体内及体外磷酸钙与蛋白质生物材料相互作用
摘要
磷酸钙生物材料因其良好的生物相容性而广泛地用作骨替换材料、药物载体材料、蛋白类生长因子吸附材料。由于它能与骨组织形成无纤维组织层的化学键合被认为是具有生物活性的骨替换材料。
人们通常认为磷酸钙的生物活性是由于磷酸钙材料在体液作用下的溶解而导致在其表面形成一类骨磷灰石层，表面类骨磷灰石层能吸附血液中特定的蛋白，促进成骨细胞的吸附、胶原的分泌和矿化，进而加速骨组织在材料表面的生长。
同时认为磷酸钙的溶解到类骨磷灰石的形成以及类骨磷灰石选择吸收蛋白等，都是在蛋白的环境中进行，其核心是类骨磷灰石层。但是到目前为止，蛋白在类骨磷灰石的形成过程起何种作用?在蛋白质环境下不同化学成份的磷酸钙表面形成的类骨磷灰石有哪些不同特征?真实生理环境与模拟体液对形成类骨磷灰石有多大差异?如何准确地表征与基体具有类似化学成份的类骨磷灰石层的结构等问题到目前都没有令人信服的答案。
因此，本文选择蛋白和不同化学成份的磷酸钙(HA、TCP)相互作用为主体来研究(1)体内体外羟基磷灰石(HA)和β-磷酸三钙(TCP)对蛋白的吸附；(2)在蛋白质影响下，HA、TCP表面体内体外形成的类骨磷灰石的特征；(3)根据体内蛋白吸附实验数据，植入吸附小牛骨形态蛋白(b-BMP)的多孔HA／TCP陶瓷于兔子脊柱，以考察复合b-BMP的多孔HA／TCP陶瓷对脊柱融合过程中骨生长以及骨／材料组织界面性能的影响。
实验结果表明：
(1)磷酸钙陶瓷的相组成对蛋白的吸附有强烈的影响：
在血清中吸附在HA和TCP表面的蛋白没有明显的差异，体内TCP上吸附的蛋白总量与种类明显多于HA，并且HA对低分子量蛋白没有吸附，而TCP能吸附低分子量蛋白。
XPS定量分析表明体内吸附的蛋白总量大于体外。蛋白吸附前后磷酸
钙的 XPS高分辨率CaZp和 PZp结合能的漂移，从实验上证明了蛋白与
磷酸钙的吸附是通过表面带电酸性氨基酸和碱性氨基酸基团与磷酸钙表
面CaZ”和PO。的结合。
蛋白在HA、TCP表面的吸附过程和机理为：蛋白质在浓度差的作用
下向材料表面扩散，当扩散至材料负电荷作用力的范围时，蛋白调整其
三维构象和表面带电氨基酸的分布。调整后的蛋白在静电作用下向材料
表面移动，最后实现表面酸性氨基酸中COO与材料表面Ca＋＋，碱性氨
基酸中NH3”与PO／－实现结合而完成整个吸附过程。
（2）体内、体外蛋白对类骨磷灰石的形成和特征具有调控作用：
蛋白质对磷酸钙材料m、TCP）表面形成的类骨磷灰石层影响最大。
对TCP而言，蛋白能影响其表面生成的类骨磷灰石层结构、成份和
形貌。对HA而言，吸附蛋白为类骨磷灰石的晶体提供结合点，形成颗粒
状的表面活性的类骨磷灰石结构。蛋白的吸附有利于在HA表面形成更多
的类骨磷灰石颗粒，但同时也明显地减小了类骨磷灰石颗粒的尺寸。在没
有蛋白影响的情况下，浸泡溶液的种类（SBF和PBS）也能影响磷酸钙表
面晶体的尺寸、形貌和结构。
本文使用根据CI－PO4－基团在光电子能谱中的特征峰o．11所建立的
XPS磷酸钙结构分析法分析类骨磷灰石层结构。
（3）蛋白质对类骨磷灰石形成和特征的调控机理
蛋白调控类骨磷灰石的形机理：吸附在HA、TCP表面的蛋白质为类
骨磷灰石提供成核点并降低其成核能，促进类骨磷灰石生成。同时，吸附
在TCP表面的蛋白将调节TCP在体液中的溶解行为，控制类骨磷灰石的
形貌。
在体外无蛋白 SBF和 PBS溶液中，TCP表面形成结晶完善的片炊‘类
骨磷灰石”。在PBS溶液中形成的晶体尺寸明显大于SBF。而HA在PBS
和 SBF中几乎没有类骨磷灰石的形成。
且互
体外无蛋白 SBF和 PBS溶液中类骨磷灰石形成机理应为：磷酸钙在
溶液中的溶解导致溶液中＊d”和PO／的变化，变化后的＊／“和P04）离子
积决定生成具有不同形貌的磷酸钙晶体。
（4）脊柱融合：在兔子脊柱横突LS16融合实验结果证明添加小牛
骨形态发生蛋白对促进脊柱融合具有明显的效果。并且，新生骨生成量与
bBMP剂量密切相关。组织学照片显示脊柱横突LS工6同时存在膜内化
骨和软骨成骨两种模式。在术后7周，同种异体骨发生重塑。多孔＊A汀＊P
没有明显的降解迹象。微观的电子显微镜照片结果显示新生松质骨组织能
与＊A汀＊P建立无间断、无纤维组织的连续界面，形成组织和材料之间的
化学键合。纳米痕印（Nanoindentation）技术分析了靠近材料／组织界面微
观区域的杨氏模量和硬度。吸附0．sllg的bBMP和未吸附bBMP的新生
松质骨的杨氏模量分别为 3刀SGPa和 2．36GPa。力学性能显示新生松质骨
在脊柱融合手术7周后矿化仍未完成，植入材料HA／TCP力学性能也在降
低。新生组织的力学性能差异、XPS微区成份分析和 WDS跨界面线扫描
成份分析Ca、P微区成份结果一致。
Background:
Owing to excellent biocompatibility, calcium phosphate (CaP) has been widely used as bone substitute, scaffold for drug delivery and growth factor, and matrix for bone tissue engineering. When used as bone substitute, CaP implants are able to form the chemical bonding between implants and biological bone. The interface between implants and biological bone exhibits the continuity in chemical composition and mechanical properties which result in the function continuity.
The mechanism of CaP bioactivity is widely accepted as: The Ca++ and PCV'dissolve from the surface CaP implant under influence of body fluid, subsequently deposits as the bone-like apatite layer on the surface of the CaP implant. This layer maybe selectively adsorb the special proteins from blood, such as vitronectin and fibronectin, and promotes the osteoblast adhesion, proliferation, and simulates the secretion of collagen fiber secreted by osteoblast and mineralization of collagen fiber, at last results in the formation of bone tissue on the surface of CaP implants. The key factor for CaP bioactivity is formation of bone-like layer on the surface of Ca-P implants.
During the mechanism mentioned above, the proteins take part in and adjust the each steps, but the interaction between CaP and proteins is not fully understood.
Up to now there are many debates and controversial answers about following questions:
(1) What are the roles of proteins in the above process?
(2) What are the effects of CaP properties, such as chemical composition, on the characteristics of bone-like layers in the biological environments?
(3) What is the difference of the bone-like apatite layers formed in simulated body fluid without proteins in vitro and the biological environments?
(4) How to characterize correctly the characteristics of bone-like layer, such as structure, chemical composition?
Objectives:
In this study, the main attention was concentrated to the interaction of proteins and CaP ceramics (hydroxyapatite (HA), tricalcium phosphate (TCP)) in vitro (immersed the samples into fetal calf serum) and in vivo (implanted the diffusion chamber enclosed the test ceramics samples into animals). We designed a series of experiments to investigate:
1. The behaviors and characterization of proteins adsorbed on the surface of HA and TCP in vivo and in vitro;
2. The process and mechanism of proteins adsorption on the surface on CaP;
3. The effects of proteins on the formation and characteristics of bone-like apatite layers formed on surface of HA and TCP in vivo and in vitro.
4. The mechanism of bone-like apatite layer formation in vitro and in vivo;
5. New characterization method of XPS for crystal structure of bone-like apatite layer.
IV
6. The implantation of porous HA/TCP ceramics adsorbed the bovine bone morphogenetic proteins in the spine fusion and the interface phenomena between bone/Implant
Results:
1. The Chemical composition significantly influence the adsorption behavior of protein:
XPS and PAGE results indicated that in vitro there was no significant difference for protein adsorption on the surface of HA and TCP in the quantity and type of proteins. In the vivo the quantity of adsorbed proteins on TCP surface was more then HA, and the HA did not show the ability to adsorb the proteins with low molecular weight, but TCP can adsorbed he proteins with low molecular weight on its surface. The overall amount of proteins adsorbed on both HA and TCP in vivo was more than in vitro. This results maybe indicate that in vivo TCP implants are able to adsorb the growth factors owing to almost all of growth factors are proteins with low molecular weight, and TCP is better delivery scaffold matrix of growth factors than HA.
This thesis first postulated the process and mechanism of charged protein adsorption on the surface of CaP with negative zeta-potentials according to the surface charge distribution on protein surface and conformation adjustment of proteins during the adsorption process.
The process of proteins adso
人们通常认为磷酸钙的生物活性是由于磷酸钙材料在体液作用下的溶解而导致在其表面形成一类骨磷灰石层，表面类骨磷灰石层能吸附血液中特定的蛋白，促进成骨细胞的吸附、胶原的分泌和矿化，进而加速骨组织在材料表面的生长。
同时认为磷酸钙的溶解到类骨磷灰石的形成以及类骨磷灰石选择吸收蛋白等，都是在蛋白的环境中进行，其核心是类骨磷灰石层。但是到目前为止，蛋白在类骨磷灰石的形成过程起何种作用?在蛋白质环境下不同化学成份的磷酸钙表面形成的类骨磷灰石有哪些不同特征?真实生理环境与模拟体液对形成类骨磷灰石有多大差异?如何准确地表征与基体具有类似化学成份的类骨磷灰石层的结构等问题到目前都没有令人信服的答案。
因此，本文选择蛋白和不同化学成份的磷酸钙(HA、TCP)相互作用为主体来研究(1)体内体外羟基磷灰石(HA)和β-磷酸三钙(TCP)对蛋白的吸附；(2)在蛋白质影响下，HA、TCP表面体内体外形成的类骨磷灰石的特征；(3)根据体内蛋白吸附实验数据，植入吸附小牛骨形态蛋白(b-BMP)的多孔HA／TCP陶瓷于兔子脊柱，以考察复合b-BMP的多孔HA／TCP陶瓷对脊柱融合过程中骨生长以及骨／材料组织界面性能的影响。
实验结果表明：
(1)磷酸钙陶瓷的相组成对蛋白的吸附有强烈的影响：
在血清中吸附在HA和TCP表面的蛋白没有明显的差异，体内TCP上吸附的蛋白总量与种类明显多于HA，并且HA对低分子量蛋白没有吸附，而TCP能吸附低分子量蛋白。
XPS定量分析表明体内吸附的蛋白总量大于体外。蛋白吸附前后磷酸
钙的 XPS高分辨率CaZp和 PZp结合能的漂移，从实验上证明了蛋白与
磷酸钙的吸附是通过表面带电酸性氨基酸和碱性氨基酸基团与磷酸钙表
面CaZ”和PO。的结合。
蛋白在HA、TCP表面的吸附过程和机理为：蛋白质在浓度差的作用
下向材料表面扩散，当扩散至材料负电荷作用力的范围时，蛋白调整其
三维构象和表面带电氨基酸的分布。调整后的蛋白在静电作用下向材料
表面移动，最后实现表面酸性氨基酸中COO与材料表面Ca＋＋，碱性氨
基酸中NH3”与PO／－实现结合而完成整个吸附过程。
（2）体内、体外蛋白对类骨磷灰石的形成和特征具有调控作用：
蛋白质对磷酸钙材料m、TCP）表面形成的类骨磷灰石层影响最大。
对TCP而言，蛋白能影响其表面生成的类骨磷灰石层结构、成份和
形貌。对HA而言，吸附蛋白为类骨磷灰石的晶体提供结合点，形成颗粒
状的表面活性的类骨磷灰石结构。蛋白的吸附有利于在HA表面形成更多
的类骨磷灰石颗粒，但同时也明显地减小了类骨磷灰石颗粒的尺寸。在没
有蛋白影响的情况下，浸泡溶液的种类（SBF和PBS）也能影响磷酸钙表
面晶体的尺寸、形貌和结构。
本文使用根据CI－PO4－基团在光电子能谱中的特征峰o．11所建立的
XPS磷酸钙结构分析法分析类骨磷灰石层结构。
（3）蛋白质对类骨磷灰石形成和特征的调控机理
蛋白调控类骨磷灰石的形机理：吸附在HA、TCP表面的蛋白质为类
骨磷灰石提供成核点并降低其成核能，促进类骨磷灰石生成。同时，吸附
在TCP表面的蛋白将调节TCP在体液中的溶解行为，控制类骨磷灰石的
形貌。
在体外无蛋白 SBF和 PBS溶液中，TCP表面形成结晶完善的片炊‘类
骨磷灰石”。在PBS溶液中形成的晶体尺寸明显大于SBF。而HA在PBS
和 SBF中几乎没有类骨磷灰石的形成。
且互
体外无蛋白 SBF和 PBS溶液中类骨磷灰石形成机理应为：磷酸钙在
溶液中的溶解导致溶液中＊d”和PO／的变化，变化后的＊／“和P04）离子
积决定生成具有不同形貌的磷酸钙晶体。
（4）脊柱融合：在兔子脊柱横突LS16融合实验结果证明添加小牛
骨形态发生蛋白对促进脊柱融合具有明显的效果。并且，新生骨生成量与
bBMP剂量密切相关。组织学照片显示脊柱横突LS工6同时存在膜内化
骨和软骨成骨两种模式。在术后7周，同种异体骨发生重塑。多孔＊A汀＊P
没有明显的降解迹象。微观的电子显微镜照片结果显示新生松质骨组织能
与＊A汀＊P建立无间断、无纤维组织的连续界面，形成组织和材料之间的
化学键合。纳米痕印（Nanoindentation）技术分析了靠近材料／组织界面微
观区域的杨氏模量和硬度。吸附0．sllg的bBMP和未吸附bBMP的新生
松质骨的杨氏模量分别为 3刀SGPa和 2．36GPa。力学性能显示新生松质骨
在脊柱融合手术7周后矿化仍未完成，植入材料HA／TCP力学性能也在降
低。新生组织的力学性能差异、XPS微区成份分析和 WDS跨界面线扫描
成份分析Ca、P微区成份结果一致。
Background:
Owing to excellent biocompatibility, calcium phosphate (CaP) has been widely used as bone substitute, scaffold for drug delivery and growth factor, and matrix for bone tissue engineering. When used as bone substitute, CaP implants are able to form the chemical bonding between implants and biological bone. The interface between implants and biological bone exhibits the continuity in chemical composition and mechanical properties which result in the function continuity.
The mechanism of CaP bioactivity is widely accepted as: The Ca++ and PCV'dissolve from the surface CaP implant under influence of body fluid, subsequently deposits as the bone-like apatite layer on the surface of the CaP implant. This layer maybe selectively adsorb the special proteins from blood, such as vitronectin and fibronectin, and promotes the osteoblast adhesion, proliferation, and simulates the secretion of collagen fiber secreted by osteoblast and mineralization of collagen fiber, at last results in the formation of bone tissue on the surface of CaP implants. The key factor for CaP bioactivity is formation of bone-like layer on the surface of Ca-P implants.
During the mechanism mentioned above, the proteins take part in and adjust the each steps, but the interaction between CaP and proteins is not fully understood.
Up to now there are many debates and controversial answers about following questions:
(1) What are the roles of proteins in the above process?
(2) What are the effects of CaP properties, such as chemical composition, on the characteristics of bone-like layers in the biological environments?
(3) What is the difference of the bone-like apatite layers formed in simulated body fluid without proteins in vitro and the biological environments?
(4) How to characterize correctly the characteristics of bone-like layer, such as structure, chemical composition?
Objectives:
In this study, the main attention was concentrated to the interaction of proteins and CaP ceramics (hydroxyapatite (HA), tricalcium phosphate (TCP)) in vitro (immersed the samples into fetal calf serum) and in vivo (implanted the diffusion chamber enclosed the test ceramics samples into animals). We designed a series of experiments to investigate:
1. The behaviors and characterization of proteins adsorbed on the surface of HA and TCP in vivo and in vitro;
2. The process and mechanism of proteins adsorption on the surface on CaP;
3. The effects of proteins on the formation and characteristics of bone-like apatite layers formed on surface of HA and TCP in vivo and in vitro.
4. The mechanism of bone-like apatite layer formation in vitro and in vivo;
5. New characterization method of XPS for crystal structure of bone-like apatite layer.
IV
6. The implantation of porous HA/TCP ceramics adsorbed the bovine bone morphogenetic proteins in the spine fusion and the interface phenomena between bone/Implant
Results:
1. The Chemical composition significantly influence the adsorption behavior of protein:
XPS and PAGE results indicated that in vitro there was no significant difference for protein adsorption on the surface of HA and TCP in the quantity and type of proteins. In the vivo the quantity of adsorbed proteins on TCP surface was more then HA, and the HA did not show the ability to adsorb the proteins with low molecular weight, but TCP can adsorbed he proteins with low molecular weight on its surface. The overall amount of proteins adsorbed on both HA and TCP in vivo was more than in vitro. This results maybe indicate that in vivo TCP implants are able to adsorb the growth factors owing to almost all of growth factors are proteins with low molecular weight, and TCP is better delivery scaffold matrix of growth factors than HA.
This thesis first postulated the process and mechanism of charged protein adsorption on the surface of CaP with negative zeta-potentials according to the surface charge distribution on protein surface and conformation adjustment of proteins during the adsorption process.
The process of proteins adso
引文
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