适于硬组织修复钙磷类生物材料的构建与性能调控
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摘要
人体硬组织的损伤修复与重建一直是现代医学力求解决的难题。本论文针对目前钙磷类组织修复材料降解速度难以调控、在体内易被冲刷而难以固化,固液两相分层而致悬浮体系失稳等关键问题,以可控降解和微创治疗为切入点,设计构建了三种适于硬组织修复的钙磷基生物材料,考察了其基本性能,并对其进行了初步的生物学研究。
多孔聚磷酸钙骨修复材料的结构及其性能研究——通过重力二次烧结法制备了多孔、高孔隙率聚磷酸钙(calcium polyphosphate, CPP);从CPP的链结构、晶相结构和聚合基团结构展开,揭示材料合成过程中影响结构变化的主要因素,并探讨聚合磷酸钙的聚合磷酸链中的端部基团(Q1)的比例、晶相结构、烧料粒径对CPP材料降解性能的调控作用；通过细胞培养研究其生物学性能。研究表明：具有线性链状结构的CPP中的Q’随着烧结温度的升高而不断下降。采用高温原位XRD并结合DI JADE XRD分析软件技术,揭示了CPP在煅烧过程中的相转变规律。材料的降解速率随烧料粒径的增大、Q1含量增高而加快,同时探讨了CPP的降解机理,将其用作骨组织支架材料显示出较好的细胞亲和性。
水相高效抗溃散快速固化可注射钙磷基骨水泥的研究——在确保浆体具有良好可注射性的基础上,制备了高效抗溃散性的可注射钙磷基骨水泥(anti-washout injectable calcium phosphate cement, aw-ICPC),同时研究抗溃散剂的加入对材料可注射性、固化时间、流变性能、抗压强度、物相成分和显微结构等的影响。研究表明：5wt%羟丙基甲基纤维素／8wt%羧甲基纤维素钠、0.8wt%黄原胶和4wt%白糊精的加入均能取得良好的抗溃散效果,其中白糊精抗溃散效果最佳。抗溃散剂不影响体系的水化固化反应,但均在不同程度上延缓了体系的固化时间。
通过在固相引入磷酸镁(magnesium phosphate cement, MPC)和调整固液比调控aw-ICPC的固化性能,并对其快速固化机理进行初步探索；通过细胞培养评价材料的体外细胞相容性。研究表明MPC的引入不仅明显缩短了浆体的固化时间,且显著改善了抗溃散性；通过调节MPC在浆体中的比例可调控体系的各项理化性能参数。液固比的增加提高了浆体的可注射性但延缓了固化时间。以新西兰大白兔为动物模型,体内实验表明制备的水相高效抗溃散快速固化可注射钙磷基骨水泥(fast setting and anti-washout injectable calcium phosphate-based cement, fa-ICMB)具有良好的细胞相容性、生物降解性并能修复股骨缺损。
非水相高效悬浮稳定的钙磷基浆体及其在牙根管填充中的应用研究——以与水互溶的非水相溶液为固化液,采用与粉末预先混合法制备出能在注射器中长期稳定保存而在体内快速固化的可注射非水相磷酸钙(non-aqueous injectable calcium-phosphate based cement, n-ICPC)。系统研究了非水相固化液性质、固相颗粒粒径、固液比对糊剂的流变性、固化时间、可注射性和抗压强度等的影响,重点探讨了n-ICPC的固化调控方式。研究表明：采用预混法制备的n-ICPC能在水中自行固化,但固化时间明显延长。以1,2-丙二醇为固化液的体系粘度低、触变性好、固化快且抗压强度高。随着颗粒粒径的减小,n-ICPC的粘固化时间缩短且可注射性、抗压强度提高,但当颗粒粒径≤75μm时,体系粘度不降反升,触变性明显提高,有利于体系的静置稳定。
采用浓缩体系全功能稳定分析仪,考察了固相颗粒粒径、气相二氧化硅及其含量对n-ICPC悬浮稳定性的影响,初步探讨了n-ICPC体系悬浮稳定的机理。结果表明：含粒径≤75μm的颗粒的体系透射光和背散射衰减变化幅度均较小,沉降速率为0.27 mm／d。加入1wt%水溶性气相二氧化硅后,体系沉降速率从0.27 mm／d降至0.029mm／d,悬浮稳定性提高10倍。
将10 wt% CTSAC加入到n-ICPC中,明显改善了n-ICPC的显影性能和可注射性,并赋予体系对常见致龈菌优异的抑菌性能。CTSAC的添加并不影响n-ICPC原有的固化特性和剪切稀化特性。
体外实验结果表明：非水相高效悬浮稳定的钙磷基浆体生物相容性好。将浆体注射到离体牙根管后,发现浆体能固化,与周围牙本质结合紧密,无收缩现象,具有优良的根封性能,有望作为一种新型的根管填充材料得到应用。
Human hard tissue repair and reconstruction has been hard to solve for modern medicine. Unfortunately, most current calcium-phosphate based biomaterials could not be biodegradated controllablly, be collapsed in the fluid and difficult to set, and loss suspension stability caused by solid-liquid separation. With controllable degradation and minimally invasive treatment for the entry point, the goal of the research presented in this dissertation is to reconstruct three kinds of calcium-phosphate based biomaterials for hard tissue repair and, the properties investigation and the preliminary research on their biological properties had been performed.
Structure and properties of poprous calcium polyphosphate (CPP)——porous, high porosity of CPP was prepared by gravity sintering. Main factors affecting the structure change were explored from the viewpoint of chain structure, crystalline structure and group structure, and the degradation mechanism of CPP was investigated. Investigation of properties showed that the proportion of hydrolytic groups (QI groups) in polyphosphate chain was decreased as the sintering temperature increased. By controlling sintering temperature and particle size and altering the proportion of Q1 groups, CPP could be obtained respectively with different degradation rate. Cell viability test results showed that porous CPP exerted biocompatibility on C2C12 cells.
Study on aqueous fast setting and anti-washout injectable calcium phosphate-based cement (fa-ICPC)——Based on the good injectability of the paste, anti-washout injectable calcium phosphate-based cement (aw-ICPC) was fabricated by introducing of anti-washout agents, and their effects on injectability, setting time, compressive strength, phase composition and microstructure were researched. The results showed that all the anti-washout agents studied in the dissertation imparted an effective anti-washout property to the ICPC, while prolonged the setting time. The aw-ICPC with white dextrin showed the best overall performance including anti-washout property.
The setting time of aw-ICPC was adjusted by incorporating magnesium phosphate cement (MPC) and altering the ratio of solid to liquid, and the fast-setting mechanism was explored. It was found that MPC not only shortened the setting time but also significantly improved the anti-washout of the paste. The physical and chemical properties of the fa-ICPC could be regulated by adjusting the ratio of MPC to CPC. The increase of the ratio of liquid to solid would improve the injectability but declay the setting time. Biological experiment indicated that fa-ICMB was biocompatible and biodegradable, and showed the good ability to repair defects of the femur.
High suspension stability of non-aqueous injectable calcium-phosphate based cement (n-ICPC) and its application in root canal filling——To meet the requirements of root canal therapy, nonaqueous but water-soluble organic liquid and calcium phosphate cement (CPC) powders were mixed to form n-ICPC which is stable in syringe and hardens in vivo. Focus on the setting mechanism, the effects of nonaqueous liquid, particle size and the ratio of solid to liquid on the setting time, injectability, rheology and compressive strength were systematic explored. It was found that n-ICPC could be set in SBF, although the setting time was much longer than that of the aqueous ICPC. n-ICPC with propylene glycol as a setting liquid had low viscosity, good thixotropy, shortened setting time which was in favor of injection. Reduction of particle size could effectively improve suspension stability of the n-ICPC.
The effects of particle size, fumed silica and its content on the suspension behavior of n-ICPC were also investigated by concentrated system with fully functional stability analyzer. The results showed that 1 wt% fumed silica increased suspension stability by 10 times. The change rates of transmission light and back-scattering attenuation in n-ICPC with particle diameter≤75μm were small, and the sedimentation rate was only 0.27 mm/d. The addition of 10 wt% CTSAC improved the radiopacity and injectability of the n-ICPC significantly. In addition, CTSAC imparted excellent antibacterial property against common bacteria induced by gingival. Furthermore, the addition of CTSAC did not affect the setting characteristic and shear thinning behavior of the n-ICPC.
In extracted teeth experiment, n-ICPC showed better sealability and were close intergrated with detine. Cytotoxicity results showed that n-ICPC were noncytotoxic and possess good biocompatibility. The developed n-ICPC could meet the requirements of root canal filling with a proper setting time, easy injection, improved suspension stability, potent radiopacity and effective antibacterial properties. The results obtained would provide basic data for the application of the n-ICPC in the root canal filling field.
多孔聚磷酸钙骨修复材料的结构及其性能研究——通过重力二次烧结法制备了多孔、高孔隙率聚磷酸钙(calcium polyphosphate, CPP);从CPP的链结构、晶相结构和聚合基团结构展开,揭示材料合成过程中影响结构变化的主要因素,并探讨聚合磷酸钙的聚合磷酸链中的端部基团(Q1)的比例、晶相结构、烧料粒径对CPP材料降解性能的调控作用；通过细胞培养研究其生物学性能。研究表明：具有线性链状结构的CPP中的Q’随着烧结温度的升高而不断下降。采用高温原位XRD并结合DI JADE XRD分析软件技术,揭示了CPP在煅烧过程中的相转变规律。材料的降解速率随烧料粒径的增大、Q1含量增高而加快,同时探讨了CPP的降解机理,将其用作骨组织支架材料显示出较好的细胞亲和性。
水相高效抗溃散快速固化可注射钙磷基骨水泥的研究——在确保浆体具有良好可注射性的基础上,制备了高效抗溃散性的可注射钙磷基骨水泥(anti-washout injectable calcium phosphate cement, aw-ICPC),同时研究抗溃散剂的加入对材料可注射性、固化时间、流变性能、抗压强度、物相成分和显微结构等的影响。研究表明：5wt%羟丙基甲基纤维素／8wt%羧甲基纤维素钠、0.8wt%黄原胶和4wt%白糊精的加入均能取得良好的抗溃散效果,其中白糊精抗溃散效果最佳。抗溃散剂不影响体系的水化固化反应,但均在不同程度上延缓了体系的固化时间。
通过在固相引入磷酸镁(magnesium phosphate cement, MPC)和调整固液比调控aw-ICPC的固化性能,并对其快速固化机理进行初步探索；通过细胞培养评价材料的体外细胞相容性。研究表明MPC的引入不仅明显缩短了浆体的固化时间,且显著改善了抗溃散性；通过调节MPC在浆体中的比例可调控体系的各项理化性能参数。液固比的增加提高了浆体的可注射性但延缓了固化时间。以新西兰大白兔为动物模型,体内实验表明制备的水相高效抗溃散快速固化可注射钙磷基骨水泥(fast setting and anti-washout injectable calcium phosphate-based cement, fa-ICMB)具有良好的细胞相容性、生物降解性并能修复股骨缺损。
非水相高效悬浮稳定的钙磷基浆体及其在牙根管填充中的应用研究——以与水互溶的非水相溶液为固化液,采用与粉末预先混合法制备出能在注射器中长期稳定保存而在体内快速固化的可注射非水相磷酸钙(non-aqueous injectable calcium-phosphate based cement, n-ICPC)。系统研究了非水相固化液性质、固相颗粒粒径、固液比对糊剂的流变性、固化时间、可注射性和抗压强度等的影响,重点探讨了n-ICPC的固化调控方式。研究表明：采用预混法制备的n-ICPC能在水中自行固化,但固化时间明显延长。以1,2-丙二醇为固化液的体系粘度低、触变性好、固化快且抗压强度高。随着颗粒粒径的减小,n-ICPC的粘固化时间缩短且可注射性、抗压强度提高,但当颗粒粒径≤75μm时,体系粘度不降反升,触变性明显提高,有利于体系的静置稳定。
采用浓缩体系全功能稳定分析仪,考察了固相颗粒粒径、气相二氧化硅及其含量对n-ICPC悬浮稳定性的影响,初步探讨了n-ICPC体系悬浮稳定的机理。结果表明：含粒径≤75μm的颗粒的体系透射光和背散射衰减变化幅度均较小,沉降速率为0.27 mm／d。加入1wt%水溶性气相二氧化硅后,体系沉降速率从0.27 mm／d降至0.029mm／d,悬浮稳定性提高10倍。
将10 wt% CTSAC加入到n-ICPC中,明显改善了n-ICPC的显影性能和可注射性,并赋予体系对常见致龈菌优异的抑菌性能。CTSAC的添加并不影响n-ICPC原有的固化特性和剪切稀化特性。
体外实验结果表明：非水相高效悬浮稳定的钙磷基浆体生物相容性好。将浆体注射到离体牙根管后,发现浆体能固化,与周围牙本质结合紧密,无收缩现象,具有优良的根封性能,有望作为一种新型的根管填充材料得到应用。
Human hard tissue repair and reconstruction has been hard to solve for modern medicine. Unfortunately, most current calcium-phosphate based biomaterials could not be biodegradated controllablly, be collapsed in the fluid and difficult to set, and loss suspension stability caused by solid-liquid separation. With controllable degradation and minimally invasive treatment for the entry point, the goal of the research presented in this dissertation is to reconstruct three kinds of calcium-phosphate based biomaterials for hard tissue repair and, the properties investigation and the preliminary research on their biological properties had been performed.
Structure and properties of poprous calcium polyphosphate (CPP)——porous, high porosity of CPP was prepared by gravity sintering. Main factors affecting the structure change were explored from the viewpoint of chain structure, crystalline structure and group structure, and the degradation mechanism of CPP was investigated. Investigation of properties showed that the proportion of hydrolytic groups (QI groups) in polyphosphate chain was decreased as the sintering temperature increased. By controlling sintering temperature and particle size and altering the proportion of Q1 groups, CPP could be obtained respectively with different degradation rate. Cell viability test results showed that porous CPP exerted biocompatibility on C2C12 cells.
Study on aqueous fast setting and anti-washout injectable calcium phosphate-based cement (fa-ICPC)——Based on the good injectability of the paste, anti-washout injectable calcium phosphate-based cement (aw-ICPC) was fabricated by introducing of anti-washout agents, and their effects on injectability, setting time, compressive strength, phase composition and microstructure were researched. The results showed that all the anti-washout agents studied in the dissertation imparted an effective anti-washout property to the ICPC, while prolonged the setting time. The aw-ICPC with white dextrin showed the best overall performance including anti-washout property.
The setting time of aw-ICPC was adjusted by incorporating magnesium phosphate cement (MPC) and altering the ratio of solid to liquid, and the fast-setting mechanism was explored. It was found that MPC not only shortened the setting time but also significantly improved the anti-washout of the paste. The physical and chemical properties of the fa-ICPC could be regulated by adjusting the ratio of MPC to CPC. The increase of the ratio of liquid to solid would improve the injectability but declay the setting time. Biological experiment indicated that fa-ICMB was biocompatible and biodegradable, and showed the good ability to repair defects of the femur.
High suspension stability of non-aqueous injectable calcium-phosphate based cement (n-ICPC) and its application in root canal filling——To meet the requirements of root canal therapy, nonaqueous but water-soluble organic liquid and calcium phosphate cement (CPC) powders were mixed to form n-ICPC which is stable in syringe and hardens in vivo. Focus on the setting mechanism, the effects of nonaqueous liquid, particle size and the ratio of solid to liquid on the setting time, injectability, rheology and compressive strength were systematic explored. It was found that n-ICPC could be set in SBF, although the setting time was much longer than that of the aqueous ICPC. n-ICPC with propylene glycol as a setting liquid had low viscosity, good thixotropy, shortened setting time which was in favor of injection. Reduction of particle size could effectively improve suspension stability of the n-ICPC.
The effects of particle size, fumed silica and its content on the suspension behavior of n-ICPC were also investigated by concentrated system with fully functional stability analyzer. The results showed that 1 wt% fumed silica increased suspension stability by 10 times. The change rates of transmission light and back-scattering attenuation in n-ICPC with particle diameter≤75μm were small, and the sedimentation rate was only 0.27 mm/d. The addition of 10 wt% CTSAC improved the radiopacity and injectability of the n-ICPC significantly. In addition, CTSAC imparted excellent antibacterial property against common bacteria induced by gingival. Furthermore, the addition of CTSAC did not affect the setting characteristic and shear thinning behavior of the n-ICPC.
In extracted teeth experiment, n-ICPC showed better sealability and were close intergrated with detine. Cytotoxicity results showed that n-ICPC were noncytotoxic and possess good biocompatibility. The developed n-ICPC could meet the requirements of root canal filling with a proper setting time, easy injection, improved suspension stability, potent radiopacity and effective antibacterial properties. The results obtained would provide basic data for the application of the n-ICPC in the root canal filling field.
引文
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