【作者】 肖桂勇；

【导师】 朱瑞富； 吕宇鹏；

【作者基本信息】 山东大学 ， 材料学， 2010， 博士

【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。更多还原

【Abstract】 Hydroxyapatite (HA) can be widely used for repair and replacement of damaged or traumatized bone tissues due to its good biocompatibility and bioactivity. With specific porous structure and good surface properties, spherical HA has been potentially applied on the field of separation and purification for bioactivity macromolecules, such as proteins, nucleic acids and enzymes. The lower crystallinity or amorphous HA microspheres (HAM) has broad application prospect not only because of its good flowability and high specific surface area, but also its biodegradable and excellent bioactivity. One of method reducing the crystallinity of HAM is through changing the properties of HA slurries and rapid solidification, which can retain much more characteristic of original HA slurry in the HAM. The low crystallinity HAM can be used for drug delivery, cell culture carrier, bone filter, bone repair and other fields because of its good properties. So far, hollow and porous HAM mainly has been fabricated primarily by the template method and then calcinated it. However, almost no degradable HAM with high crystallinity could be prepared using this mehod. The relevant reports about preparation and characterization of lower crystallinity of HAM are very rare.In this study, HAM with low crystallinity and high porosity were fabricated by the flame-drying method with self-made device using methane flame as the drying medium. The microstructure, phase component and other performances of HAM were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), transmission electron microscopy (TEM), laser diffraction particle size analyzer (LDPSA), surface area analyzer (SSA), and fourier transform infrared spectrum analyzer (FTIR). Besides, the parameters of the flame-drying method were systematically optimized in the process of HAM fabrication. Three kinds of HAM were fabricated by that flame-drying method using HA slurries with ammonia (AHAs), without ammonia (NAHAs) and under ice-water condition (IWHAs), respectively. The in vitro and vivo experiments of HAM were discussed for studying the biological behavior differences with different crystallinity and microstructure. Furthermore, taking the HAM by the NAHAs as the supplement, porous PLLA/HAM composite scaffolds were fabricated by the thermally induced phase separation technology. Then the effect of content of HAM in the composite scaffolds on their mechanical and biological properties was systematically researched in this paper.The results indicated that HAM has been retained more performances of HA crystal in the slurry under higher drying medium using self-made flame-drying device. According to these results, it could reduce the crystallinity of HAM and improve its structural properties through changing HA slurries parameters. In this paper, HAM by the AHAs has the regular spherical structure, smooth surface morphology, smaller particle size range and highest crystallinity which were compared with other two kinds of HAM. HAM by the NAHAs showed some hollow and burst open structural and has lower crystallinity. HAM by the IWHAs has the porous structure, larger particle size range and lowest crystallinity (29.8%). In addition, the specific surface area (167.16 m2/g) and the total pore volume (0.5054 ml/g) of HAM by the IWHAs were much higher than that of HAM by AHAs (52.523 m2/g and 0.2331 ml/g, respectively).Because the characteristic of HA slurry can significantly impact the properties of HAM by the flame-drying method, it is necessary to research the crystal process during procedure of synthesis HA. The results showed that HA crystallization and its crystal grain microstructure in the slurry were affected by the reaction time and temperature as well as sedimentation time. Among them, the reaction temperature is the main parameters of crystalline HA. High temperature could provide more free energy, so that HA crystallinity increased with the temperature increasing.The TEM microstructure of HAM by the flame-drying method showed that the properties of HA crystal in the original slurry would affect the HAM microstructure. The component particles of HAM by the NAHAs had the high crystallinity, and distributed as rod or strip like. For the HAM by AHAs, there were some non-transparent crystal existents in the microstructure morphologies. The TEM results indicated that some bubble-like defects were existed in the nano-particles of HAM by the IWHAs. In addition, there were lots of nano-pores included in the HAM by the IWHAs, which resulted in its soft microstructure.Three kinds of HAM by the flame-drying method showed different impacts at different calcining temperature. A little change took place on the surface morphologies, crystallinity and BET of HAM after sintering under 600℃. The grain of HAM became coarsening and fusion with the calcining temperature increasing above 800℃. Moreover, impure phases, such asα-TCP andβ-TCP, appeared when HA decomposed at 1000℃. And the BET and porosity of HAM were significantly decreased because small particles united together when calcined at higher temperature. However, for HAM by the IWHAs, obviously changes appeared, including the BET and total pore volume markedly decreased, even though at lower calcining temperature.In vitro experiment indicated that HAM fabricated by the flame-drying method has the good bioactivity and the potential application of adsorption and control release for bovine serum albumin (BSA). Three kinds of HAM showed different impacts in the physiological environment. Among them, the HAM by the IWHAs had the best bioactivity because of its lowest crystallinity and high total pore volume. In vivo data showed that HAM by the flame-drying method had non-toxic effect and could be well integrate with bone. Histology results demonstrated that the HAM had well osteoconduction and biodegradation after implanting into New Zealand rabbit femur for 4 months. The degradability of HAM by the IWHAs was much more serious compared with the other two kinds of HAM. In addition, SEM morphologies showed that some connective tissue grew into the HAM implants after implanting into rabbit’s femur for 4 months.Polymer combined with HAM could form composite biomaterials which would modify the acidic environment due to degradability of HA and improve biological and mechanical properties. In this study, porous PLLA/HAM composite scaffolds were fabricated by the thermally induced phase separation technology. The results demonstrated that the HAM was uniformly incorporated into the PLLA/HAM composite porous scaffolds. As the HAM ratio was increased the porous composite scaffolds changed from ladder-like into isotropic structure. In addition, the mechanical property of PLLA/HAM composite scaffolds improved with increasing HAM ratio in the scaffolds. The compressive modulus reached to maximum (9.1MPa) when the HAM ratio was 30% in the composite scaffolds. While the compressive of plain PLLA scaffolds was only 4.4MPa. Bone-like apatites would be formed onto the surface of composite scaffolds after incubated into simulated body fluid (SBF) for a period of time. And the amount of deposition on the surface of scaffolds was increased with the HAM ratio increasing. Meanwhile, BSA adsorption of composite scaffolds was also improved as adding into HAM.In vitro experiment indicated that PLLA/HAM composite scaffolds improved the attachment, migration and differentiation of MC3T3 osteoblastic cells after culture for 4 weeks. It demonstrated that the PLLA/HAM composite scaffolds were superior to plain PLLA scaffold for bone tissue engineering. The histologies morphologies also showed that the cells could grow into and well integrate with PLLA/HAM composite scaffolds.更多还原