含锶磷酸钙中空微球的生物模板法仿生合成研究
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摘要
磷酸钙生物陶瓷材料由于与天然骨的无机质具有化学组分和晶体结构的相似性，表现出良好的生物活性、生物相容性和骨传导性，是研究和开发骨组织再生修复材料的重要研究方向和应用种类之一。但是目前传统制备技术无法实现对产物的形貌、结构和组分的精确控制与仿生制备，所获得的磷酸钙材料与生物磷灰石在组成、结构和性能上仍存在较大的差异，存在力学性能、降解速率等新骨形成不匹配的问题，这极大地限制了其对骨缺损再生修复的应用。而开发高性能的骨组织再生修复材料，需要研究更有效可控的磷酸钙生物陶瓷材料仿生制备技术。
本研究采用生物模板法仿生合成技术，创新性地利用酵母细胞作为生物模板，实现对多孔中空微球结构和对微量元素的离子含量与离子富集位置的有效调控，研制出可用于骨组织再生修复的含锶多孔磷酸钙中空微球。该材料的多孔中空微球结构具有密度低、稳定性高、流动性好、表面渗透能力强、可容纳大量客体分子等优点，使负载和释放具有促进新骨形成的功能性分子成为可能。在此基础上，进一步研究低剂量微量元素锶对磷酸钙材料的离子改性，可明显改善材料的生物相容性、骨传导性和生物可降解性，有效促进新骨的形成，提高骨缺损再生修复的效率。
通过研究酵母细胞种类、聚电解质浓度、自组装次数、温度、pH值、离子浓度等关键技术参数对产物组分和结构的影响，确定制备磷酸钙中空微球的最优化条件，实现对多孔中空微球结构的精确控制，并探讨了磷酸钙中空微球的仿生合成机理。最终获得的多孔磷酸钙中空微球，主晶相为β-TCP，微球平均粒径约为5.85μm，壁厚约为0.5~1.0μm，并且微球壁上存在大量不规则的多级微纳米孔隙结构（孔径≤500nm）。
在生物模板法合成多孔中空微球结构的基础上，进一步研究微量元素对磷酸钙材料的离子改性所引起的组分不均匀、离子突释等问题，利用聚电解质和酵母细胞对Sr~(2+)的特殊吸附作用，达到对离子含量和离子富集位置的有效控制，研制出含锶磷酸钙中空微球（Sr-CPMC），并研究了Sr~(2+)对Sr-CPMC组成和结构的影响，以及Sr~(2+)含量与Sr~(2+)富集位置对产物的离子释放行为、矿化、蛋白质吸附作用和细胞生物学性能的影响。
研究结果表明，利用聚电解质吸附作用制备的多孔Sr-CPMC，其Sr~(2+)预先进入无定形磷酸钙中，特定热处理后形成Sr-TCP，Sr~(2+)主要富集在微球外层， Sr~(2+)含量与初始锶钙摩尔比成正比例关系。而利用酵母细胞的生物吸附作用制备的Sr-CPMC，其Sr~(2+)被负载在生物模板内核，在热处理阶段掺入TCP晶体中，Sr~(2+)主要富集在微球内层，Sr~(2+)含量随着初始Sr~(2+)浓度先升高后下降。生物模板法可调控磷酸钙材料的微量元素离子改性，有效控制CPMC的Sr~(2+)含量以及Sr~(2+)在微球内/外层的富集。酵母细胞作为一种智能的离子掺杂的初级筛选装置，可实现对磷酸钙的低剂量微量元素的离子改性。
Sr-CPMC具有Sr~(2+)缓释性能以及良好的矿化和蛋白质吸附性能，Sr~(2+)含量和Sr~(2+)富集位置是影响Sr~(2+)释放行为、矿化和蛋白质吸附性能的关键技术参数。Sr-CPMC的多孔中空微球结构对Sr~(2+)的缓释起到关键控制作用。Sr-CPMC与人骨髓间充质干细胞（hMSCs）共培养过程中表现出良好的细胞相容性。Sr-CPMC具有促进hMSCs增殖和成骨分化的生物学性能，这主要得益于多孔中空微球结构和Sr-Ca组分的协同效应。与不含锶的磷酸钙空白样对比，当Sr~(2+)含量为0.18±0.04at%且Sr~(2+)富集在微球内层的Sr-CPMC，其细胞增值率提高了约40%。Sr~(2+)富集在微球内层的Sr-CPMC，在成骨诱导培养过程中使hMSCs的ALP酶活性提高了约50%，显著地促进了hMSCs的ALP、Collagen-I和Runx-2基因表达，以及ALP酶的分泌和钙结节的形成。Sr-CPMC与hMSCs经过30天成骨诱导共培养，形成了粉体-细胞三维复合团，在复合团内外hMSCs的黏附与增殖情况良好，并向成骨分化产生了大量的Collagen-I，该三维复合团具有向类骨组织转化的潜能。
本研究采用生物模板法制备的含锶磷酸钙材料，表现出良好的离子缓释、矿化、蛋白质吸附和细胞生物学性能，有望作为药物载体或者填充料、钙磷系统可注射自固化骨水泥或者骨组织工程支架，在生物医学方面具有广阔的应用前景。
Calcium phosphate bioceramic exhibits excellent biological activity, biocompatibilityand osteoconductivity, because of the similarity with the inorganic component of the naturalbone in the chemical composition and crystal structure. It is one of the important researchesand application categories in the study and development of bone regeneration materials.However, the traditional techniques can not be achieved on the precise control and biomimeticpreparation toward the product morphology, structure and composition at present. There arestill large differences between the artificial calcium phosphate and biological apatite in thecomposition, structure and properties, existed the problems such as the mechanical propertiesand biodegradation rate mismatching with the new bone formation, which greatly limits itsapplication in the regeneration and repair of bone defects. Besides to research the moreeffectively controllable biomimetic technology to prepare calcium phosphate bioceramic isrequired for developing the high-performance materials for bone regeneration and repair.
In this study, biotemplate biomimetic technology was used. We innovatively used theyeast-based biotemplate to implement the effective control toward the porous hollowmicrosphere structure and the ionic content and ionic enriching position of the trace elements,and prepared the porous hollow storntium-contained calcium phospahte microspheres for thebone tissue regeneration and repair. The porous hollow microsphere structure has theadvantages, such as low density, high stability, good fluidity, good surface penetration abilityand accommodating a large number of guest molecules, etc. The structure makes the load andrelease with the functional molecules for the promotion of the new bone formation becomepossible. On this basis, we further researched on the ionic modification of the low dosesstorntium in calcium phospahte, which can significantly improve the biocompatibility,osteoconductivity and biodegradation, and effectively promote the new bone formation, aswell as increased the bone defect regeneration efficiency.
The effect of the key parameters on the product component and structure was researched,such as the cell types, polyelectrolyte concentration, the self-assembly times, temperature, pHvalue, ion concentration, etc. We determined the optimal conditions for preparing the poroushollow calcium phospahte microspheres, and achieved on the precise control of the poroushollow microsphere structure. The biomimetic mechanism was discussed. The main phase ofthe finally product was β-TCP. The microsphere average diameter was about5.85μm. Theshell thickness was among0.5-1.0μm. There were numbers of micro-and nano-sized pore inthe shell wall (The pore diameter≤500nm).
Based on the use of the biotemplate for preparing porous calcium phosphate hollowmicrospheres, we further researched the ionic modification of the trace element on calciumphosphate and the problems of the component uneven and ion burst release. The specificadsorption of polyelectrolyte and microbial cells to the Sr~(2+)was utilized to achieve on theeffective control of the ion content and ion enriching position. The porousstrontium-sbustituted calcium phosphate hollow microsphere/microcapsule (Sr-CPMC) wassuccessfully synthesized. The study was carried out for the effect of Sr~(2+)on the hollowmicrosphere component and structure, as well as the infection of Sr~(2+)content and Sr~(2+)enriching position to the product's ion release behavior, mineralization, protein adsorption andbiological performance.
The results showed that the Sr-CPMC was obtained by using the polyelectrolyteadsorption. The Sr~(2+)pre-substitued in the amorphous calcium phosphate, which became theSr-TCP after sintered. The Sr~(2+)enriched in the outer layer of microspheres. The Sr~(2+)contentand the initial Sr/(Sr+Ca) molar ratio was a proportional relationship. However, the theSr-CPMC prepared by using the yeast cells biosorption loaded the Sr~(2+)on the innerbiotemplates. Sr~(2+)incorporated in the TCP crystal lattice through the heat treatment, whichmainly enriched in the inner microspheres. The Sr~(2+)content increased with the initial Sr~(2+)concentration increased and then decreased. The biotemplate method can regulate the ionicmodification of the trace element in calcium phosphate and effectively control the Sr~(2+)content, Sr~(2+)enriching in outer/inner microsphere. Yeast cells as an intelligent primary devicefor the ion doping can achieve on the low dose ionic modification of the trace element.
Sr-CPMC has the Sr~(2+)slow release properties and good mineralization and proteinadsorption properties. The Sr~(2+)content and Sr~(2+)enriching location were the key parameters toaffect the Sr~(2+)release behavior, mineralization and protein adsorption performance. Theporous hollow microsphere structure has the key function to control the Sr~(2+)slow release.Sr-CPMC showed good compatibility during co-culturing wiht the human marrow stromalcells (hMSCs). Sr-CPMC can promote the hMSCs proliferation and osteogenic differentiationdue to the synergies of the porous hollow microsphere structure and Sr-Ca component.Compared to the control, the cell proliferation rate of the Sr-CPMC sample increased about40%, when the Sr~(2+)content was0.18±0.04at%and the Sr~(2+)was enriching in the innermicrosphere. During co-culturing osteogenic process, the sample of the Sr~(2+)enriching in theinner microsphere increased the hMSCs ALP activity about50%and significantly promotedthe hMSCs ALP, Collagen-I and Runx-2gene expression, as well as the ALP enzymesecretion and calcium nodule formation. Sr-CPMC and hMSCs were cultured to turn to be a powder-cells three-dimensional composite group after30days' osteogenic process. HMSCsshowed good adhesion and proliferation condition both inside and outside of the compositegroup and produced a large number of Collagen-I after the osteogenic differentiation. Thethree-dimensional composite group has the potential of transforming to the bone tissue.
In this work, the biotemplate method was used to prepare Sr-CPMC. The product hadgood ion slow release, mineralization, protein adsorption and cell biological properties. Weproposed the product could be made for the drug carrier, packing material, injectableself-setting calcium phosphate bone cement, or bone tissue engineering scaffold. The producthas broad prospects for the biomedical application.
本研究采用生物模板法仿生合成技术，创新性地利用酵母细胞作为生物模板，实现对多孔中空微球结构和对微量元素的离子含量与离子富集位置的有效调控，研制出可用于骨组织再生修复的含锶多孔磷酸钙中空微球。该材料的多孔中空微球结构具有密度低、稳定性高、流动性好、表面渗透能力强、可容纳大量客体分子等优点，使负载和释放具有促进新骨形成的功能性分子成为可能。在此基础上，进一步研究低剂量微量元素锶对磷酸钙材料的离子改性，可明显改善材料的生物相容性、骨传导性和生物可降解性，有效促进新骨的形成，提高骨缺损再生修复的效率。
通过研究酵母细胞种类、聚电解质浓度、自组装次数、温度、pH值、离子浓度等关键技术参数对产物组分和结构的影响，确定制备磷酸钙中空微球的最优化条件，实现对多孔中空微球结构的精确控制，并探讨了磷酸钙中空微球的仿生合成机理。最终获得的多孔磷酸钙中空微球，主晶相为β-TCP，微球平均粒径约为5.85μm，壁厚约为0.5~1.0μm，并且微球壁上存在大量不规则的多级微纳米孔隙结构（孔径≤500nm）。
在生物模板法合成多孔中空微球结构的基础上，进一步研究微量元素对磷酸钙材料的离子改性所引起的组分不均匀、离子突释等问题，利用聚电解质和酵母细胞对Sr~(2+)的特殊吸附作用，达到对离子含量和离子富集位置的有效控制，研制出含锶磷酸钙中空微球（Sr-CPMC），并研究了Sr~(2+)对Sr-CPMC组成和结构的影响，以及Sr~(2+)含量与Sr~(2+)富集位置对产物的离子释放行为、矿化、蛋白质吸附作用和细胞生物学性能的影响。
研究结果表明，利用聚电解质吸附作用制备的多孔Sr-CPMC，其Sr~(2+)预先进入无定形磷酸钙中，特定热处理后形成Sr-TCP，Sr~(2+)主要富集在微球外层， Sr~(2+)含量与初始锶钙摩尔比成正比例关系。而利用酵母细胞的生物吸附作用制备的Sr-CPMC，其Sr~(2+)被负载在生物模板内核，在热处理阶段掺入TCP晶体中，Sr~(2+)主要富集在微球内层，Sr~(2+)含量随着初始Sr~(2+)浓度先升高后下降。生物模板法可调控磷酸钙材料的微量元素离子改性，有效控制CPMC的Sr~(2+)含量以及Sr~(2+)在微球内/外层的富集。酵母细胞作为一种智能的离子掺杂的初级筛选装置，可实现对磷酸钙的低剂量微量元素的离子改性。
Sr-CPMC具有Sr~(2+)缓释性能以及良好的矿化和蛋白质吸附性能，Sr~(2+)含量和Sr~(2+)富集位置是影响Sr~(2+)释放行为、矿化和蛋白质吸附性能的关键技术参数。Sr-CPMC的多孔中空微球结构对Sr~(2+)的缓释起到关键控制作用。Sr-CPMC与人骨髓间充质干细胞（hMSCs）共培养过程中表现出良好的细胞相容性。Sr-CPMC具有促进hMSCs增殖和成骨分化的生物学性能，这主要得益于多孔中空微球结构和Sr-Ca组分的协同效应。与不含锶的磷酸钙空白样对比，当Sr~(2+)含量为0.18±0.04at%且Sr~(2+)富集在微球内层的Sr-CPMC，其细胞增值率提高了约40%。Sr~(2+)富集在微球内层的Sr-CPMC，在成骨诱导培养过程中使hMSCs的ALP酶活性提高了约50%，显著地促进了hMSCs的ALP、Collagen-I和Runx-2基因表达，以及ALP酶的分泌和钙结节的形成。Sr-CPMC与hMSCs经过30天成骨诱导共培养，形成了粉体-细胞三维复合团，在复合团内外hMSCs的黏附与增殖情况良好，并向成骨分化产生了大量的Collagen-I，该三维复合团具有向类骨组织转化的潜能。
本研究采用生物模板法制备的含锶磷酸钙材料，表现出良好的离子缓释、矿化、蛋白质吸附和细胞生物学性能，有望作为药物载体或者填充料、钙磷系统可注射自固化骨水泥或者骨组织工程支架，在生物医学方面具有广阔的应用前景。
Calcium phosphate bioceramic exhibits excellent biological activity, biocompatibilityand osteoconductivity, because of the similarity with the inorganic component of the naturalbone in the chemical composition and crystal structure. It is one of the important researchesand application categories in the study and development of bone regeneration materials.However, the traditional techniques can not be achieved on the precise control and biomimeticpreparation toward the product morphology, structure and composition at present. There arestill large differences between the artificial calcium phosphate and biological apatite in thecomposition, structure and properties, existed the problems such as the mechanical propertiesand biodegradation rate mismatching with the new bone formation, which greatly limits itsapplication in the regeneration and repair of bone defects. Besides to research the moreeffectively controllable biomimetic technology to prepare calcium phosphate bioceramic isrequired for developing the high-performance materials for bone regeneration and repair.
In this study, biotemplate biomimetic technology was used. We innovatively used theyeast-based biotemplate to implement the effective control toward the porous hollowmicrosphere structure and the ionic content and ionic enriching position of the trace elements,and prepared the porous hollow storntium-contained calcium phospahte microspheres for thebone tissue regeneration and repair. The porous hollow microsphere structure has theadvantages, such as low density, high stability, good fluidity, good surface penetration abilityand accommodating a large number of guest molecules, etc. The structure makes the load andrelease with the functional molecules for the promotion of the new bone formation becomepossible. On this basis, we further researched on the ionic modification of the low dosesstorntium in calcium phospahte, which can significantly improve the biocompatibility,osteoconductivity and biodegradation, and effectively promote the new bone formation, aswell as increased the bone defect regeneration efficiency.
The effect of the key parameters on the product component and structure was researched,such as the cell types, polyelectrolyte concentration, the self-assembly times, temperature, pHvalue, ion concentration, etc. We determined the optimal conditions for preparing the poroushollow calcium phospahte microspheres, and achieved on the precise control of the poroushollow microsphere structure. The biomimetic mechanism was discussed. The main phase ofthe finally product was β-TCP. The microsphere average diameter was about5.85μm. Theshell thickness was among0.5-1.0μm. There were numbers of micro-and nano-sized pore inthe shell wall (The pore diameter≤500nm).
Based on the use of the biotemplate for preparing porous calcium phosphate hollowmicrospheres, we further researched the ionic modification of the trace element on calciumphosphate and the problems of the component uneven and ion burst release. The specificadsorption of polyelectrolyte and microbial cells to the Sr~(2+)was utilized to achieve on theeffective control of the ion content and ion enriching position. The porousstrontium-sbustituted calcium phosphate hollow microsphere/microcapsule (Sr-CPMC) wassuccessfully synthesized. The study was carried out for the effect of Sr~(2+)on the hollowmicrosphere component and structure, as well as the infection of Sr~(2+)content and Sr~(2+)enriching position to the product's ion release behavior, mineralization, protein adsorption andbiological performance.
The results showed that the Sr-CPMC was obtained by using the polyelectrolyteadsorption. The Sr~(2+)pre-substitued in the amorphous calcium phosphate, which became theSr-TCP after sintered. The Sr~(2+)enriched in the outer layer of microspheres. The Sr~(2+)contentand the initial Sr/(Sr+Ca) molar ratio was a proportional relationship. However, the theSr-CPMC prepared by using the yeast cells biosorption loaded the Sr~(2+)on the innerbiotemplates. Sr~(2+)incorporated in the TCP crystal lattice through the heat treatment, whichmainly enriched in the inner microspheres. The Sr~(2+)content increased with the initial Sr~(2+)concentration increased and then decreased. The biotemplate method can regulate the ionicmodification of the trace element in calcium phosphate and effectively control the Sr~(2+)content, Sr~(2+)enriching in outer/inner microsphere. Yeast cells as an intelligent primary devicefor the ion doping can achieve on the low dose ionic modification of the trace element.
Sr-CPMC has the Sr~(2+)slow release properties and good mineralization and proteinadsorption properties. The Sr~(2+)content and Sr~(2+)enriching location were the key parameters toaffect the Sr~(2+)release behavior, mineralization and protein adsorption performance. Theporous hollow microsphere structure has the key function to control the Sr~(2+)slow release.Sr-CPMC showed good compatibility during co-culturing wiht the human marrow stromalcells (hMSCs). Sr-CPMC can promote the hMSCs proliferation and osteogenic differentiationdue to the synergies of the porous hollow microsphere structure and Sr-Ca component.Compared to the control, the cell proliferation rate of the Sr-CPMC sample increased about40%, when the Sr~(2+)content was0.18±0.04at%and the Sr~(2+)was enriching in the innermicrosphere. During co-culturing osteogenic process, the sample of the Sr~(2+)enriching in theinner microsphere increased the hMSCs ALP activity about50%and significantly promotedthe hMSCs ALP, Collagen-I and Runx-2gene expression, as well as the ALP enzymesecretion and calcium nodule formation. Sr-CPMC and hMSCs were cultured to turn to be a powder-cells three-dimensional composite group after30days' osteogenic process. HMSCsshowed good adhesion and proliferation condition both inside and outside of the compositegroup and produced a large number of Collagen-I after the osteogenic differentiation. Thethree-dimensional composite group has the potential of transforming to the bone tissue.
In this work, the biotemplate method was used to prepare Sr-CPMC. The product hadgood ion slow release, mineralization, protein adsorption and cell biological properties. Weproposed the product could be made for the drug carrier, packing material, injectableself-setting calcium phosphate bone cement, or bone tissue engineering scaffold. The producthas broad prospects for the biomedical application.
引文
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