雾化干燥法制备羟基磷灰石微球的结构表征及其生物学行为研究

发布时间：2018-05-11 23:28

本文选题：羟基磷灰石 + 微球　；参考：《山东大学》2010年博士论文

【摘要】：羟基磷灰石(HA)材料具有优异的生物相容性和生物活性,被广泛应用在生物材料的多个领域。球形HA具有特殊的多孔结构和良好的表面性能,对蛋白质、核酸、生物酶等生物活性大分子的分离和纯化具有较好的选择性。而低结晶度乃至非晶的HA微球,不仅具有流动性好、比表面积大等优点,还有较好的可降解性及优越的生物活性,能更好的应用在药物载体、细胞培养载体、骨填充、骨修复等领域。降低HA微球结晶度的途径之一就是通过改变HA料浆的参数,并快速干燥,使HA微球更多地保留原始料浆的性质。目前,空心和多孔HA微球主要通过模板法进而烧结处理制备,这导致其结晶度较高,降解速率过低。而对低结晶度、高降解速率HA微球的相关研究较少。本文利用自制的火焰-喷雾干燥装置,以高于500℃的甲烷火焰为干燥介质,通过调整原始HA料浆的参数,得到结晶度较低、孔隙度较高的HA微球。利用扫描电子显微镜(SEM)、透射电子显微镜(TEM)、高分辨透射电镜(HRTEM)、场发射电子显微镜(FE-SEM)、X射线衍射仪(XRD)、比表面积分析仪(SSA)、激光粒度分析仪(LDPSA)、红外光谱分析仪(FTIR)等检测手段,对所得HA微球的形貌、相组成、粒度分布、比表面积、孔径分布、微观结构特征以及基团分布等进行了系统的分析研究,优化出了利用火焰-雾化干燥法制备HA微球的工艺参数。以去氨水处理后的料浆、未去氨水料浆和冰水混合物料浆为试验对象,对采用火焰-雾化干燥法制备出的三种HA微球进行体外、体内试验,研究不同结晶度和结构形态的HA微球在生物学行为上的差异。进而以未去氨水料浆火焰干燥所得的HA微球为例,利用热致相分离技术制备多孔PLLA/HA微球复合支架,系统研究了不同比例的HA微球对复合支架力学性能和生物学性能的影响。结果表明,利用自制的火焰-喷雾干燥装置,在甲烷火焰高温干燥介质中得到的HA微球,能更多的保留HA在原料浆中的性质。原始料浆中的氨水在干燥过程中分解的氨气气体会导致HA微球结构疏松,比表面积增加,粒度分布不均。低温原始料浆干燥后,可得到低结晶度、结构疏松且粒度不均的HA微球。本试验中,冰水混合物料浆所得HA微球的结晶度为29.8%,远低于典型HA颗粒90%以上的结晶程度,且比表面积(167.16 m2/g)和总孔体积(0.5054ml/g)远高于去氨水处理后料浆所得微球(52.523 m2/g,0.2331 ml/g)。本研究可以通过调节HA料浆的参数来降低所得HA微球的结晶度,改善其结构和性能。 HA料浆的性质对火焰干燥后所得HA微球的性能有重要影响。HA在其料浆中的结晶程度和晶粒微观形貌受反应时间、沉化时间以及反应温度等因素的影响。在常温条件下,随反应时间的延长,HA的结晶程度越来越高。料浆沉化一段时间后,其中的HA纳米晶体呈针状生长。反应温度越高料浆中HA的结晶度越高。火焰-雾化干燥HA微球的结晶程度随原始料浆中HA的结晶度升高而增加。TEM结果表明,组成去氨水料浆所得的HA微球的微颗粒呈棒状或条状分布；未去氨水料浆所得HA微球中有部分非透明的非晶存在；而冰水混合物料浆所得HA微球的晶粒存在泡状半透明球状非晶磷酸钙。另外,冰水混合物料浆所得HA微球表面存在较多疏松的微孔结构。煅烧温度对不同性质HA料浆所得HA微球的影响程度也不相同。中低温处理(低于600℃)对去氨水处理后的料浆所得HA微球的形貌、结晶度以及比表面积没有明显影响。高温煅烧处理(800℃-1000℃)会使微球的晶粒粗化长大,并在微球内部熔合,进而明显降低其比表面积和孔体积。但是600℃低温煅烧后,冰水混合物料浆所得的微球比表面积和孔体积也会从原来的167.16m2/g,0.5054 ml/g分别急剧降至65.985 m2/g和0.1952 ml/g。火焰干燥所得HA微球在体外生物模拟实验中的结果表明其具有较好的生物活性,并对BSA蛋白质有一定的吸附和缓释作用。SBF浸泡后HA微球的质量变化呈先减小后增加的趋势。HA微球结晶度越低,质量变化越明显。HA微球对BSA的吸附量并不是单纯地随结晶度的降低而升高,还受孔隙度和降解速率的影响。在体内生物体液作用一段时间后,火焰-雾化干燥后的HA微球无毒副作用,并和活体骨结合良好。从组织形貌分析来看,HA微球植入体具有良好的骨引导能力。植入新西兰大白兔的股骨中4个月后,三种植入体周围都形成了新的生物组织,且有不同程度的降解,降解程度随结晶度的降低而增加；未去氨水料浆所对应的HA微球植入体内部有结缔组织长入。 HA微球加入到高分子材料中能形成复合生物材料,改善高分子材料由于降解而产生的酸性环境,提高复合材料的生物学性能和力学性能。本研究将未去氨水料浆经火焰-雾化干燥法所得HA微球均匀的混入PLLA的二氧六环溶液中,利用热致相分离技术成功制备出连通度较好的多孔PLLA/HA微球复合支架。结果表明,HA微球和PLLA材料能较好的结合,且随着HA微球加入量的增加,复合支架结构的不规则程度加剧,力学性能提高。相对于纯PLLA多孔支架的压缩模量(4.4MPa),HA微球的质量比占复合支架的30%时,复合支架的压缩模量(9.1MPa)最高。在SBF中浸泡一段时间后,支架表面会形成类骨磷灰石,并且其沉积量随HA微球含量的增加而增加。同时,HA微球的加入还能提高复合支架对BSA蛋白质的吸附能力。大鼠的MC3T3成骨细胞4周的培养后,在PLLA/HA微球复合支架上能较好的粘附、分化和繁殖。由于HA微球具有优异的生物活性和良好的润湿性,加入后能使细胞更好的在复合支架材料上分化和繁殖。组织学切片也表明,PLLA/HA微球复合支架中的部分细胞能够长入到支架内部,和材料融合状态良好。
[Abstract]:Hydroxyapatite (HA) materials have excellent biocompatibility and biological activity, and are widely used in many fields of biological materials. Spherical HA has special porous structure and good surface properties. It has good selectivity for the separation and purification of biological active macromolecules, such as protein, nucleic acid, biological enzyme and so on. The crystal HA microspheres not only have the advantages of good fluidity and larger surface area, but also have good biodegradability and superior biological activity. It can be better used in the fields of drug carrier, cell culture carrier, bone filling, bone repair and so on. One of the ways to reduce the crystallinity of HA microspheres is to change the parameters of HA slurry and quickly dry and make HA micro At present, hollow and porous HA microspheres are mainly prepared by template method and then sintered, which results in high crystallinity and low degradation rate, but less research on low crystallinity and high degradation rate of HA microspheres.
In this paper, a self-made flame spray drying device was used to obtain HA microspheres with low crystallinity and high porosity by adjusting the parameters of the original HA slurry by adjusting the parameters of the methane flame above 500 degrees C. Using scanning electron microscope (SEM), transmission electron microscope (TEM), high resolution transmission electron microscopy (HRTEM), field emission electron microscope (FE-SEM). X ray diffractometer (XRD), specific surface area analyzer (SSA), laser particle size analyzer (LDPSA), infrared spectrum analyzer (FTIR) and other detection methods, the morphology, phase composition, particle size distribution, specific surface area, pore size distribution, microstructure characteristics and group distribution of the obtained HA microspheres were systematically analyzed and studied, and the flame atomization was optimized. The process parameters of HA microspheres were prepared by drying method. Three kinds of HA microspheres, which were prepared by flame atomization drying method, were carried out in vitro and in vivo. The differences in biological behavior of HA microspheres with different crystallinity and structure form were studied in vitro. A porous PLLA/HA microsphere composite scaffold was prepared by thermotropic phase separation technology without the HA microspheres obtained from ammonia slurry flame drying. The effects of different proportion of HA microspheres on the mechanical properties and biological properties of the composite scaffold were systematically studied.
The results show that the HA microspheres obtained by the self-made flame spray drying device in the high temperature drying medium of methane flame can retain more of the properties of HA in the raw material pulp. The ammonia gas decomposed during the drying process in the raw slurry will lead to the loose structure of HA microspheres, the increase of specific surface area and the uneven distribution of grain size. After drying, HA microspheres with low crystallinity, loose structure and uneven grain size can be obtained. In this experiment, the crystallinity of the HA microspheres obtained by the mixture slurry of ice water mixture is 29.8%, far below the crystallinity of more than 90% of the typical HA particles, and the specific surface area (167.16 m2/g) and the total pore volume (0.5054ml/g) are much higher than that of the slurry after the ammonia removal (52.523). M2/g, 0.2331 ml/g). This study can reduce the crystallinity and improve the structure and properties of HA microspheres by adjusting the parameters of HA slurry.
The properties of HA slurry have an important effect on the properties of HA microspheres obtained after the flame drying. The crystallization degree of.HA in the slurry and the microstructure of the grain are influenced by the reaction time, precipitation time and reaction temperature. Under the condition of normal temperature, the crystallization of HA is getting higher and higher with the prolongation of the reaction time. The HA nanocrystals showed needle growth. The higher the reaction temperature, the higher the crystallinity of HA in the slurry.
The crystallization of HA microspheres with flame atomization drying increased with the increase of the crystallinity of HA in the raw slurry. The results showed that the microparticles of the HA microspheres composed of ammonia water slurry were bar or strip, and there were some amorphous amorphous particles in the HA microspheres without ammonia water slurry; and the crystals of HA microspheres obtained from the mixture slurry of ice water mixture were crystal microspheres. There is a bubble like semitransparent globular amorphous calcium phosphate in the grains. Moreover, there are many loose micropores on the surface of HA microspheres obtained from ice water mixture.
The influence of calcining temperature on the HA microspheres obtained by HA slurry with different properties is different. Low temperature treatment (below 600 C) has no obvious influence on the morphology, crystallinity and specific surface area of the HA microspheres obtained from the slurry treated with ammonia water. The high temperature calcination treatment (800 C) will make the grain size of the microspheres grow up and melt in the microspheres. The specific surface area and pore volume are obviously reduced, but the specific surface area and pore volume of the microspheres obtained by the ice water mixture slurry will also drop sharply from the original 167.16m2/g and 0.5054 ml/g to 65.985 m2/g and 0.1952 ml/g., respectively, after the calcination at 600 C
The results of HA microspheres obtained by flame drying in vitro showed that the microspheres had better bioactivity and had a certain adsorption and sustained release effect on BSA protein. After.SBF immersion, the quality changes of HA microspheres were decreased first and then increased, the lower the crystallinity of.HA microspheres, the more obvious the mass change, the adsorption capacity of.HA microspheres to BSA was not the same. Only with the decrease of crystallinity, it is also affected by the porosity and degradation rate. After a period of time in vivo, the HA microspheres after the flame atomization drying have no toxic side effects and have good combination with the living bone. From the analysis of tissue morphology, the HA microsphere implants have good bone guidance ability. After 4 months of the white rabbit's femur, new biological tissues were formed around the three implant, and there were different degrees of degradation. The degree of degradation increased with the decrease of the crystallinity, and the connective tissue in the HA microsphere implants corresponding to the non ammonia water slurry had a connective tissue.
HA microspheres can form compound biomaterials in polymer materials, improve the acid environment produced by the degradation of polymer materials and improve the biological and mechanical properties of the composites. This study made the HA microspheres mixed into the two oxygen six ring solution of PLLA by the method of flame atomization drying. The porous PLLA/HA microspheres composite scaffolds with better connectivity have been successfully prepared by phase separation technology. The results show that the HA microspheres and PLLA materials can be well combined. With the increase of the amount of HA microspheres, the irregular degree of the composite scaffold structure increases and the mechanical properties are improved. Relative to the compressive modulus (4.4MPa) of the pure PLLA porous scaffold (4.4MPa), the quality of the HA microsphere The compression modulus (9.1MPa) of the composite bracket (9.1MPa) is the highest when the volume ratio is occupied by the composite support. After a period of immersion in SBF, the surface of the scaffold will form bone like apatite, and the amount of the deposition increases with the increase of the content of HA microspheres. At the same time, the addition of HA microspheres can improve the adsorption capacity of the composite scaffold to BSA protein.
After 4 weeks of culture, the rat MC3T3 osteoblast can adhere, differentiate and propagate on the PLLA/HA microsphere composite scaffold. Because of the excellent bioactivity and good wettability of HA microspheres, the cells can be better differentiated and propagated on the composite scaffold material. Histological section also shows that the PLLA/HA Microsphere Composite Scaffold Some cells can grow into the scaffold and fuse well with the material.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R318.08

【引证文献】