羟基磷灰石/胶原复合涂层的共沉积制备、表征及孔状结构的形成机理研究
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摘要
羟基磷灰石(HA)化学式为Ca_(10)(PO_4)_6(OH)_2是人体硬组织的主要无机成分,具有良好的生物相容性和生物活性,临床植入后能在短时间内与人体组织形成骨性结合。胶原是结缔组织的主要成份,存在于细胞外间质内,有良好的生物相容性和完全的生物降解性,且免疫抗原性很低,具有保护、支持人体组织及增强骨骼张力强度等特性,表现出特别的生物力学性质。自然人骨是由纳米HA和胶原蛋白有序组装而成的一种无机/有机高分子复合物,骨中HA晶粒直径约20nm,长约60nm,HA的c轴是沿着胶原蛋白的三维螺旋轴方向生长,具有特定的纳-微米二级结构。目前,关于羟基磷灰石与胶原复合材料的制备已有不少研究,主要采用共沉积法、生物仿生法等,但尚不能满足负重部位应用的临床要求。本论文从仿生学的角度出发,制备具有类似自然骨的成分和结构的复合涂层,综合利用HA涂层的生物性能和钛的机械性能,以期获得具有优异力学性能和生物性能的骨替代材料。
本论文在低浓度体系下采用电化学恒电流共沉积技术,在纯钛表面沉积制备羟基磷灰石/胶原复合涂层和羟基磷灰石/壳聚糖复合涂层,通过改变影响形成多孔状结构的几种因素,对多孔状结构的形成机理进行探讨,并提出“气泡模板”概念。主要研究结果如下:
1.控制电解液钙磷物种浓度为10~(-4)mol/L,Ca/P为1.67,并添加少量胶原蛋白(0.1~0.3mg/mL),pH值主要范围为6.0~6.3,在医用纯钛表面电沉积制备HA/胶原复合涂层,运用SEM、AFM、XRD、FTIR及XPS等方法对涂层的表面形貌、晶体结构、组分构成等理化性质进行了一系列的表征并对涂层的力学性能和生物学性能进行表征。SEM和AFM结果表明:复合涂层呈特定的纳-微米二级多孔结构,从断截面电镜照片可看到孔状结构之间是完全贯通的;XRD图谱显示添加胶原可使HA在电极表面的择优生长受到抑制;FTIR谱图研究表明,复合膜层中出现胶原蛋白特征峰酰胺Ⅰ带和酰胺Ⅱ带,酰胺Ⅰ带和酰胺Ⅱ带发生了一定程度的位移,表明涂层中HA与胶原蛋白在一定程度上发生化学键合;XPS谱图显示,涂层表面碳元素和氮元素的原子含量明显增大,涂层内部也有这两种元素的的存在。采用体外细胞的培养实验和扫描电镜观察种植于不同材料表面的细胞贴壁及生长形态,发现细胞与电化学共沉积的HA/胶原复合涂层的表面接触良好,细胞培养12h即有伪足长出,伪足有向材料内部生长的趋势,表明胶原蛋白的掺杂增加了涂层的生物相容性。MTT实验发现,细胞培养三天,掺杂有胶原的涂层的细胞增值情况最好,也进一步说明复合涂层具有良好的生物相容性。此外,HA/胶原蛋白的复合还有利于生物材料膜层力学性能的改善,粘结拉伸实验表明,胶原的掺杂使涂层与基地材料和涂层内部的结合力增大了一倍,通过观察拉伸过程力与时间的关系,发现复合涂层具有一定的韧性。通过本实验使涂层具有类似于自然骨的结构和成分,获得力学性能和生物性能得到一定改善的HA/胶原复合涂层。
2.从电沉积的基底材料、电沉积液的黏度和温度及沉积时间等方面研究低浓度条件下影响形成多孔状结构的原因,提出了“气泡模板”概念,合理的解释了纳微米二级结构的形成机理。胶原蛋白、牛血清白蛋白和壳聚糖的加入使电沉积液的黏度增大,黏度增大后,可以在电极表面获得稳定生成的气泡,从而为孔状结构的形成提供了“模板”;同样,电极表面的粗糙度也对气泡的形成有较大的影响,表面非常平整的溅射有金膜的硅片很容易得到多孔状的结构,锗晶表面也可得到典型的多孔结构,而粗糙度最大的钛板表面较难得到该结构,说明电极表面的粗糙度对气泡的稳定形成也至关重要;低浓度体系下,温度对这种结构的形成亦非常关键。
3.初步探索了通过在电沉积液中添加少量壳聚糖制备HA/壳聚糖复合涂层,并对涂层的相关性质进行了表征,结果表明:复合涂层的主要成分是具有钙离子缺陷的羟基磷灰石涂层,表面形貌与HA/胶原复合涂层相似呈纳-微米二级结构,壳聚糖的添加对涂层力学性能的进一步改善起到一定作用。
Hydroxyapatite (Ca_(10)(PO_4)_6(OH)_2, HA) is the main mineral component of human hard tissues. It has great bioactivity and biocompatibility,and after being implanted, it can form a kind of osteal bond with the organic tissues within a short time. Collagen existing in the extracellular matrix is an essential component of the connective tissue and it has excellent biocompatibility, complete biodegradability, low immunity and low antigenicity. And it can protect and support human tissue, enhance the tensile strength of bond and it shows special biomechanics. Natural bone consists of a kind of inorganic/organic composite assembled by ordered nano-HA and collagen protein, in which the HA crystal diameter is about 20 nm and its length is about 60 nm. The c-axis of the HA crystal grow along the collagen 3D helical axis and it has special nano-micro two level of structure. At present, some groups have done a mass of research about HA/collagen composites by using coprecipitation methods, biomimic methods, and so on. But these composites cannot meet the needs of the heavy load part in the body completely. In this work, we mimic the basic composition and structure of bone and combine the advantages of the HA coating and the Ti substrate to achieve an excellent bioactive and mechanical bone substitute material.
In this work, an electrochemical co-deposition technique in low concentration system has been developed to prepare HA/collagen and HA/chitosan composite coatings on Ti substrate. Meanwhile, we also tried to change reaction conditions to find formation mechanism of the nano-micro porous structure, which is explained by a proposed concept of“Bubble Template”. The main work is summaried as follows:
1. The electrolyte contained 10-4 mol/L CaCl_2 and NaH_2PO_4 and 0.1 mol/L NaCl with Ca/P ratio 1.67, in which collagen concentration was 0.1~0.3mg/mL and pH value was adjusted to 6.0~6.3 by 0.1 mol/L NaOH. The SEM, AFM, XRD, FTIR and XPS spectroscopic measurements were performed to characterize the morphologies, chemical composition and crystalline structures of the composite coating. It is found that the composite coating exhibited a homogeneous and porous morphology with nano-micro structure. Compared with the pure HA coating, the peak intensity of the (002) crystal face of the composite coating in the XRD measurements is greatly weakened, due to the block effect of collage during the nucleation and growth of HA crystallizing process. The FTIR spectroscopy exhibits the typical peaks of the collagen: the amideⅠband and the amideⅡband, and their peaks derives a little from the same peaks of the pure collagen, indicating that there is a kind of chemical bond between collagen and HA. The XPS results indicate that the C and N atom content increases greatly. The cell culture results in vitro and MTT tests reveal that, compared with pure HA and pure Ti, the HA/collagen composite coating exhibits best biological properties. And the bonding strength of the hybrid coating is improved markedly.
2. The effects of substrate materials, additives, reaction temperature and deposition time on nano-micro porous structure were also studied. The addition of additives(collagen, BSA and chitosan) makes the viscosity of the electrolyte increased and from silicon slice with gold film, Ge wafer to pure Ti plate, the smoothness decreases in sequence. The experiments indicate that viscosity of the electrolyte, the smoothness of the substrate, reaction temperature and deposition time play important roles in the formation of nano-micro porous structure. A concept of“Bubble Template”is proposed to explain the formation mechanism of nano-micro porous structured HA/collagen coating.
3. The composite coating of HA/chitosan has also been prepared by electrochemical co-deposition method. The preliminary characterization has been carried out by SEM and AFM images, and it is shown that the HA/chitosan composite coating has ordered uniform porous tow-level structure. The EDS spectrum and the sharp peaks of the XRD patterns indicate that the main components is high crystal HA. The bonding strengths between the HA/chitosan coating and the titanium substrate has been improved in a certain extent.
本论文在低浓度体系下采用电化学恒电流共沉积技术,在纯钛表面沉积制备羟基磷灰石/胶原复合涂层和羟基磷灰石/壳聚糖复合涂层,通过改变影响形成多孔状结构的几种因素,对多孔状结构的形成机理进行探讨,并提出“气泡模板”概念。主要研究结果如下:
1.控制电解液钙磷物种浓度为10~(-4)mol/L,Ca/P为1.67,并添加少量胶原蛋白(0.1~0.3mg/mL),pH值主要范围为6.0~6.3,在医用纯钛表面电沉积制备HA/胶原复合涂层,运用SEM、AFM、XRD、FTIR及XPS等方法对涂层的表面形貌、晶体结构、组分构成等理化性质进行了一系列的表征并对涂层的力学性能和生物学性能进行表征。SEM和AFM结果表明:复合涂层呈特定的纳-微米二级多孔结构,从断截面电镜照片可看到孔状结构之间是完全贯通的;XRD图谱显示添加胶原可使HA在电极表面的择优生长受到抑制;FTIR谱图研究表明,复合膜层中出现胶原蛋白特征峰酰胺Ⅰ带和酰胺Ⅱ带,酰胺Ⅰ带和酰胺Ⅱ带发生了一定程度的位移,表明涂层中HA与胶原蛋白在一定程度上发生化学键合;XPS谱图显示,涂层表面碳元素和氮元素的原子含量明显增大,涂层内部也有这两种元素的的存在。采用体外细胞的培养实验和扫描电镜观察种植于不同材料表面的细胞贴壁及生长形态,发现细胞与电化学共沉积的HA/胶原复合涂层的表面接触良好,细胞培养12h即有伪足长出,伪足有向材料内部生长的趋势,表明胶原蛋白的掺杂增加了涂层的生物相容性。MTT实验发现,细胞培养三天,掺杂有胶原的涂层的细胞增值情况最好,也进一步说明复合涂层具有良好的生物相容性。此外,HA/胶原蛋白的复合还有利于生物材料膜层力学性能的改善,粘结拉伸实验表明,胶原的掺杂使涂层与基地材料和涂层内部的结合力增大了一倍,通过观察拉伸过程力与时间的关系,发现复合涂层具有一定的韧性。通过本实验使涂层具有类似于自然骨的结构和成分,获得力学性能和生物性能得到一定改善的HA/胶原复合涂层。
2.从电沉积的基底材料、电沉积液的黏度和温度及沉积时间等方面研究低浓度条件下影响形成多孔状结构的原因,提出了“气泡模板”概念,合理的解释了纳微米二级结构的形成机理。胶原蛋白、牛血清白蛋白和壳聚糖的加入使电沉积液的黏度增大,黏度增大后,可以在电极表面获得稳定生成的气泡,从而为孔状结构的形成提供了“模板”;同样,电极表面的粗糙度也对气泡的形成有较大的影响,表面非常平整的溅射有金膜的硅片很容易得到多孔状的结构,锗晶表面也可得到典型的多孔结构,而粗糙度最大的钛板表面较难得到该结构,说明电极表面的粗糙度对气泡的稳定形成也至关重要;低浓度体系下,温度对这种结构的形成亦非常关键。
3.初步探索了通过在电沉积液中添加少量壳聚糖制备HA/壳聚糖复合涂层,并对涂层的相关性质进行了表征,结果表明:复合涂层的主要成分是具有钙离子缺陷的羟基磷灰石涂层,表面形貌与HA/胶原复合涂层相似呈纳-微米二级结构,壳聚糖的添加对涂层力学性能的进一步改善起到一定作用。
Hydroxyapatite (Ca_(10)(PO_4)_6(OH)_2, HA) is the main mineral component of human hard tissues. It has great bioactivity and biocompatibility,and after being implanted, it can form a kind of osteal bond with the organic tissues within a short time. Collagen existing in the extracellular matrix is an essential component of the connective tissue and it has excellent biocompatibility, complete biodegradability, low immunity and low antigenicity. And it can protect and support human tissue, enhance the tensile strength of bond and it shows special biomechanics. Natural bone consists of a kind of inorganic/organic composite assembled by ordered nano-HA and collagen protein, in which the HA crystal diameter is about 20 nm and its length is about 60 nm. The c-axis of the HA crystal grow along the collagen 3D helical axis and it has special nano-micro two level of structure. At present, some groups have done a mass of research about HA/collagen composites by using coprecipitation methods, biomimic methods, and so on. But these composites cannot meet the needs of the heavy load part in the body completely. In this work, we mimic the basic composition and structure of bone and combine the advantages of the HA coating and the Ti substrate to achieve an excellent bioactive and mechanical bone substitute material.
In this work, an electrochemical co-deposition technique in low concentration system has been developed to prepare HA/collagen and HA/chitosan composite coatings on Ti substrate. Meanwhile, we also tried to change reaction conditions to find formation mechanism of the nano-micro porous structure, which is explained by a proposed concept of“Bubble Template”. The main work is summaried as follows:
1. The electrolyte contained 10-4 mol/L CaCl_2 and NaH_2PO_4 and 0.1 mol/L NaCl with Ca/P ratio 1.67, in which collagen concentration was 0.1~0.3mg/mL and pH value was adjusted to 6.0~6.3 by 0.1 mol/L NaOH. The SEM, AFM, XRD, FTIR and XPS spectroscopic measurements were performed to characterize the morphologies, chemical composition and crystalline structures of the composite coating. It is found that the composite coating exhibited a homogeneous and porous morphology with nano-micro structure. Compared with the pure HA coating, the peak intensity of the (002) crystal face of the composite coating in the XRD measurements is greatly weakened, due to the block effect of collage during the nucleation and growth of HA crystallizing process. The FTIR spectroscopy exhibits the typical peaks of the collagen: the amideⅠband and the amideⅡband, and their peaks derives a little from the same peaks of the pure collagen, indicating that there is a kind of chemical bond between collagen and HA. The XPS results indicate that the C and N atom content increases greatly. The cell culture results in vitro and MTT tests reveal that, compared with pure HA and pure Ti, the HA/collagen composite coating exhibits best biological properties. And the bonding strength of the hybrid coating is improved markedly.
2. The effects of substrate materials, additives, reaction temperature and deposition time on nano-micro porous structure were also studied. The addition of additives(collagen, BSA and chitosan) makes the viscosity of the electrolyte increased and from silicon slice with gold film, Ge wafer to pure Ti plate, the smoothness decreases in sequence. The experiments indicate that viscosity of the electrolyte, the smoothness of the substrate, reaction temperature and deposition time play important roles in the formation of nano-micro porous structure. A concept of“Bubble Template”is proposed to explain the formation mechanism of nano-micro porous structured HA/collagen coating.
3. The composite coating of HA/chitosan has also been prepared by electrochemical co-deposition method. The preliminary characterization has been carried out by SEM and AFM images, and it is shown that the HA/chitosan composite coating has ordered uniform porous tow-level structure. The EDS spectrum and the sharp peaks of the XRD patterns indicate that the main components is high crystal HA. The bonding strengths between the HA/chitosan coating and the titanium substrate has been improved in a certain extent.
引文
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