数字化载银珊瑚羟基磷灰石人工骨的制备及生物学研究
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摘要
研究背景
在世界范围内,由于创伤,肿瘤和感染等原因造成的骨缺损每年都在折磨着众多的患者,需要进行植骨移植。然而,临床上对于大面积污染性骨缺损的治疗使用常规的植骨材料有可能因继发感染而致骨移植失败。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,从而对抗菌剂提出了更高的要求。近年来,无机抗菌材料以其有效的抗菌性、较高的安全性和稳定性引起了广泛关注。无机抗菌剂与有机天然抗菌剂相比,具有抗菌谱广、长效持久、不产生耐药性、无毒副作用等优点。Ag+的抗菌性能是金属离子中最强的,亦是应用最早、最成熟的无机抗菌剂。
长期以来,研制具有抗菌功能的生物材料替代自体骨修复骨缺损一直是医学和材料学领域的重要课题。而骨组织工程为骨缺损,特别是复杂结构的骨缺损的修复带来了新的期盼。骨组织工程包括三个关键因素：信号分子(骨生长因子、骨诱导因子)、支架材料和靶细胞。其中支架材料的选择是骨组织工程的核心问题。目前的支架材料主要有天然的支架材料、人工合成的支架材料以及复合材料。
珊瑚羟基磷灰石(CHA)是由天然珊瑚通过“水热反应”转变而来的羟基磷灰石,保留了天然珊瑚的多孔结构,生物相容性好,具有较大的孔径、较高的孔隙率和孔隙交通率。大量的文献表明珊瑚类人工骨为一类较为理想的骨替代材料,并因为它的良好组织相容性和三维多孔结构,也被认为是有前途的组织工程支架材料和载体。但CHA人工骨脆性较大,无法根据骨缺损的形状个性化地制备出人工骨支架,存在一定不足。
左旋-聚乳酸(L-polylactic acid, L-PLA)是一种已在医学上广泛应用的有机高分子材料,作为人体的植入物已经通过美国食品药品管理局的认证,并已被广泛用于内固定装置例如骨板、骨钉、手术缝合线等。其优点是可降解性,良好的细胞相容性,容易塑形。
然而,单一类型材料(天然支架或人工合成支架)一般难以满足骨组织工程用于细胞外支架材料的要求。天然材料具有生物相容性好、细胞识别信号、利于细胞黏附增殖；人工材料虽然缺乏细胞信号,但具有天然材料所不足的可以大规模生产、可以设计和控制结构、机械性能和降解时间等优点。通过一定的方法将由几种不同材料相结合形成,或者由生物材料与生长因了复合而成,复合材料在性能上互相取长补短,在实际应用中已经取得了良好效果。天然生物衍生材料、高分子材料、陶瓷材料既可以作为复合材料的基材,又可作为其增强体和填料,他们互相搭配和组合形成大量性质各异的复合材料。
快速成形技术(Rapid Prototyping Technology, RPT)是种基于离散堆积成型思想的新型成形技术,可快速、高精度地从CT数据完成快速成形生物模型的制造。它自20世纪80年代产生以来得到了迅速的发展,是制造技术领域出现的一次重大突破,其对制造业的影响完全可以与数控技术相媲美。近年来,已广泛应用于医学领域,可以制造任意复杂形状的三维实体模型,为骨组织工程支架的个性化制造提供了可能。
如果能够利用快速成形技术,在保留珊瑚羟基磷灰石的天然微孔样结构的基础上,载入银离子,根据骨缺损的形态,个性化地制备出具有抗菌性人工骨支架修复材料,这将极大地促进抗菌性骨组织工程支架的发展。
本研究将珊瑚羟基磷灰石(CHA),通过溶液离子交换法,成功载入银离子,制备出不同含银量的载银羟基磷灰石抗菌粉末,与聚乳酸(PLA)混合后,通过选择性激光烧结快速成形制备出具有特殊形状的数字化载银抗菌人工骨(CHA-Ag)支架材料。通过系列实验,研究CHA/PLA-Ag人工骨支架材料的理化学特性、抗菌性、生物相容性,通过修复动物大段污染性骨缺损模型,探讨其应用于感染性骨缺损的可行性。
研究目的
1、研究载银数字化珊瑚羟基磷灰石／聚乳酸人工骨的制备及性能表征。
2、研究载银数字化珊瑚羟基磷灰石／聚乳酸人工骨的细胞相容性。
3、研究载银数字化珊瑚羟基磷灰石／聚乳酸人工骨体外抑菌性。
4、研究载银数字化珊瑚羟基磷灰石／聚乳酸人工骨载银量及体外释放速率。
5、探讨载银数字化珊瑚羟基磷灰石／聚乳酸人工骨的组织相容性与修复大段污染性骨缺损的动物实验。
研究方法
1.将珊瑚羟基磷灰石,经球磨机球磨后成细颗粒粉末状,过目筛后取颗粒大小为140-200μ m,分别放在含有10-2、10-3、10-4、10-5mmol/LAgNO3溶液避光浸泡,制备出含不同质量银的载银羟基磷灰石粉末,然后将其与L-PLA以3：1质量比混均后,采用Simpleware3.1软件,通过CAD构建一数字化的圆柱形模型,通过数控转换,将其生成STL格式的文件,导入快速成型机中,采用选择性激光烧结快速成型的工艺,将数字化的模型制备成制备出含不同质量银的载银羟基磷灰石人工骨支架材料(CHA/PLA-Ag)。采用扫描电镜、红外光谱仪、X射线衍射仪对数字化载银羟基磷灰石抗菌人工骨进行理化、结构分析。
2.体外抗菌性能实验
2.1抑菌圈实验：复苏金黄色葡萄球菌ATCC25923及大肠杆菌ATCC25922,接种于含LB液体培养基试管中,于37℃恒温摇床中培养1d备用。取浸泡10-2、10-3、10-4、10-5mmol/LAgNO3所制备的5种珊瑚羟基磷灰石人工骨圆柱状材料各1粒,分别置于接种有金黄色葡萄球菌(接种量为1.5×107CFU)及大肠杆菌(接种量为1.5×107CFU)的90mm的LB琼脂培养基中,青霉素液滴作为阳性对照组；于37℃恒温培养箱中培养24小时,测量抑菌圈,记录抑菌圈大小。每个菌株重复6次。
2.2菌落计数实验：将金黄色葡萄球菌及大肠杆菌配制成浓度为3×108CFU/mL的细菌悬液。取100μ L细菌悬液分别滴于载银及未载银珊瑚羟基磷灰石人工骨材料表面,于37℃恒温培养箱中培养24小时。将材料置于5mL PBS液中,在漩涡振荡仪上振荡5min,洗脱细菌。取100μ L洗脱液用PBS稀释至适当浓度,推平板,于37℃恒温培养箱中培养3d后计平板菌落数及计算抗菌率。每个菌种实验重复6次。
3.参照GB/T16886.1-2001idt ISO10993-1:1997和GB/T16175-2008,对载银羟基磷灰石人工骨支架材料,进行细胞毒性实验、溶血反应、急性毒性实验、迟发性超敏反应实验的生物学评价。
3.1细胞毒性实验：采用体外细胞培养技术,观察载银羟基磷灰石人工骨支架材料浸提液对L-929细胞形态的变化,采用MTT法检测对L-929细胞增殖情况的影响,以评价其细胞毒性。
3.2溶血反应：检测载银羟基磷灰石人工骨支架材料浸提液的对新西兰大白兔溶血率,评价材料的溶血反应。
3.3急性毒性实验：小鼠腹腔注射载银羟基磷灰石人工骨支架材料的浸提液,观察小鼠的毒性反应,评价材料的毒性。
3.4迟发性超敏反应实验：豚鼠皮内注射载银羟基磷灰石人工骨支架材料浸提液,观察豚鼠皮肤过敏反应情况,评价材料的致敏反应。
4.将不同质量银的载银羟基磷灰石人工骨支架材料,采用原子吸收光谱仪
对材料进行Ag+含量分析。将10q组载银羟基磷灰石人工骨支架材料分别放置于初始pH值为7.4的15mL模拟体液(stimulated body fluid, SBF)中,在37℃恒温箱中降解,并分别于1d、4d、7d、14d、21d、28d、49d检测其在模拟体液中的银离了释放量和速度。
5.于新西兰大白兔胫骨干骺端构建5mm×15mm的污染性腔隙性骨缺损,实验组植入10-3mmol/L AgNO3载银羟基磷灰石人工骨支架材料修复骨缺损,对照组骨缺损区植入未载银的羟基磷灰石／聚乳酸人工骨支架材料,空白对照组骨缺损区不植入任何材料。于术后第1天,第4周、8周、12周行X线摄片,观察骨缺损修复及骨塑形情况。参照Lane-Sandhu X线评分标准对各组胫骨缺损的骨修复程度评分,标本行HE染色组织学观察以了解骨修复情况。
结果
1.采用珊瑚羟基磷灰石(CHA)粉末浸泡不同浓度的硝酸银溶液,通过溶液离子交换法,制备出不同含银量的载银羟基磷灰石,再将不同的载银CHA粉末与左旋聚乳酸(L-PLA)按3：1质量比混均,并通过CAD和选择性激光烧结快速成形技术,可以成功制备出具有特殊形状的数字化载银抗菌人工骨支架材料。数字化CHA-Ag人工骨制备后为灰白色,其中浸泡10-2mol/L AgNO3载银珊瑚羟基磷灰石人工骨为棕黑色,其粉末制备成型前仍为灰白色,考虑为其载银量相对较高,制备成型时银离子经激光束强光照射后与氧结合成氧化银所致。制备的人工骨支架表面粗糙,呈规整的小圆柱体状,粗细均匀,可见微孔结构。各组材料的抗压强度均大于1Mpa。
2.体外抗菌实验。抑菌圈实验：浸泡10-4mmol/AgNO3、10-5mmol/L AgNO3浓度硝酸银所制备的数字化CHA-Ag人工骨材料在整个实验过程中对金黄色葡萄球菌和大肠杆菌均无抑菌圈产生。浸泡10-2mmol/L AgNO3、10-3mmol/L AgNO3浓度硝酸银所制备的数字化CHA-Ag人工骨材料24h对2种细菌均产生抑菌圈,其中浸泡10-2mmol/L AgNO3载银人工骨材料对金黄色葡萄球菌和大肠杆菌的抑菌圈直径分别为(13.00±0.71)mm及(12.30±0.71)mm,浸泡10-33mmol/LAgNO3载银人工骨材料分别平均为(11.51±0.09)mm及(11.00±0.15)mm。金黄色葡萄球菌各材料间以及与青霉素对照组比较,单因素方差分析统计结果显示各组方差不齐(F=5.149,P=0.020),组间差异有显著性统计学意义(F=95370.727,P=0.000),两两比较均有显著性统计学意义(P=0.000)。大肠杆菌各材料间以及与青霉素对照组比较,单因素方差分析统计结果显示各组方差齐性(F=1.585,P=0.237),组间差异有显著性统计学意义(F=2692.000,P=0.000),两两比较均有显著性统计学意义(P=0.000)。
菌落数实验：金黄色葡萄球菌、大肠杆菌与浸泡10-2mmol/L AgNO3载银CHA-Ag人工骨材料接触后无生长繁殖,抗菌率为100%；与浸泡10-3、10-4、10-5mmol/L AgNO3载银CHA-Ag人工骨材料接触后生长减缓,表明有抗菌作用,对金黄色葡萄球菌抗菌率分别为99.99±0.00%、49.42±0.28%及22.18±0.26%；对大肠杆菌抗菌率分别为99.99±0.00%、44.66±0.44%及21.09±0.32%。金黄色葡萄球菌各材料间比较,单因素方差分析统计结果显示各组方差不齐(F=6.154,P=0.004),组间差异有显著性统计学意义(F=2E+007,P=0.000),两两比较均有显著性统计学意义(P=0.000)。大肠杆菌各材料间比较,单因素方差分析统计结果显示各组方差不齐(F=15.792,P=0.000),组间差异有显著性统计学意义(F=1E+007,P=0.000),两两比较均有显著性统计学意义(P=0.000)。
3.100%浸泡10-2mol/L AgNO3的CHA-Ag人工骨浸提液作用于L929细胞2、4、7d,大部分细胞死亡,呈圆形,生长欠佳,对细胞有毒性,毒性分级为3级；而10-3、10-4、10-5mmol/L AgNO3的CHA-Ag人工骨浸提液作用于L929细胞2、4、7d后,细胞生长旺盛,倒置显微镜下观察,细胞膜完整,细胞呈典形的梭形,贴壁良好,毒性分级分别为1级、0级、0级。实验结果表明10-mmol/LAgNO3的CHA-Ag人工骨材料浸提液的细胞毒性均为0级；溶血率为：2.22%,小于5%,符合生物医学要求；急性毒性程度分级均为0级；致敏反应记分均为0分。表明10-3mmol/L AgNO3的CHA-Ag人工骨材料符合生物材料医用标准。
4.10-2、10-4、10-4、10-5组Ag+含量分别为2310.520±33.418、318.692±1.763、67.535±0.191及6.050±0.028μ g/g,单因素方差分析统计结果显示各组方差不齐(F=9.005,P=0.001),组间差异有显著性统计学意义(F=25847.567,P=0.000),两两比较均有显著性统计学意义(P=0.000)。10-3材料在体外SBF中Ag+呈缓慢释放趋势,在最初浸泡7d内释放速度较快,之后释放速度逐渐减慢。
5.各组实验动物术后进食基本正常,2周后均能自由活动。其中10-3CHA/PLA-Ag组术口无明显红肿,无裂开,无渗液流脓,愈合良好；CHA/PLA组术口愈合差,裂开,术口红肿渗液流脓,取分泌物行细菌培养,为金黄色葡萄球菌,术后2周术口软组织肿胀减轻,逐渐愈合；空白对照组术口度红肿,无明显渗液流脓,1周后术口愈合良好。术后3月未出现动物死亡,未出现病理性骨折。大体观实验组及CHA/PLA组兔胫骨缺损完全愈合；空白对照组缺损基本无骨修复作用,由纤维组织充填。实验组、CHA/PLA组及空白对照组的放射学检查评分同一时间点比较,单因素方差分析统计结果显示各组方差齐性(P>0.05),组间差异有显著性统计学意义(P<0.05),其中实验组、CHA/PLA组各时间点评分差别无显著性统计学意义(P>0.05),说明实验组、CHA/PLA组的骨修复作用无显著差异,而实验组、CHA/PLA组与空白对照组各时间点评分差异有显著性统计学意义(R<0.05),实验组、CHA/PLA两组均高于空白对照组的评分,说明实验组、CHA/PLA组有明显的骨修复作用。
结论
1.本研究采用珊瑚羟基磷灰石(CHA)粉末浸泡不同浓度的硝酸银溶液,通过溶液离子交换法,制备出不同含银量的载银羟基磷灰石,再将不同的载银CHA粉末与左旋聚乳酸(L-PLA)按3：1质量比混均,并通过CAD和选择性激光烧结快速成形技术,成功制备出具有特殊形状的数字化载银抗菌人工骨支架材料,其工艺简便易行。并通过一系列方法对数字化载银的珊瑚羟基磷灰石/聚乳酸人工骨支架材料进行表征发现,本方法充分利用和保留了珊瑚天然的微孔样结构,各组材料的抗压强度均大于1Mpa。
2.载银CHA/PLA-Ag人工骨材料体外抗菌实验表明,其对金黄色葡萄球菌及大肠杆菌均有抗菌作用,其中浸泡10-4、10-5mmo/L AgNO3载银CHA-Ag人工骨材料体外对上述2种细菌抑菌圈实验为阴性,菌落数实验表明其抗菌率均低于90%,不符合医用抗菌材料标准。而浸泡10-2、10-3mol/L AgNO3的载银人工骨材料体外对上述2种细菌产生明显的抑菌圈,菌落数实验表明其抗菌作用明显,抗菌率均大于90%,具有良好的抗菌性能,符合医用抗菌材料标准。
3.载银人工骨支架材料体外生物学试验表明浸泡10-2mmol/L AgNO3所制备的CHA/PLA-Ag人工骨材料浸提液有细胞毒性,不符合生物材料医用标准。而10-3、10-4、10-5mol/L AgNO3的CHA/PLA-Ag人工骨浸提液对细胞毒性分级分别为1级、0级、0级。表明材料随着载银量增高,对细胞毒性越大。10-1、10-1、10-5mol/L AgNO3的CHA/PLA-Ag人工骨材料符合生物材料医用标准,其中10-3mmol/L AgNO3载银人工骨支架材料无溶血反应、无全身毒性及致敏性,具有良好的生物相容性。
4.载银CHA-Ag人工骨材料的载银量及释放实验表明人工骨材料的载银量随着溶液中银离子浓度的增加而增加,支架材料中银离子能保持长时间的缓释特性,从而能有效地避免银离子早期大量释放,保持长时间的持续缓慢释放,有效地减少银离子对组织细胞的毒性作用。
5.在修复兔胫骨干骺端污染性腔隙性骨缺损模型中,10-3mmol/L AgN3,所制备的载银人工骨支架材料具有良好地抑制骨感染及修复骨缺损效果,表明羟基磷灰石人工骨载银后与左旋聚乳酸复合后具有良好的抗菌性、生物相容性和骨传导作用,未明显影响成骨作用,是一种具有良好应用前景的新型抗菌性骨修复替代材料。
Background
In the worldwide, due to trauma, tumor and infection causes bone defects each year afflicts many patients, the need for bone graft transplantation. However, clinical treatment for bone defects of large-scale pollution using conventional bone graft material may caused to bone transplantation failure due to the secondary infection. And the growing problem of bacterial resistance to antibiotics in the clinical, graft material loaded antibiotics become increasingly localized, then put forward higher requirements for antimicrobial. In recent years, inorganic antibacterial materials for its effective antibacterial, higher security and stability has aroused widespread concern, compared with the organic and natural antibacterial agent, Inorganic antimicrobial agent has the advantages of broad spectrum antimicrobial, long lasting, does not produce drug-resistant, non-toxic side effects and so on. The antibacterial properties of Ag+is the strongest in the metal ion and also the earliest and most mature application of inorganic antibacterial agent.
Over the years, the development of biological materials with antibacterial function instead of autologous bone repair of bone defects has been an important issue in medicine and materials science.. Bone tissue engineering for bone defects, especially of complex structures has brought new hope to repair bone defects. Bone tissue engineering consists of three key factors:signaling molecules (bone growth factors, bone inducing factor), scaffolds and target cells. The choice of scaffold materials is the core issue of the bone tissue engineering. At present,the scaffolds include the natural scaffolds, synthetic scaffolds and composite materials.
Coral hydroxyapatite (CHA) is a natural coral by "hydrothermal reaction", the transition from the hydroxyapatite to retain the porous structure of the natural coral, good biocompatibility, with a larger aperture, higherporosity and pore transport rate. Large body of literature show that coral artificial bone is an ideal bone substitute materials, and because of its good compatibility and three-dimensional porous structure, is also considered the future of tissue engineering scaffolds and carriers.
However, the CHA artificial bone is brittle, and can not be customized to the shape of the bone defect preparation of artificial stents, there is a certain shortage of its.
L-polylactic acid (L-polylactic acid, of L-PLA) is an organic polymer material of one kind have been widely used in medicine, As the body of the implant has been certified by the U.S. Food and Drug Administration, and has been widely used in fixtures such as bone plates, bone screws, surgical suture lines. The advantage of it is the biodegradability, good biocompatibility, and easy shaping.
However, a single type of material (natural scaffold or synthetic stent) is generally difficult to meet the requirements of scaffolds for bone tissue engineering for cell. Natural materials has good biocompatibility, cell recognition signal, which will help the proliferation of cell adhesion; artificial materials, compare to natural materials, Artificial material, although lacking in cell signaling, can be large-scale production,design and control structure, mechanical properties and degradation time, etc The composite material is a combination of several different materials through a certain method will be formed, or a compound formed by the biological material and growth factor,the materials can complement each other in performance, have already achieved good results in practical applications. Natural bio-derived materials, polymers, ceramic materials, either as a composite substrate, but also as a reinforcement and filler, with each other and combined to form the diverse nature of composite materials
Rapid prototyping technology (Rapid Prototyping Technology, RPT) is a new forming technology based on the forming ideas of discrete accumulation, it and complete manufacturing rapid prototyping of biological model with high fast and accuracy from the CT data. It has been a rapid development since the1980s. It is a major breakthrough in the field of manufacturing technology and its impact on the manufacturing sector can be comparable with numerical control technology technology. In recent years, it has been widely used in the medical field, three-dimensional solid model can be made arbitrarily complex shapes, provides the possibility for bone tissue engineering scaffolds personalized manufacturing.
If we can take advantage of rapid prototyping technology, to retain the natural coralline hydroxyapatite porous-like structure on the basis of the load silver ions, according to the morphology of bone defects, personalized preparation with anti-bacterial artificial bone scaffold repair material, whichwill greatly contribute to the development of antibacterial bone tissue engineering scaffolds.
In this study. We use coral hydroxyapatite (CHA) by solution ion exchange method successfully loaded silver ions, to prepare different antibacterial silver content of silver hydroxyapatite powder, and mixed with polylactic acid (PLA) rapid prototyping and prepared the special shape of the digital silver bactericide artificial scaffolds by selective laser sintering. Through a series of experiments, study the Physicochemical properties, antimicrobial, biocompatibility of the artificial bone scaffolds. Through the repair Animal paragraph pollution bone defect model to investigate the feasibility applied to infected bone defects.
Objections:
1. To study the preparation methods of the digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds, and analyze their structural characteristics, physical and chemical properties.
2. To study the Cell compatibility of the digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds.
3. To study the antimicrobial activity in vitro of the digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds.
4. To explore silver content and in vitro release rate of digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds.
5. To explore the histocompatibility and repair a large section of pollution bone defects in animal experiments of the digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds.
Methods and materials
1. Coralline hydroxyapatite was mill into CHA particles after ball milling,take the particle size of140-200μm by sieved, soaked in solution of containing1O-2,10-310-4,10-5mmol/L AgN03from light, prepared silver hydroxyapatite powder with different quality of silver, and then with the L-PLA3:1quality mixed, With Simpleware3.1software, a digital cylindrical model was built by computer-aided design (CAD). Through the digital conversion, the model was transformed into STL file format, and then was imported into rapid prototyping machine. The digital silver coralline hydroxyapatite artificial bone scafflold (CHA/PLA-Ag) was prepared by selective laser sintering rapid prototyping (SLS RP) process. Use scanning electron microscopy, infrared spectroscopy, X-ray diffraction to detect the physical and chemical character and analyze the structure of the digital silver anti-bacterial artificial.
2. Antibacterial properties of experiments in virto
2.1Zone of inhibition experiments
Recover the Staphylococcus aureus ATCC25923and E. coli ATCC25922, Inoculated in a test tube containing LB liquid medium at37℃shaking culture Id. Take five kinds different cylindrical digital silver coralline hydroxyapatite artificial bone scafflold (CHA/PLA-Ag) prepared from soaking10-2,10-3,10-4,10-5mmol/L AgN03, the placed them in the inoculation of Staphylococcus aureus (inoculum of1.5×107CFU) and Escherichia coli (inoculum1.5×107CFU)90mm LB agar medium respectively, penicillin droplets as a positive control group. Cultured at37°C incubator for24hours, measuring the inhibition zone, and record the size of the zone of inhibition.Each strain was repeated six times.
2.2Colony count experiments:prepare the Staphylococcus aureus and Escherichia to a final concentration of3×108CFU/mL bacterial suspension,the take100μL bacterial suspension drops containing silver and does not contain silver coral hydroxyapatite artificial bone surface and cultured for24h at37℃incubator. Material placed in5mL PBS for5min oscillation in the vortex instrument, eluting bacteria after24h.
Take100μL eluate diluted with PBS to the appropriate concentration, to push the tablet at37℃incubator for3d,then count the colony of of plate culture, and calculate the antibacterial rate. The experiment was repeated six times for each species.
3.Refer to the standard of GB/T16886.1-2001idt ISO10993-1:1997and GB/T 16175-2008, Cell toxicity test, hemolytic reaction, and biological evaluation of acute toxicity test, delayed hypersensitivity experiment were performed for biological evaluation of the contained silver hydroxyapatite artificial bone scaffolds.
3.1Cytotoxicity:Take In vitro cell culture techniques to observe the changes of the silver hydroxyapatite artificial bone scaffolds extract on L-929cell morphology. MTT assay was used on the L-929cell proliferation, and to evaluate its cytotoxicity.
3.2Hemolytic reaction:Detect the hemolytic rate after silver hydroxyapatite artificial bone scaffolds extract on New Zealand white rabbits, and to evaluate the hemolytic reaction.
3.3Acute toxicity test:Mice by intraperitoneal injection of the extract of silver hydroxyapatite artificial bone scaffolds, and observed toxicity in mice, then to evaluate the toxicity of the materials.
3.4Delayed hypersensitivity experiment:Guinea pig intradermal injection of Silver hydroxyapatite artificial bone scaffolds extracts to observe the allergic reactions of the skin of guinea pigs, then to evaluate the sensitization of the materials.
4. use atomic absorption spectrometer to analyze Ag+content of the silver coral hydroxyapatite artificial bone scaffolds containing different quality of silver.10-3group digital silver hydroxyapatite artificial bone scaffolds were placed in the initial pH of7.451mL simulated body fluid (SBF).degradation in37℃incubator,1d,4d,7d,14d,21d,28d,49d to detect the release of silver ions in simulated body fluid and speed respectively.
5.5mm x15mm pollution lacunar bone defect built in New Zealand white rabbit tibial metaphysis.The experimental group was implanted10-3mmol/L AgNO3silver hydroxyapatite artificial bone scaffold materials for repair of bone defects, the control group bone defects implanted coral hydroxyapatite did not contain silver,while the blank control group bone defect was not implanted in any material.
Results:
1.ral hydroxyapatite (CHA) powder soaked in different concentrations of silver nitrate solution, by solution ion exchange method, prepared silver hydroxyapatite containing different silver content, then mixed the different containing silver CHA powder with PLLA according to the quality of3:1,and through the CAD and selective laser sintering rapid prototyping technology, can be successfully prepared digital silver bactericide and has a special shape of the artificial bone scaffolds. The color of digital CHA-Ag artificial bone is gray, but containing silver coral hydroxyapatite artificial bone soaked in10-2mol/L of AgN03is brownish black,its powder prior to molding is still gray, we think it becaude of its contained silver content is relatively high, during the preparation of molding laser beam glareing, Silver ions icombine oxygen to form silver oxide. Surface of artificial stent is roughness, regular cylindrical in shape, thickness uniformity, visible porous structure. The compressive strength of every materials are greater than1Mpa.
2. In vitro antibacterial experiments.Zone of inhibition experiments:During the whole experiment of the digital CHA/PLA-Ag artificial bone material soaked10-4,10-5mmol/L of AgNO3concentration of silver nitrate, Staphylococcus aureus and Escherichia coli had no zone of inhibition,but the digital CHA/PLA-Ag artificial bone material for24hours on two kinds of bacteria Soaked10-2,10-3mmol/L of AgN03concentration of silver nitrate produced inhibition zone. The average inhibition zone diameter of silver artificial bone material soaked10-2mmol/L of AgN03to Staphylococcus aureus and Escherichia coli was13.00±0.71mm and12.30±0.71mm.respectively. while that of10-3CHA/PLA-Ag was11.51±0.09mm and11.00±0.15mm,respectively. To Staphylococcus aureus,various materials as well as with penicillin in the control group, One-way ANOVA analysis of variance statistics showed that each group heterogeneity of variance (F=5.149, P=0.020), the difference between the groups was significant statistical significance (F95370.727, P=0.000),pairwise comparisons were significant statistically significant (P=0.000). To E. coli, various materials as well as with penicillin in the control group, One-way ANOVA analysis of variance statistics showed that the homogeneity of variance (F=1.585, P=0.237),the difference between the groups was significant statistical significance (F=2692.000, P=0.000), pairwise comparisons were significant statisticallysignificance (P=0.000).
Colony count experiments:There is no growth and reproduction of Staphylococcus aureus, Escherichia coli after exposure to silver CHA-Ag artificial bone material soaked10-2mmol/L of AgNO3, the antibacterial rate was100%; but the growth and reproduction of Staphylococcus aureus, Escherichia coli was reduced after exposure to silver CHA-Ag artificial bone material soaked10-3,10-4,10-5mmol/L of AgNO3, the antibacterial rate of Staphylococcus aureus was99.99%±0.00%,49.42%±0.28%and22.18%±0.26%respectively.,while that of Escherichia coli was99.99%±0.00%,44.66%±0.44%and21.09%±0.32%respectively. To Staphylococcus aureus, Comparison between various materials, One-way ANOVA analysis of variance statistics showed that each group heterogeneity of variance (F=6.154, P=0.004), the difference between the groups was significant statistical significance (F=2E+007, P=0.000),pairwise comparisons were significant statistically significant (P=0.000). To E. coli. Comparison between various materials, One-way ANOVA analysis of variance statistics showed that each group heterogeneity of variance (F=15.792, P=0.000),the difference between the groups was significant statistical significance (F=1E+007, P=0.000), pairwise comparisons were significant statistically significance (P-0.000).
3. The majority of cell death, rounded growth and poor after extracts of100% soaked10-2mmol/L AgNO3CHA/PLA-Ag artificial bone cultured with L929cells2,4,7d.the cell was toxic and the toxicity grade was3. While there was strong growth of cells, observed under inverted microscope, cell membrane integrity, cells showed the typical shape of the spindle, adherent, after extracts of100%soaked10-3,10-4,10-5mmol/L AgNO3CHA/PLA-Ag artificial bone cultured with L929cells2,4,7d, the toxicity degree were1,0,0respectively. The The experimental results show that the cytotoxicity degree of10-3mmol/L AgNO3CHA/PLA-Ag artificial bone material extracts was0; the hemolysis rate was2.22%, less than5%, in line with the requirements of biomedical; The degree of acute toxicity grading was0; the sensitization scoring was0points,it showed that10-3mmol/L AgNO3CHA/PLA-Ag artificial bone material meet the medical standards for biological materials.
4. The Ag+content of10-2,10-3,10-4,10-5group is2310.520±33.418,318.692±1.763,67.535±0.191and6.050±0.028μg/g respectively,.One-way ANOVA analysis of statistics showed that each group heterogeneity of variance (F=9.005, P=0.001),the difference between the groups was significant statistical significance (F=25847.567, P=0.000), pairwise comparisons were significant statistically significance (P=0.000). Ag+content of10-3materials in vitro SBF showed the trend of slow release, fast release within7days of the initial soak, after the release rate gradually slowed down.
5.Each group of experimental animals eat normally, and can free activity after2weeks after operation. The wound of10-3CHA/PLA-Ag group animals has no significant swelling, no crack, no exudate pus,it healed well; The wound of CHA/PLA group animals was poor, dehiscence, swelling and exudate pus, the secretions of bacterial culture was Staphylococcus aureus, two weeks after surgery soft tissue around wood swelling reduced.and gradually healed; While that of the blank control group was mild swelling, no significant exudate pus, healing after one week.During 3months,there was no death of animals and pathologic fracture. General observation showed the rabbit tibia defects of experimental group and CHA/PLA group healed completely,but that of blank control group almost no bone repair by fibrous tissue filling. The same time-point comparison of radiological examination score of experimental group, CHA/PLA group and blank control group,One-way ANOVA analysis of statistical results showed that each group homogeneity of variance (P>0.05),the difference between the groups was significant statistical significance (P<0.05), pairwise comparisons were significant statisticallysignificance (P=0.000). Among them, the difference of scores at each time point of experimental group and CHA/PLA group has no statistical significance (P>0.05). This showed the bone repair capacity was no significant difference between the experimental group and CHA/PLA group, while the experimental group, CHA/PLA group and control group at each time point differences in scores have significant statistical significance (P<0.05), the scores of the experimental group and CHA/PLA group were higher than the control group, demonstrating the experimental group and CHA/PLA group have a significant role in bone repair.
Conclusion
1. In this study, coral hydroxyapatite (CHA) powder soaked in different concentrations of silver nitrate solution, by solution ion exchange method, prepared silver hydroxyapatite containing different silver content, then mixed the different containing silver CHA powder with PLLA according to the quality of3:1,and through the CAD and selective laser sintering rapid prototyping technology, can successfully prepared digital silver bactericide and has a special shape of the artificial bone scaffolds,the process is simple and easy. The compressive strength of materials are greater than1Mpa.
2. In vitro antibacterial tests of CHA/PLA-Ag artificial bone material showed that the it has an antibacterial effect against to Staphylococcus aureus and Escherichia coli.
The zone of inhibition experiments of CHA/PLA-Ag artificial bone material soaked10-4,10-5mmol/L of AgN03against to the above two kinds of bacterial were negative in vitro, Colony number of experiments show that the antibacterial rate of less than90%,they do not meet the standards of medical antibacterial materials,While that of the materials soaked10-2,10-3mmol/L of AgNO3against to the above-mentioned two kinds of bacteria was Obvious. The colony number of experiments show its antibacterial effect is obvious, antibacterial rate of more than90%, has good antibacterial properties, in line with the standards of medical antibacterial materials.
3. Contained silver artificial bone scaffolds in vitro biological tests showed that soaking10-2mmol/L of AgNO3material extract has cytotoxicity, does not meet the medical standards for biological materials. But the cell toxicity grading of CHA/PLA-Ag artificial extracts soaked10"3,10-4,10-5mmol/L AgNO3were1,0,0. It show that with the material as containing silver content increased, the greater the cytotoxicity. The CHA/PLA-Ag artificial bone material soaked10-3,10"4,10-5mmol/L AgNO3meet the medical standards for biological materials, Which CHA/PLA-Ag silver artificial bone scaffolds soaked10-3mmol/L has no hemolytic reaction, no systemic toxicity and allergenicity, has good biocompatibility.
4. The containing silver and release experiments of contained silver CHA/PLA-Ag artificial materials show that the artificial bone material containing silver increased with increasing silver ion concentration in solution, Silver ions in the scaffolds can maintain prolonged release characteristics, so that it can effectively avoid the early massive release of silver ions, to maintain a long period of sustained slow release, and effectively reduce the toxic effects of silver ions on the tissue cells.
5. In the repair of rabbit tibial metaphysis pollution lacunar bone defect model, contained silver CHA/PLA-Ag artificial materials prepared by soaked10-3mmol/L AgNO3can significantly inhibited the bone infection and repair the bone defects, the results showed that hydroxyapatite artificial bone combined with PLLA after containing silver did not significantly affect the osteogenesis,and has antimicrobial, biocompatibility and bone conduction,.It is a good prospect of new antibacterial bone repair alternative materials.
在世界范围内,由于创伤,肿瘤和感染等原因造成的骨缺损每年都在折磨着众多的患者,需要进行植骨移植。然而,临床上对于大面积污染性骨缺损的治疗使用常规的植骨材料有可能因继发感染而致骨移植失败。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,从而对抗菌剂提出了更高的要求。近年来,无机抗菌材料以其有效的抗菌性、较高的安全性和稳定性引起了广泛关注。无机抗菌剂与有机天然抗菌剂相比,具有抗菌谱广、长效持久、不产生耐药性、无毒副作用等优点。Ag+的抗菌性能是金属离子中最强的,亦是应用最早、最成熟的无机抗菌剂。
长期以来,研制具有抗菌功能的生物材料替代自体骨修复骨缺损一直是医学和材料学领域的重要课题。而骨组织工程为骨缺损,特别是复杂结构的骨缺损的修复带来了新的期盼。骨组织工程包括三个关键因素：信号分子(骨生长因子、骨诱导因子)、支架材料和靶细胞。其中支架材料的选择是骨组织工程的核心问题。目前的支架材料主要有天然的支架材料、人工合成的支架材料以及复合材料。
珊瑚羟基磷灰石(CHA)是由天然珊瑚通过“水热反应”转变而来的羟基磷灰石,保留了天然珊瑚的多孔结构,生物相容性好,具有较大的孔径、较高的孔隙率和孔隙交通率。大量的文献表明珊瑚类人工骨为一类较为理想的骨替代材料,并因为它的良好组织相容性和三维多孔结构,也被认为是有前途的组织工程支架材料和载体。但CHA人工骨脆性较大,无法根据骨缺损的形状个性化地制备出人工骨支架,存在一定不足。
左旋-聚乳酸(L-polylactic acid, L-PLA)是一种已在医学上广泛应用的有机高分子材料,作为人体的植入物已经通过美国食品药品管理局的认证,并已被广泛用于内固定装置例如骨板、骨钉、手术缝合线等。其优点是可降解性,良好的细胞相容性,容易塑形。
然而,单一类型材料(天然支架或人工合成支架)一般难以满足骨组织工程用于细胞外支架材料的要求。天然材料具有生物相容性好、细胞识别信号、利于细胞黏附增殖；人工材料虽然缺乏细胞信号,但具有天然材料所不足的可以大规模生产、可以设计和控制结构、机械性能和降解时间等优点。通过一定的方法将由几种不同材料相结合形成,或者由生物材料与生长因了复合而成,复合材料在性能上互相取长补短,在实际应用中已经取得了良好效果。天然生物衍生材料、高分子材料、陶瓷材料既可以作为复合材料的基材,又可作为其增强体和填料,他们互相搭配和组合形成大量性质各异的复合材料。
快速成形技术(Rapid Prototyping Technology, RPT)是种基于离散堆积成型思想的新型成形技术,可快速、高精度地从CT数据完成快速成形生物模型的制造。它自20世纪80年代产生以来得到了迅速的发展,是制造技术领域出现的一次重大突破,其对制造业的影响完全可以与数控技术相媲美。近年来,已广泛应用于医学领域,可以制造任意复杂形状的三维实体模型,为骨组织工程支架的个性化制造提供了可能。
如果能够利用快速成形技术,在保留珊瑚羟基磷灰石的天然微孔样结构的基础上,载入银离子,根据骨缺损的形态,个性化地制备出具有抗菌性人工骨支架修复材料,这将极大地促进抗菌性骨组织工程支架的发展。
本研究将珊瑚羟基磷灰石(CHA),通过溶液离子交换法,成功载入银离子,制备出不同含银量的载银羟基磷灰石抗菌粉末,与聚乳酸(PLA)混合后,通过选择性激光烧结快速成形制备出具有特殊形状的数字化载银抗菌人工骨(CHA-Ag)支架材料。通过系列实验,研究CHA/PLA-Ag人工骨支架材料的理化学特性、抗菌性、生物相容性,通过修复动物大段污染性骨缺损模型,探讨其应用于感染性骨缺损的可行性。
研究目的
1、研究载银数字化珊瑚羟基磷灰石／聚乳酸人工骨的制备及性能表征。
2、研究载银数字化珊瑚羟基磷灰石／聚乳酸人工骨的细胞相容性。
3、研究载银数字化珊瑚羟基磷灰石／聚乳酸人工骨体外抑菌性。
4、研究载银数字化珊瑚羟基磷灰石／聚乳酸人工骨载银量及体外释放速率。
5、探讨载银数字化珊瑚羟基磷灰石／聚乳酸人工骨的组织相容性与修复大段污染性骨缺损的动物实验。
研究方法
1.将珊瑚羟基磷灰石,经球磨机球磨后成细颗粒粉末状,过目筛后取颗粒大小为140-200μ m,分别放在含有10-2、10-3、10-4、10-5mmol/LAgNO3溶液避光浸泡,制备出含不同质量银的载银羟基磷灰石粉末,然后将其与L-PLA以3：1质量比混均后,采用Simpleware3.1软件,通过CAD构建一数字化的圆柱形模型,通过数控转换,将其生成STL格式的文件,导入快速成型机中,采用选择性激光烧结快速成型的工艺,将数字化的模型制备成制备出含不同质量银的载银羟基磷灰石人工骨支架材料(CHA/PLA-Ag)。采用扫描电镜、红外光谱仪、X射线衍射仪对数字化载银羟基磷灰石抗菌人工骨进行理化、结构分析。
2.体外抗菌性能实验
2.1抑菌圈实验：复苏金黄色葡萄球菌ATCC25923及大肠杆菌ATCC25922,接种于含LB液体培养基试管中,于37℃恒温摇床中培养1d备用。取浸泡10-2、10-3、10-4、10-5mmol/LAgNO3所制备的5种珊瑚羟基磷灰石人工骨圆柱状材料各1粒,分别置于接种有金黄色葡萄球菌(接种量为1.5×107CFU)及大肠杆菌(接种量为1.5×107CFU)的90mm的LB琼脂培养基中,青霉素液滴作为阳性对照组；于37℃恒温培养箱中培养24小时,测量抑菌圈,记录抑菌圈大小。每个菌株重复6次。
2.2菌落计数实验：将金黄色葡萄球菌及大肠杆菌配制成浓度为3×108CFU/mL的细菌悬液。取100μ L细菌悬液分别滴于载银及未载银珊瑚羟基磷灰石人工骨材料表面,于37℃恒温培养箱中培养24小时。将材料置于5mL PBS液中,在漩涡振荡仪上振荡5min,洗脱细菌。取100μ L洗脱液用PBS稀释至适当浓度,推平板,于37℃恒温培养箱中培养3d后计平板菌落数及计算抗菌率。每个菌种实验重复6次。
3.参照GB/T16886.1-2001idt ISO10993-1:1997和GB/T16175-2008,对载银羟基磷灰石人工骨支架材料,进行细胞毒性实验、溶血反应、急性毒性实验、迟发性超敏反应实验的生物学评价。
3.1细胞毒性实验：采用体外细胞培养技术,观察载银羟基磷灰石人工骨支架材料浸提液对L-929细胞形态的变化,采用MTT法检测对L-929细胞增殖情况的影响,以评价其细胞毒性。
3.2溶血反应：检测载银羟基磷灰石人工骨支架材料浸提液的对新西兰大白兔溶血率,评价材料的溶血反应。
3.3急性毒性实验：小鼠腹腔注射载银羟基磷灰石人工骨支架材料的浸提液,观察小鼠的毒性反应,评价材料的毒性。
3.4迟发性超敏反应实验：豚鼠皮内注射载银羟基磷灰石人工骨支架材料浸提液,观察豚鼠皮肤过敏反应情况,评价材料的致敏反应。
4.将不同质量银的载银羟基磷灰石人工骨支架材料,采用原子吸收光谱仪
对材料进行Ag+含量分析。将10q组载银羟基磷灰石人工骨支架材料分别放置于初始pH值为7.4的15mL模拟体液(stimulated body fluid, SBF)中,在37℃恒温箱中降解,并分别于1d、4d、7d、14d、21d、28d、49d检测其在模拟体液中的银离了释放量和速度。
5.于新西兰大白兔胫骨干骺端构建5mm×15mm的污染性腔隙性骨缺损,实验组植入10-3mmol/L AgNO3载银羟基磷灰石人工骨支架材料修复骨缺损,对照组骨缺损区植入未载银的羟基磷灰石／聚乳酸人工骨支架材料,空白对照组骨缺损区不植入任何材料。于术后第1天,第4周、8周、12周行X线摄片,观察骨缺损修复及骨塑形情况。参照Lane-Sandhu X线评分标准对各组胫骨缺损的骨修复程度评分,标本行HE染色组织学观察以了解骨修复情况。
结果
1.采用珊瑚羟基磷灰石(CHA)粉末浸泡不同浓度的硝酸银溶液,通过溶液离子交换法,制备出不同含银量的载银羟基磷灰石,再将不同的载银CHA粉末与左旋聚乳酸(L-PLA)按3：1质量比混均,并通过CAD和选择性激光烧结快速成形技术,可以成功制备出具有特殊形状的数字化载银抗菌人工骨支架材料。数字化CHA-Ag人工骨制备后为灰白色,其中浸泡10-2mol/L AgNO3载银珊瑚羟基磷灰石人工骨为棕黑色,其粉末制备成型前仍为灰白色,考虑为其载银量相对较高,制备成型时银离子经激光束强光照射后与氧结合成氧化银所致。制备的人工骨支架表面粗糙,呈规整的小圆柱体状,粗细均匀,可见微孔结构。各组材料的抗压强度均大于1Mpa。
2.体外抗菌实验。抑菌圈实验：浸泡10-4mmol/AgNO3、10-5mmol/L AgNO3浓度硝酸银所制备的数字化CHA-Ag人工骨材料在整个实验过程中对金黄色葡萄球菌和大肠杆菌均无抑菌圈产生。浸泡10-2mmol/L AgNO3、10-3mmol/L AgNO3浓度硝酸银所制备的数字化CHA-Ag人工骨材料24h对2种细菌均产生抑菌圈,其中浸泡10-2mmol/L AgNO3载银人工骨材料对金黄色葡萄球菌和大肠杆菌的抑菌圈直径分别为(13.00±0.71)mm及(12.30±0.71)mm,浸泡10-33mmol/LAgNO3载银人工骨材料分别平均为(11.51±0.09)mm及(11.00±0.15)mm。金黄色葡萄球菌各材料间以及与青霉素对照组比较,单因素方差分析统计结果显示各组方差不齐(F=5.149,P=0.020),组间差异有显著性统计学意义(F=95370.727,P=0.000),两两比较均有显著性统计学意义(P=0.000)。大肠杆菌各材料间以及与青霉素对照组比较,单因素方差分析统计结果显示各组方差齐性(F=1.585,P=0.237),组间差异有显著性统计学意义(F=2692.000,P=0.000),两两比较均有显著性统计学意义(P=0.000)。
菌落数实验：金黄色葡萄球菌、大肠杆菌与浸泡10-2mmol/L AgNO3载银CHA-Ag人工骨材料接触后无生长繁殖,抗菌率为100%；与浸泡10-3、10-4、10-5mmol/L AgNO3载银CHA-Ag人工骨材料接触后生长减缓,表明有抗菌作用,对金黄色葡萄球菌抗菌率分别为99.99±0.00%、49.42±0.28%及22.18±0.26%；对大肠杆菌抗菌率分别为99.99±0.00%、44.66±0.44%及21.09±0.32%。金黄色葡萄球菌各材料间比较,单因素方差分析统计结果显示各组方差不齐(F=6.154,P=0.004),组间差异有显著性统计学意义(F=2E+007,P=0.000),两两比较均有显著性统计学意义(P=0.000)。大肠杆菌各材料间比较,单因素方差分析统计结果显示各组方差不齐(F=15.792,P=0.000),组间差异有显著性统计学意义(F=1E+007,P=0.000),两两比较均有显著性统计学意义(P=0.000)。
3.100%浸泡10-2mol/L AgNO3的CHA-Ag人工骨浸提液作用于L929细胞2、4、7d,大部分细胞死亡,呈圆形,生长欠佳,对细胞有毒性,毒性分级为3级；而10-3、10-4、10-5mmol/L AgNO3的CHA-Ag人工骨浸提液作用于L929细胞2、4、7d后,细胞生长旺盛,倒置显微镜下观察,细胞膜完整,细胞呈典形的梭形,贴壁良好,毒性分级分别为1级、0级、0级。实验结果表明10-mmol/LAgNO3的CHA-Ag人工骨材料浸提液的细胞毒性均为0级；溶血率为：2.22%,小于5%,符合生物医学要求；急性毒性程度分级均为0级；致敏反应记分均为0分。表明10-3mmol/L AgNO3的CHA-Ag人工骨材料符合生物材料医用标准。
4.10-2、10-4、10-4、10-5组Ag+含量分别为2310.520±33.418、318.692±1.763、67.535±0.191及6.050±0.028μ g/g,单因素方差分析统计结果显示各组方差不齐(F=9.005,P=0.001),组间差异有显著性统计学意义(F=25847.567,P=0.000),两两比较均有显著性统计学意义(P=0.000)。10-3材料在体外SBF中Ag+呈缓慢释放趋势,在最初浸泡7d内释放速度较快,之后释放速度逐渐减慢。
5.各组实验动物术后进食基本正常,2周后均能自由活动。其中10-3CHA/PLA-Ag组术口无明显红肿,无裂开,无渗液流脓,愈合良好；CHA/PLA组术口愈合差,裂开,术口红肿渗液流脓,取分泌物行细菌培养,为金黄色葡萄球菌,术后2周术口软组织肿胀减轻,逐渐愈合；空白对照组术口度红肿,无明显渗液流脓,1周后术口愈合良好。术后3月未出现动物死亡,未出现病理性骨折。大体观实验组及CHA/PLA组兔胫骨缺损完全愈合；空白对照组缺损基本无骨修复作用,由纤维组织充填。实验组、CHA/PLA组及空白对照组的放射学检查评分同一时间点比较,单因素方差分析统计结果显示各组方差齐性(P>0.05),组间差异有显著性统计学意义(P<0.05),其中实验组、CHA/PLA组各时间点评分差别无显著性统计学意义(P>0.05),说明实验组、CHA/PLA组的骨修复作用无显著差异,而实验组、CHA/PLA组与空白对照组各时间点评分差异有显著性统计学意义(R<0.05),实验组、CHA/PLA两组均高于空白对照组的评分,说明实验组、CHA/PLA组有明显的骨修复作用。
结论
1.本研究采用珊瑚羟基磷灰石(CHA)粉末浸泡不同浓度的硝酸银溶液,通过溶液离子交换法,制备出不同含银量的载银羟基磷灰石,再将不同的载银CHA粉末与左旋聚乳酸(L-PLA)按3：1质量比混均,并通过CAD和选择性激光烧结快速成形技术,成功制备出具有特殊形状的数字化载银抗菌人工骨支架材料,其工艺简便易行。并通过一系列方法对数字化载银的珊瑚羟基磷灰石/聚乳酸人工骨支架材料进行表征发现,本方法充分利用和保留了珊瑚天然的微孔样结构,各组材料的抗压强度均大于1Mpa。
2.载银CHA/PLA-Ag人工骨材料体外抗菌实验表明,其对金黄色葡萄球菌及大肠杆菌均有抗菌作用,其中浸泡10-4、10-5mmo/L AgNO3载银CHA-Ag人工骨材料体外对上述2种细菌抑菌圈实验为阴性,菌落数实验表明其抗菌率均低于90%,不符合医用抗菌材料标准。而浸泡10-2、10-3mol/L AgNO3的载银人工骨材料体外对上述2种细菌产生明显的抑菌圈,菌落数实验表明其抗菌作用明显,抗菌率均大于90%,具有良好的抗菌性能,符合医用抗菌材料标准。
3.载银人工骨支架材料体外生物学试验表明浸泡10-2mmol/L AgNO3所制备的CHA/PLA-Ag人工骨材料浸提液有细胞毒性,不符合生物材料医用标准。而10-3、10-4、10-5mol/L AgNO3的CHA/PLA-Ag人工骨浸提液对细胞毒性分级分别为1级、0级、0级。表明材料随着载银量增高,对细胞毒性越大。10-1、10-1、10-5mol/L AgNO3的CHA/PLA-Ag人工骨材料符合生物材料医用标准,其中10-3mmol/L AgNO3载银人工骨支架材料无溶血反应、无全身毒性及致敏性,具有良好的生物相容性。
4.载银CHA-Ag人工骨材料的载银量及释放实验表明人工骨材料的载银量随着溶液中银离子浓度的增加而增加,支架材料中银离子能保持长时间的缓释特性,从而能有效地避免银离子早期大量释放,保持长时间的持续缓慢释放,有效地减少银离子对组织细胞的毒性作用。
5.在修复兔胫骨干骺端污染性腔隙性骨缺损模型中,10-3mmol/L AgN3,所制备的载银人工骨支架材料具有良好地抑制骨感染及修复骨缺损效果,表明羟基磷灰石人工骨载银后与左旋聚乳酸复合后具有良好的抗菌性、生物相容性和骨传导作用,未明显影响成骨作用,是一种具有良好应用前景的新型抗菌性骨修复替代材料。
Background
In the worldwide, due to trauma, tumor and infection causes bone defects each year afflicts many patients, the need for bone graft transplantation. However, clinical treatment for bone defects of large-scale pollution using conventional bone graft material may caused to bone transplantation failure due to the secondary infection. And the growing problem of bacterial resistance to antibiotics in the clinical, graft material loaded antibiotics become increasingly localized, then put forward higher requirements for antimicrobial. In recent years, inorganic antibacterial materials for its effective antibacterial, higher security and stability has aroused widespread concern, compared with the organic and natural antibacterial agent, Inorganic antimicrobial agent has the advantages of broad spectrum antimicrobial, long lasting, does not produce drug-resistant, non-toxic side effects and so on. The antibacterial properties of Ag+is the strongest in the metal ion and also the earliest and most mature application of inorganic antibacterial agent.
Over the years, the development of biological materials with antibacterial function instead of autologous bone repair of bone defects has been an important issue in medicine and materials science.. Bone tissue engineering for bone defects, especially of complex structures has brought new hope to repair bone defects. Bone tissue engineering consists of three key factors:signaling molecules (bone growth factors, bone inducing factor), scaffolds and target cells. The choice of scaffold materials is the core issue of the bone tissue engineering. At present,the scaffolds include the natural scaffolds, synthetic scaffolds and composite materials.
Coral hydroxyapatite (CHA) is a natural coral by "hydrothermal reaction", the transition from the hydroxyapatite to retain the porous structure of the natural coral, good biocompatibility, with a larger aperture, higherporosity and pore transport rate. Large body of literature show that coral artificial bone is an ideal bone substitute materials, and because of its good compatibility and three-dimensional porous structure, is also considered the future of tissue engineering scaffolds and carriers.
However, the CHA artificial bone is brittle, and can not be customized to the shape of the bone defect preparation of artificial stents, there is a certain shortage of its.
L-polylactic acid (L-polylactic acid, of L-PLA) is an organic polymer material of one kind have been widely used in medicine, As the body of the implant has been certified by the U.S. Food and Drug Administration, and has been widely used in fixtures such as bone plates, bone screws, surgical suture lines. The advantage of it is the biodegradability, good biocompatibility, and easy shaping.
However, a single type of material (natural scaffold or synthetic stent) is generally difficult to meet the requirements of scaffolds for bone tissue engineering for cell. Natural materials has good biocompatibility, cell recognition signal, which will help the proliferation of cell adhesion; artificial materials, compare to natural materials, Artificial material, although lacking in cell signaling, can be large-scale production,design and control structure, mechanical properties and degradation time, etc The composite material is a combination of several different materials through a certain method will be formed, or a compound formed by the biological material and growth factor,the materials can complement each other in performance, have already achieved good results in practical applications. Natural bio-derived materials, polymers, ceramic materials, either as a composite substrate, but also as a reinforcement and filler, with each other and combined to form the diverse nature of composite materials
Rapid prototyping technology (Rapid Prototyping Technology, RPT) is a new forming technology based on the forming ideas of discrete accumulation, it and complete manufacturing rapid prototyping of biological model with high fast and accuracy from the CT data. It has been a rapid development since the1980s. It is a major breakthrough in the field of manufacturing technology and its impact on the manufacturing sector can be comparable with numerical control technology technology. In recent years, it has been widely used in the medical field, three-dimensional solid model can be made arbitrarily complex shapes, provides the possibility for bone tissue engineering scaffolds personalized manufacturing.
If we can take advantage of rapid prototyping technology, to retain the natural coralline hydroxyapatite porous-like structure on the basis of the load silver ions, according to the morphology of bone defects, personalized preparation with anti-bacterial artificial bone scaffold repair material, whichwill greatly contribute to the development of antibacterial bone tissue engineering scaffolds.
In this study. We use coral hydroxyapatite (CHA) by solution ion exchange method successfully loaded silver ions, to prepare different antibacterial silver content of silver hydroxyapatite powder, and mixed with polylactic acid (PLA) rapid prototyping and prepared the special shape of the digital silver bactericide artificial scaffolds by selective laser sintering. Through a series of experiments, study the Physicochemical properties, antimicrobial, biocompatibility of the artificial bone scaffolds. Through the repair Animal paragraph pollution bone defect model to investigate the feasibility applied to infected bone defects.
Objections:
1. To study the preparation methods of the digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds, and analyze their structural characteristics, physical and chemical properties.
2. To study the Cell compatibility of the digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds.
3. To study the antimicrobial activity in vitro of the digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds.
4. To explore silver content and in vitro release rate of digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds.
5. To explore the histocompatibility and repair a large section of pollution bone defects in animal experiments of the digital silver coralline hydroxyapatite/polylactic acid artificial bone scafflolds.
Methods and materials
1. Coralline hydroxyapatite was mill into CHA particles after ball milling,take the particle size of140-200μm by sieved, soaked in solution of containing1O-2,10-310-4,10-5mmol/L AgN03from light, prepared silver hydroxyapatite powder with different quality of silver, and then with the L-PLA3:1quality mixed, With Simpleware3.1software, a digital cylindrical model was built by computer-aided design (CAD). Through the digital conversion, the model was transformed into STL file format, and then was imported into rapid prototyping machine. The digital silver coralline hydroxyapatite artificial bone scafflold (CHA/PLA-Ag) was prepared by selective laser sintering rapid prototyping (SLS RP) process. Use scanning electron microscopy, infrared spectroscopy, X-ray diffraction to detect the physical and chemical character and analyze the structure of the digital silver anti-bacterial artificial.
2. Antibacterial properties of experiments in virto
2.1Zone of inhibition experiments
Recover the Staphylococcus aureus ATCC25923and E. coli ATCC25922, Inoculated in a test tube containing LB liquid medium at37℃shaking culture Id. Take five kinds different cylindrical digital silver coralline hydroxyapatite artificial bone scafflold (CHA/PLA-Ag) prepared from soaking10-2,10-3,10-4,10-5mmol/L AgN03, the placed them in the inoculation of Staphylococcus aureus (inoculum of1.5×107CFU) and Escherichia coli (inoculum1.5×107CFU)90mm LB agar medium respectively, penicillin droplets as a positive control group. Cultured at37°C incubator for24hours, measuring the inhibition zone, and record the size of the zone of inhibition.Each strain was repeated six times.
2.2Colony count experiments:prepare the Staphylococcus aureus and Escherichia to a final concentration of3×108CFU/mL bacterial suspension,the take100μL bacterial suspension drops containing silver and does not contain silver coral hydroxyapatite artificial bone surface and cultured for24h at37℃incubator. Material placed in5mL PBS for5min oscillation in the vortex instrument, eluting bacteria after24h.
Take100μL eluate diluted with PBS to the appropriate concentration, to push the tablet at37℃incubator for3d,then count the colony of of plate culture, and calculate the antibacterial rate. The experiment was repeated six times for each species.
3.Refer to the standard of GB/T16886.1-2001idt ISO10993-1:1997and GB/T 16175-2008, Cell toxicity test, hemolytic reaction, and biological evaluation of acute toxicity test, delayed hypersensitivity experiment were performed for biological evaluation of the contained silver hydroxyapatite artificial bone scaffolds.
3.1Cytotoxicity:Take In vitro cell culture techniques to observe the changes of the silver hydroxyapatite artificial bone scaffolds extract on L-929cell morphology. MTT assay was used on the L-929cell proliferation, and to evaluate its cytotoxicity.
3.2Hemolytic reaction:Detect the hemolytic rate after silver hydroxyapatite artificial bone scaffolds extract on New Zealand white rabbits, and to evaluate the hemolytic reaction.
3.3Acute toxicity test:Mice by intraperitoneal injection of the extract of silver hydroxyapatite artificial bone scaffolds, and observed toxicity in mice, then to evaluate the toxicity of the materials.
3.4Delayed hypersensitivity experiment:Guinea pig intradermal injection of Silver hydroxyapatite artificial bone scaffolds extracts to observe the allergic reactions of the skin of guinea pigs, then to evaluate the sensitization of the materials.
4. use atomic absorption spectrometer to analyze Ag+content of the silver coral hydroxyapatite artificial bone scaffolds containing different quality of silver.10-3group digital silver hydroxyapatite artificial bone scaffolds were placed in the initial pH of7.451mL simulated body fluid (SBF).degradation in37℃incubator,1d,4d,7d,14d,21d,28d,49d to detect the release of silver ions in simulated body fluid and speed respectively.
5.5mm x15mm pollution lacunar bone defect built in New Zealand white rabbit tibial metaphysis.The experimental group was implanted10-3mmol/L AgNO3silver hydroxyapatite artificial bone scaffold materials for repair of bone defects, the control group bone defects implanted coral hydroxyapatite did not contain silver,while the blank control group bone defect was not implanted in any material.
Results:
1.ral hydroxyapatite (CHA) powder soaked in different concentrations of silver nitrate solution, by solution ion exchange method, prepared silver hydroxyapatite containing different silver content, then mixed the different containing silver CHA powder with PLLA according to the quality of3:1,and through the CAD and selective laser sintering rapid prototyping technology, can be successfully prepared digital silver bactericide and has a special shape of the artificial bone scaffolds. The color of digital CHA-Ag artificial bone is gray, but containing silver coral hydroxyapatite artificial bone soaked in10-2mol/L of AgN03is brownish black,its powder prior to molding is still gray, we think it becaude of its contained silver content is relatively high, during the preparation of molding laser beam glareing, Silver ions icombine oxygen to form silver oxide. Surface of artificial stent is roughness, regular cylindrical in shape, thickness uniformity, visible porous structure. The compressive strength of every materials are greater than1Mpa.
2. In vitro antibacterial experiments.Zone of inhibition experiments:During the whole experiment of the digital CHA/PLA-Ag artificial bone material soaked10-4,10-5mmol/L of AgNO3concentration of silver nitrate, Staphylococcus aureus and Escherichia coli had no zone of inhibition,but the digital CHA/PLA-Ag artificial bone material for24hours on two kinds of bacteria Soaked10-2,10-3mmol/L of AgN03concentration of silver nitrate produced inhibition zone. The average inhibition zone diameter of silver artificial bone material soaked10-2mmol/L of AgN03to Staphylococcus aureus and Escherichia coli was13.00±0.71mm and12.30±0.71mm.respectively. while that of10-3CHA/PLA-Ag was11.51±0.09mm and11.00±0.15mm,respectively. To Staphylococcus aureus,various materials as well as with penicillin in the control group, One-way ANOVA analysis of variance statistics showed that each group heterogeneity of variance (F=5.149, P=0.020), the difference between the groups was significant statistical significance (F95370.727, P=0.000),pairwise comparisons were significant statistically significant (P=0.000). To E. coli, various materials as well as with penicillin in the control group, One-way ANOVA analysis of variance statistics showed that the homogeneity of variance (F=1.585, P=0.237),the difference between the groups was significant statistical significance (F=2692.000, P=0.000), pairwise comparisons were significant statisticallysignificance (P=0.000).
Colony count experiments:There is no growth and reproduction of Staphylococcus aureus, Escherichia coli after exposure to silver CHA-Ag artificial bone material soaked10-2mmol/L of AgNO3, the antibacterial rate was100%; but the growth and reproduction of Staphylococcus aureus, Escherichia coli was reduced after exposure to silver CHA-Ag artificial bone material soaked10-3,10-4,10-5mmol/L of AgNO3, the antibacterial rate of Staphylococcus aureus was99.99%±0.00%,49.42%±0.28%and22.18%±0.26%respectively.,while that of Escherichia coli was99.99%±0.00%,44.66%±0.44%and21.09%±0.32%respectively. To Staphylococcus aureus, Comparison between various materials, One-way ANOVA analysis of variance statistics showed that each group heterogeneity of variance (F=6.154, P=0.004), the difference between the groups was significant statistical significance (F=2E+007, P=0.000),pairwise comparisons were significant statistically significant (P=0.000). To E. coli. Comparison between various materials, One-way ANOVA analysis of variance statistics showed that each group heterogeneity of variance (F=15.792, P=0.000),the difference between the groups was significant statistical significance (F=1E+007, P=0.000), pairwise comparisons were significant statistically significance (P-0.000).
3. The majority of cell death, rounded growth and poor after extracts of100% soaked10-2mmol/L AgNO3CHA/PLA-Ag artificial bone cultured with L929cells2,4,7d.the cell was toxic and the toxicity grade was3. While there was strong growth of cells, observed under inverted microscope, cell membrane integrity, cells showed the typical shape of the spindle, adherent, after extracts of100%soaked10-3,10-4,10-5mmol/L AgNO3CHA/PLA-Ag artificial bone cultured with L929cells2,4,7d, the toxicity degree were1,0,0respectively. The The experimental results show that the cytotoxicity degree of10-3mmol/L AgNO3CHA/PLA-Ag artificial bone material extracts was0; the hemolysis rate was2.22%, less than5%, in line with the requirements of biomedical; The degree of acute toxicity grading was0; the sensitization scoring was0points,it showed that10-3mmol/L AgNO3CHA/PLA-Ag artificial bone material meet the medical standards for biological materials.
4. The Ag+content of10-2,10-3,10-4,10-5group is2310.520±33.418,318.692±1.763,67.535±0.191and6.050±0.028μg/g respectively,.One-way ANOVA analysis of statistics showed that each group heterogeneity of variance (F=9.005, P=0.001),the difference between the groups was significant statistical significance (F=25847.567, P=0.000), pairwise comparisons were significant statistically significance (P=0.000). Ag+content of10-3materials in vitro SBF showed the trend of slow release, fast release within7days of the initial soak, after the release rate gradually slowed down.
5.Each group of experimental animals eat normally, and can free activity after2weeks after operation. The wound of10-3CHA/PLA-Ag group animals has no significant swelling, no crack, no exudate pus,it healed well; The wound of CHA/PLA group animals was poor, dehiscence, swelling and exudate pus, the secretions of bacterial culture was Staphylococcus aureus, two weeks after surgery soft tissue around wood swelling reduced.and gradually healed; While that of the blank control group was mild swelling, no significant exudate pus, healing after one week.During 3months,there was no death of animals and pathologic fracture. General observation showed the rabbit tibia defects of experimental group and CHA/PLA group healed completely,but that of blank control group almost no bone repair by fibrous tissue filling. The same time-point comparison of radiological examination score of experimental group, CHA/PLA group and blank control group,One-way ANOVA analysis of statistical results showed that each group homogeneity of variance (P>0.05),the difference between the groups was significant statistical significance (P<0.05), pairwise comparisons were significant statisticallysignificance (P=0.000). Among them, the difference of scores at each time point of experimental group and CHA/PLA group has no statistical significance (P>0.05). This showed the bone repair capacity was no significant difference between the experimental group and CHA/PLA group, while the experimental group, CHA/PLA group and control group at each time point differences in scores have significant statistical significance (P<0.05), the scores of the experimental group and CHA/PLA group were higher than the control group, demonstrating the experimental group and CHA/PLA group have a significant role in bone repair.
Conclusion
1. In this study, coral hydroxyapatite (CHA) powder soaked in different concentrations of silver nitrate solution, by solution ion exchange method, prepared silver hydroxyapatite containing different silver content, then mixed the different containing silver CHA powder with PLLA according to the quality of3:1,and through the CAD and selective laser sintering rapid prototyping technology, can successfully prepared digital silver bactericide and has a special shape of the artificial bone scaffolds,the process is simple and easy. The compressive strength of materials are greater than1Mpa.
2. In vitro antibacterial tests of CHA/PLA-Ag artificial bone material showed that the it has an antibacterial effect against to Staphylococcus aureus and Escherichia coli.
The zone of inhibition experiments of CHA/PLA-Ag artificial bone material soaked10-4,10-5mmol/L of AgN03against to the above two kinds of bacterial were negative in vitro, Colony number of experiments show that the antibacterial rate of less than90%,they do not meet the standards of medical antibacterial materials,While that of the materials soaked10-2,10-3mmol/L of AgNO3against to the above-mentioned two kinds of bacteria was Obvious. The colony number of experiments show its antibacterial effect is obvious, antibacterial rate of more than90%, has good antibacterial properties, in line with the standards of medical antibacterial materials.
3. Contained silver artificial bone scaffolds in vitro biological tests showed that soaking10-2mmol/L of AgNO3material extract has cytotoxicity, does not meet the medical standards for biological materials. But the cell toxicity grading of CHA/PLA-Ag artificial extracts soaked10"3,10-4,10-5mmol/L AgNO3were1,0,0. It show that with the material as containing silver content increased, the greater the cytotoxicity. The CHA/PLA-Ag artificial bone material soaked10-3,10"4,10-5mmol/L AgNO3meet the medical standards for biological materials, Which CHA/PLA-Ag silver artificial bone scaffolds soaked10-3mmol/L has no hemolytic reaction, no systemic toxicity and allergenicity, has good biocompatibility.
4. The containing silver and release experiments of contained silver CHA/PLA-Ag artificial materials show that the artificial bone material containing silver increased with increasing silver ion concentration in solution, Silver ions in the scaffolds can maintain prolonged release characteristics, so that it can effectively avoid the early massive release of silver ions, to maintain a long period of sustained slow release, and effectively reduce the toxic effects of silver ions on the tissue cells.
5. In the repair of rabbit tibial metaphysis pollution lacunar bone defect model, contained silver CHA/PLA-Ag artificial materials prepared by soaked10-3mmol/L AgNO3can significantly inhibited the bone infection and repair the bone defects, the results showed that hydroxyapatite artificial bone combined with PLLA after containing silver did not significantly affect the osteogenesis,and has antimicrobial, biocompatibility and bone conduction,.It is a good prospect of new antibacterial bone repair alternative materials.
引文
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