工骨的细胞相容性研究
目的：研制载银珊瑚羟基磷灰石抗菌性人工骨支架材料,观察其在体外与小鼠胚胎成骨细胞株(MC3T3-E1)的细胞相容性。
方法：实验于2009-06/09在广州军区广州总医院实验科完成。采用体外细胞培养技术,将MC3T3-E1细胞种植于10-3、8×10-5、10-5mol/L Ag+-CHA, CHA及纯珊瑚支架材料上。实验评估：①MTT法测定MC3T3-E1细胞接种在不同支架材料上生长1,3,5d后的吸光光度值,绘制细胞在不同骨材料上的生长曲线,评价材料上细胞的增殖情况；②碱性磷酸酶(ALP)活性法测定细胞接种在不同支架材料上2,4,6d的吸光光度值,计算培养液中碱性磷酸酶值；③倒置相差显微镜下直接观察培养板上MC3T3-E1细胞的生长形貌；④应用激光共聚焦显微镜(Confocal laser scanning microscopes, CLSM)直接观察支架材料上MC3T3-E1细胞的生长形貌；⑤应用扫描电镜(SEM)观察细胞在材料上生长的微观形貌。应用SPSS 13.0软件对各框架材料上的细胞生长情况结果行析因方差分析及LSD法两两比较,(α=0.05)。
结果：①成骨细胞在各组材料上的增殖能力有显著性差异(F=76.457,P=0.000)。CHA组与8×10-5mol/L组比较有显著性差异(P=0.019),而与10-5mol/L组及纯珊瑚组两两比较均无显著性差异(P值分别为0.177、0.428),成骨细胞在CHA组上的活性最高,增殖能力最强,增长速度最快,其次为纯珊瑚组、10-5mol/L组及8×10-5mol/L组；而细胞在10-3mol/L组上的增殖能力最低,材料上细胞数目最少,与其他组比较均有显著性差异(P值分别为0.000、0.000、0.000、0.000、0.000),各组与对照组相比较均有显著性差异(P值分别为0.000、0.000、0.000、0.000、0.000),表明8×10-5、10-5mol/L Ag+-CHA及其他非载银材料适合用于培养成骨细胞,对细胞无明显毒性,可诱导细胞迅速增长,而10-3mol/L Ag+-CHA不适合成骨细胞在其上生长。②各组之间比较有显著性差异(F=15.582,P=0.000)；10-3mol/L组与其他各组之间比较有显著性差异(P值分别为0.000、0.000、0.000、0.000、0.000),说明细胞在此组材料上生长最慢,分泌ALP最少；8×10-5mol/L组与对照组比较无显著性差异(P值为0.292),而8×10-5mol/L组与10-5mol/L组及CHA组两两比较无显著性差异(P值分别为0.340、0.068),说明随着材料载银量的减少,上述载银材料对细胞已无明显毒性作用；10-5mol/L组、CHA组、纯珊瑚组三组之间比较无显著性差异(P值分别为0.366、0.292、0.880),而与对照组比较有显著性差异(P值分别为0.049、0.006、0.004),说明三组材料适合细胞在其表面粘附生长,同时材料的三维多孔空间结构拓展了细胞粘附的表面积,有利于细胞快速增殖,其结果与MTT试验结果一致。③MC3T3-E1细胞与不同框架材料联合培养1d时的生长形貌：10-3mol/L Ag+-CHA培养板上细胞未贴壁生长,为透明的圆球形,呈游离状态,未能很好舒展,且有些皱缩,生长活力也不旺盛；细胞与8×10-5mol/L组、10-5mol/L组、CHA组及纯珊瑚组联合培养的生长情况要明显好于10-3mol/L组,细胞相互融合成片,多呈长梭形,细胞紧密排列成束,聚集生长的趋势更明显,这些结果与MTT及ALP结果相一致。④CLSM图像显示三组材料上的细胞数量及形貌无明显差别。⑤SEM图像显示材料上细胞呈梭形,较扁平,表面有细小绒毛与皱褶,细胞在与材料接触部位伸出伪足,形成鸭蹼状粘附并包绕材料,表面可见其分泌的骨基质。
结论：通过MTT、ALP活性、倒置相差显微镜、激光共聚焦及扫描电镜全方位多角度观察了细胞在天然框架材料上的反应。细胞增殖、生长良好,表明材料具有良好的细胞相容性。MTT、ALP活性的定量结果表明低载银量珊瑚羟基磷灰石材料与非载银骨替代材料的生物相容性基本一致。
课题研究第四部分：载银珊瑚羟基磷灰石人工骨修复兔桡骨污染性骨缺损的实验研究
目的：通过体内动物实验检测该抗菌性人工骨植入动物体内的组织相容性、骨传导性和材料的生物降解等组织自愈合生物学性能。
方法：新西兰大白兔36只,随机分为4组,每组9只。选用新西兰大白兔制作兔桡骨15mm节段性污染性骨与骨膜缺损模型,将36只大白兔称重,按体重大小顺序编号,按随机数目表法随机分为A-D组。A组植入载银珊瑚羟基磷灰石材料；B组植入珊瑚羟基磷灰石材料；C组植入原位自体骨材料；D组无任何植入,做为对照组。分别于术后2,6,10周分三次处死动物取材,每次每组3只,通过大体观察、影像学检查、组织学检查观察比较各组骨缺损修复情况,应用SPSS13.0软件对X线观察结果行重复测量方差分析及LSD法两两比较,α=0.05,比较上述各组对大段骨缺损的修复作用。
结果：不同组材料对骨缺损修复的大体观察结果可见2w时A、B两组移植物表面有肉芽组织和部分半透明骨质覆盖,与宿主骨断端间有部分骨痂连接,移植物无明显移位；C组移植物与宿主骨有部分骨痂连接,移植物无活动；D组骨缺损区填充以肌纤维组织。6w时A、B两组移植物表面有薄层骨质覆盖,与宿主骨断端间有大量骨痂连接,较稳固；C组移植物与宿主骨皮质连续,但不均匀,移植物无明显移位；D组缺损区仍填充以肌纤维组织。10w时A、B两组移植物表面有明显连续骨质覆盖,移植物稳固；C组移植物与宿主骨皮质连续成一体；D组缺损区仍填充以肌纤维组织。X线检查结果可见不同时间点各组之间比较有显著性差异(F=11.537,P=0.000)；自体骨成骨情况最好,与其他各组之间比较有显著性差异(P值分别为0.004、0.010、0.000),说明自体骨组织在大段骨缺损修复时仍然是最好的材料；Ag+-CHA组与CHA组两组比较无显著性差异(P值分别为0.524),说明珊瑚羟基磷灰石经一定量载银后其成骨作用未受明显影响；对照组与其他各组之间比较有显著性差异(P=0.000、0.000、0.000),说明污染性兔桡骨大段骨缺损后其成骨作用明显受到抑制,其模型制作是成功的。
结论：根据仿生的思路和以往对骨修复框架材料的研究,我们制备了一种新型的抗菌性框架材料用于污染性骨缺损修复。此材料成分和结构均与天然骨无机成分有相似性,多孔结构与松质骨类似。通过体内动物实验,证明了此材料具有良好的生物相容性和骨传导性,是有效的骨修复框架材料,可以达到与自体骨材料相同的效果,有希望成为污染性骨缺损修复的优选材料,具有广阔的市场前景。
综上所述,抗菌性载银珊瑚羟基磷灰石骨替代材料的制备过程简单,抗菌剂银离子的缓释性能较为理想,在污染性大段骨缺损处有良好的骨传导性及成骨作用,可以预想该抗菌人工骨材料是一种治疗污染性骨缺损的良好骨修复材料。
The most ideal biological material is the personal bone tissue from oneself. As the evolution of natural biological materials for billions of years, biological materials are formed by a structure-complicated and sophisticated, wonderful principles and mechanism. As a complex, fresh autogenous bone is ingeniously combined with the inorganic and organic materials together. Hydroxyapatite mostly in inorganic materials are of highly osteoinductive, non-immunological rejection response. But autogenuous bone graft will increase the surgical trauma, and lead to local pain, paresthesia, or allergies and other complications in patients, and the source of bone is very limited. In recent years, the use of tissue-engineering bone to repair bone defects, not only has provided a method of repairing bone defects, but also has provided a new idea. New hope has been brought for the treatment of bone defects because of bone tissue engineering research. At present, artificial bones constructed by tissue engineering in vitro still remain in the laboratory and animal-experimental segments, but with the development of the related subjects, such as cell biology, engineering, immunology, material science, the research of bone tissue engineering will make a breakthrough progress and there is an extremely broad application prospects in industrial production and clinical applications. It will become a great potential for the development of high-tech industries in the 21st century, and offer enormous economic and social benefits. As the main inorganic component of human bone tissue, hydroxyapatite bioceramic is cheap, and has good biocompatibility, biodegradability, bone conductivity, and bone fusion. Particularly the porous coral hydroxyapatite can provide a lot of surface area/volume ratio, can ensure cell proliferation, differentiation, and high metabolic activity. Then it is particularly suitable for bone tissue engineering. With the increased mechanization of modern industrialization, a large number of significantly infectious bone defects increase caused by trauma, infection, cancer, accidents, wars, earthquakes. The traditional methods of treatment is that there is a debridement at first until the wound closed, and no signs of infection for 3 to 6 months, then there is a bone repairment secondly. The higher infective rate of bone graft in one-stage often occurs, and it has been regarded as taboos. And the clinical bacterial resistance to antibiotics is a growing problem, the role of fighting against microbial bone in antibiotics-loaded graft material is taken more and more limit, and microbial agent has been put forward higher requirements. To solve the traditional treatment methods to bring a range of issues, such as increasing treatment time, the difficulty, the patient's suffering and economic burden, traditional attitudes have been changed in recent years. Domestic and foreign scholars has made some progress for the anti-infective bone graft materials in basic experiments and clinical practice. To the end, we are cooperating with the Department of Materials Science and Engineering in Tsinghua University to develop an anti-infective bone scaffold materials, and a series of experimental research have shown that:The antibacterial bone repairment material has good biocompatibility, and can release slowly silver ions to effectively inhibit local residual bacteria in infective bone defect after debridement, as follow.
This experiment divides into four parts:
Section one:Development and Characterization of Silver-loaded coralline hydroxyapatite (Ag+-CHA) artificial bone.
Objective:To develop Ag+-CHA artificial bone and study the physical and chemical characterization.
Methods:The experiment was accomplished in Tsinghua University, Laboratory of Materials Science and Engineering from May to August in 2008. Experimental material:Ag+-CHA was prepared by immersing coralline hydroxyapatite (Coral Hydroxyapatite, CHA) particles into the some concentration of silver nitrate (Silver nitrate, AgNO3) solution, and frozen in freezed dryer after a certain time. X-ray diffraction method is applied on the Ag+-CHA, CHA and pure coral to investigate their crystal phase, structure and phase analysis; FT-IR spectroscopy is applied to analyze the composition of the sample material; SEM micrograph is applied to materials morphology and the aperture; Compression tests and three-point bending test are used to evaluate mechanical properties of bone repair scaffold. The experimental data is analyzed with One-Way ANOVA and LSD on mechanical properties by the software SPSS13.0.
Results:①X-ray diffraction results revealed the characteristic peaks of Ag+-CHA, CHA, and pure coral, and there was no secondary phases, indicating that there was no other derivatives generated in the reaction between silver ions and the CHA, remaining the original crystalline structure; the same crystal plane phase at (211), (300) peak were lower than the CHA's. It showed that silver ions might react with the location of the HA crystal surface.②FT-IR spectroscopy are shown that they have a certain similarity,1456 cm-1 and 1413cm-1 are the material characteristic peaks of CO32-group, the same as pure coral. It showed that the most component of Ag+-CHA and CHA was CaCO3. The characteristic peaks of HA are 1031 cm-1,604 cm-1 and 563cm-1, shows that HA exists in the part of the Ag+-CHA and CHA already. The broadened HA features peaks on the FT-IR spectrum may be related to the transformation between silver ion of Ag+-CHA and the calcium ion of CHA, because of Ag+ ionic radius is larger than the Ca2+.③The SEM and BSE images showed that the morphology of the SLCHAs depended on the content of Ag+, and the silver ions were uniformly distributed on the surface of SLCHAs. EDS results demonstrated that the silver content of the SLCHAs decreased along with the decrease of the concentration of silver nitrate.;④compression test method was used to analyze the compressive strength among the three kinds of materials, and there was no significant difference (F=3.147, P=0.196), the P values were 0.323,0.695,0.403 compared among them; The compression modulus among three kinds of material was also no significant difference (F=0.827, P=0.482), the P values were 0.491,0.604,0.247 compared among them; and the broken power in the three-point bending test was no significant difference (F=2.543, P=0.159), the P values were 0.080,0.731,0.131 compared.
Conclusion:The physical and chemical properties of coralline hydroxyapatite still was maintained after loading silver ion and a three-dimensional porous framework has not been changed; The principle of Preparation is mainly the replacement reaction between silver ions and calcium, followed by the dissolution of silver ions-deposition. And with the increase of the concentration of silver nitrate solution, the content of silver in Ag+-CHA increased.
Section two:Study on the content of silver in Ag+-CHA and releasing character in vitro.
Objective:To develop antimicrobial Ag+-CHA artificial bone scaffold material, and the knowledge of Ag+-CHA is taken by silver content and releasing experiment in vitro.
Methods:The total silver content in the SLCHAs and the average concentration in the releasing test were determined by inductively coupled plasma emission spectroscopy (ICP-OES), calculated the content of silver loading refer to the standard curve of realationship between the content of Ag and the concentration of AgN03. Simulated body fluid (SBF, pH=7.25) was used in the releasing test in vitro, and we try to reveal the relationship between the materials' strucure and the silver releasing. The experimental data is analyzed with Linear Regression and Curve Fit by the SPSS13.0.
Results:The equation of Linear Regression:y=3E+007 x2+126863.3x+ 12.845 (R Square Change=0.997). According to Linear regression test, F=959.625, P=0.000, there is statistical significance. The Ag+-CHA has a well silver delivery capability, and the samples each are soaked into the liqiud of SBF, the titer of silver releasing after the 24 hours is 885.27±122.49μg/L at its peak, lower than that reported in the literature minimum inhibitory concentration of silver ions (MIC) 1.25μg/mL. Then gradually reduced,7 to 14 days to release the maintenance of a plateau, followed by a slow releasing in the following days a lower concentration. After 28 days, there are 79.80±6.69μg/L of silver ion release.
Conclusion:The Ag+-CHA has been able to remain a long time the release of silver ions closely related to the 3D porous structure itself. The reversible ion exchange process shows more visiblely releasing performance and more effectively avoid the sudden release of silver ions to maintain the sustained release for a long time, effectively reducing the use of metallic silver to achieve its effect.
Section three:Study on cytocompatibility of Silver-loaded coralline hydroxya-patite artificial bone
Objective:To develop antimicrobial Ag+-CHA artificial bone scaffold material and study on the cytocompatibility of mouse embryonic osteoblasts line (MC3T3-E1) in vitro.
Methods:The experiment was accomplished in Department of Orthopaedics, Guangzhou General Hospital of Guangzhou Military Command from June to September in 2009. Using cell culture technique in vitro, MC3T3-E1 cells were seeded on composites 10-3,8×10-5,10-5 mol/L Ag+-CHA, CHA and pure coral scaffold materials. Cell growth and reciprocity were monitored using MTT assay, ALP activity measurement, Inverted phase contrast microscope, Confocal laser scanning microscopes (CLSM), and SEM. The experimental data is analyzed with Univariate ANOVA on proliferation of osteoblasts by the SPSS 13.0.
Results:①The results of MTT assay can be seen that significant differences-exist among the different groups (F=76.457, P=0.000). Proliferation of MC3T3-E1 cells is not significantly different from that on CHA, pure coral, and the lower silver concentrations (P=0.177、0.428) at days 1,3,5, but is significantly higher on the control group and 170μg/ml Ag+/CHA group (P=0.000、0.000、0.000、0.000、0.000). Compared to blank control group, the proliferation of the cells cultured in each scaffold increased with the increasing of culture time besides the CHA soaked with 170μg/ml Ag+ ions;②The results of ALP can be seen significant differences exist among the different groups(F=15.582, P=0.000). The group 10-3mol/L has more significant difference than other groups(P=0.000,0.000,0.000,0.000,0.004), indicating that cells have been growing slowest in this group, and ALP has been secreting at least; 8×10-5mol/L group,10-5mol/L group have no significant difference compared with the control group (P=0.340、0.068); CHA group showed no significant difference compared with pure coral group(P=0.292), and no significant difference exist in 8×10-5mol/L group and 10-5mol/L group(P=0.366、0.292、0.880), but there was significant difference between the control group (P=0.049、0.006、0.004, respectively), indicating that 8×10-5mol/L group,10-5mol/L group had no significant toxicity on cells, suitable for cell growth and adhesion on its surface. Meanwhile, the spatial structure of 3D porous material expands the surface area of cell adhesion, and is bebefit for the rapid proliferation of cell. The results are consistent with the MTT test.③Surface topography of MC3T3-E1 cells seeding for 1 days:The transparent and round cells seeding on 10-3mol/L Ag+-CHA were free, folded and unadhered, presenting shrinkage and low proliferation; The osteoblasts were proliferated much better on the 8×10-5mol/L group,10-5mol/L group than that on the surface 10-3mol/L. The confluent cells were shaped in fusiform and arranged tightly in fasciculation or finger print, with the narrow interval and gathering growth.④CLSM image shows the morphology of the 8×10-5mol/L group is the same as the other two groups.⑤SEM image shows material cells were spindle-shaped than the flat surface of the tiny villi and folds, the cell contact area with the material protruding pseudopods formed duck webbed adhesive and wrapping materials, the surface shows that their secretion of bone matrix.
Conclusion:The MTT, ALP activity, inverted phase contrast microscopy, laser confocal and scanning electron microscopy observation of the full range of multi-angle cells in response to the natural framework of the material. The cell proliferation, growth and well, indicates that material has good cytocompatibility. The results of MTT and ALP activity quantitatively show that the low-silver content loaded in Ag+-CHA and non-loaded materials are basically the same in the biocompatibility.
Section four:Study on the repairment with Ag+-CHA bone graft in infected radial bone defects in rabbits
Objective:To investigate the therapeutic effect of the Ag+-CHA artificial bone on a large number of bone defects.
Methods:36 New Zealand white rabbits were selected, and randomly assigned to four groups with nine rabbits in each group. A rabbit model of infected and defect radial bone segment with 15mm. The animals all were followed by implant of Ag+-CHA in group A, CHA in group B, situ autogenous bone in group C and those in group D were left without anything grafting, as a control group. Respectively, specimens were harvestd after 2,6,10 weeks after the above procedures and were then subjected to gross observation, radiographic examination, histological observation to compare their therapeutic effect on bone tissue repairment. The experimental data is analyzed with Repeated-Measures ANOVA and LSD on the results of radiographic examination by the SPSS 13.0.
Results:The different groups of materials for repairing_bone defects:2w general observations can be seen when the A, B groups the surface of granulation tissue graft and some translucent bone coverage, with the host bone callus stump some connection between the graft-free apparent shift; C group graft and host bone callus, some connected to graft inactivity; D group to muscle bone defect filled with fibrous tissue.6w, when A, B groups the surface of thin bone graft covered stump with the host bone callus between the large number of connections, a more solid; C group of cortical bone graft and host continuous, but uneven, no significant graft shift; D group in order to muscle defect was still filled with fibrous tissue. 10w, when A, B two consecutive graft bone covering the surface of a clear, stable graft; C group graft and host bone cortex into one row; D group in order to muscle defect was still filled with fibrous tissue. X-ray results can be seen among the groups at different time points, there was a significant difference (F=11.537, P=0.000); Autogenous bone was the best than the other groups compared with the significant difference (P=0.004,0.010, 0.000), indicating that autologous bone tissue in large segmental bone defect repair is still the best materials; Ag+-CHA group and the CHA group showed no significant difference(P=0.524), indicating that lower silver content of Ag+-CHA was the same as non-loaded silver; Control group had significant differences compared with other groups (P=0.000,0.000,0.000), and the role of bone repairment was obviously inhibited owing to the infection of large segment of rabbit radial bone the bone defect, and the animal model is successful.
Conclusion:Based on the idea of bionics and previous study on scaffold material for bone repair, we prepared a new type of antimicrobial scaffold materials for infected bone defects. The composition and structure of bone material are similar to natural bone inorganic ingredients and porous structure of cancellous bone. The animal experiments in vivo proved that this material has good biocompatibility and bone conductivity, and is an effective bone repair-scaffold materials, can be achieved the same effect as non-silver materials for bone repairment, and there is hope to become the preferred antiinfected bone repair material and has broad market prospects.
In summary, the preparation process of anti-bacterial Ag+-CHA bone substitute materials is simple, and antimicrobial properties of silver ions is achieved more desirablely by slow-release. There are good bone conductivity and osteogenic effect in the large infective bone defect, it is conceivable that the anti-bacterial artificial bone material may have a widespread application prospect in treating infective bone defect to prevent infection as a good-assisted treatment.
引文
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