摘要
骨组织工程的提出和发展改变了传统的骨缺损修复治疗模式,同时也对支架的制备提出了更高的要求。然而,在当前的骨组织工程陶瓷支架研究中,存在着强度较低、影响支架性能的微细结构的制备不可控,以及烧结与性能之间的矛盾等问题,极大地限制了支架的临床应用。支架作为骨组织工程的关键要素之一,其三维精细结构的成形严重依赖于制备技术。随着自由成形技术的发展,以挤出沉积成形技术为代表的直写技术以其极高的材料适用性和柔性显示了在微纳米生物材料成形方面的巨大优势,结合微波快速烧结技术,为解决上述问题提供了有力的手段。
为满足骨组织工程支架在结构、力学和生物学等方面的要求,本文以多孔磷酸钙支架为研究对象,建立基于电机助推挤出沉积成形技术与微波快速烧结技术相结合的陶瓷支架可控制备工艺体系。通过材料学测试与生物学评价,结合计算机模拟及理论分析,探讨支架微观结构、烧结工艺等与力学性能、降解性、生物学性能的关系,总结并提出材料微观结构可控制备及其性能优化的途径和方法。
根据挤出成形工艺对浆料性能的要求,制备了具有良好分散性、稳定性的高浓度纳米羟基磷灰石(HA)浆料,阐述了纳米陶瓷粒子的稳定分散机理。结果表明,在PH=9,分散剂PAA-NH4含量为1%(与粉体质量相比)的条件下,平均粒径为20nm的HA陶瓷颗粒在浆料能够获得良好的分散稳定性,浓度为25-40vo1%的纳米HA浆料具有良好的自支持能力与合适的凝固速度,可用于挤出沉积成形。
发展了基于电机助推式挤出沉积成形技术与微波快速烧结工艺相结合的支架可控制备技术,建立了浆料在喷嘴内流动的数学模型和工艺参数匹配模型,优化了支架的微波烧结工艺。制备了具有不同解剖外形(立方体、圆柱、胫骨轮廓)与不同内部孔结构(三角形、四边形、六边形等)的多孔HA支架,支架结构可控,孔隙率可调,且连通性良好。经1200℃(40℃/min,30min)微波烧结后,支架收缩均匀,平均收缩率为31.8%,致密度高、晶粒细小均匀(1.12±0.23μm)。强度测试表明,微波烧结有利于提高多孔支架的力学强度,孔隙率约50%的支架平均抗压强度为45.57MPa,远远大于常规烧结工艺所得的支架的强度。
有限元分析与实验研究结果表明,支架结构特征通过影响支架内应力的大小与分布,从而影响支架的承载能力。结构特征对支架力学性能影响的权重关系为：孔隙率>孔洞大小>孔洞形状。对于孔洞结构均匀的HA支架,抗压强度与孔隙率的关系为：lnσ=ln432.3-4.825p。为进一步提高支架强度,设计并可控制备了带有“微加强筋”的多孔支架,平均抗压强度为84.2MPa(孔隙率约50%),其强度与相同孔隙率条件下未设置加强筋的支架相比提高了约80%,与皮质骨的强度相当。同时,该结构支架在降解过程中保持了良好的力学稳定性,能够为细胞的分化与组织的生长提供更长时间的强度支持。解理是多孔陶瓷支架的主要断裂机理,在拉应力的作用下支架将从挤出丝的搭接处发生断裂。
体外模拟降解实验与细胞-支架的联合培养实验表明,多孔HA支架在生理盐水中能够缓慢降解释放出Ca2+,并能在SBF中沉积形成类骨磷灰石层,具有良好的生物相容性,细胞能够迅速而紧密地在支架的表面与侧壁形成生物学黏附,并能够进入支架的内部,实现细胞的贴附与生长。微波烧结支架因烧结时间短,升温速率快,晶粒结构细小,其降解速率快于常规烧结支架,有利于成骨细胞的黏附、生长和增殖。
本文研究表明,以挤出沉积成形技术为代表的直写技术结合微波烧结工艺能够实现植入支架的个性化制造,并能同时提高支架的力学性能与生物学性能,为解决磷酸钙陶瓷支架临床应用瓶颈提供了依据,丰富和发展了生物制造方法。
The statement and development of bone tissue engineering have changed the traditional treatment modes for bone defects, and also put forward higher requirements for scaffolds fabrication. However, the ceramics scaffolds fabricated so far exsit some problems, such as relatively low strength, uncontrollable inner structures and the contradition between sintering and performances, which greatly limit their clinical applications. Scaffold is one of the key elements of bone tissue engineering. The fabrication of its three-dimensional fine structures is largely depended on preparation techniques. With the development of solid freeform fabrication, extrusion deposition technique (EDT), a representative of direct writing, shows great advantages on manufacturing micro-nano bio-devices due to its highly applicability and flexibility. Combined with microwave rapid sintering, EDT provides a powerful mean to solve the above problems in bone scaffolds fabrication.
The objective of this paper is to establish a controllable process for bone scaffold fabrication based on motor assisted microsyringe extrusion deposition technique and microwave sintering, to meet the structural, mechanical and biological requirements of porous calcium phosphate bone scaffolds. The effects of structure and sintering process on the mechanical, degradative and biological properties of scaffold were investaged, according to materials test, biological evaluation, computer simulation and theoretical analysis. Methods for scaffold controllable preparation and performance optimization were proposed.
According to the requirements of slurry for EDT, nano HA slurry with high concentration, good dispersibility and stability were prepared. The dispersal mechanism of nano particles was expounded. The results showed that the slurry prepared from20nm HA ceramic powder was able to obtain good dispersion stability under the conditions of PH=9.0,1wt%dispersant agent PAA-NH4(compared to the weight of HA). The slurry with volume concentration of25-40vol%has good self-support capability and proper solidification rate, that can be used for extrusion deposition process.
A new process based on motor assisted microsyringe extrusion deposition technique and microwave sintering was developed for scaffold controllable fabrication. The mathematical model of the slurry flow in the nozzle and the parameters matching model of EDT were established. The sintering process was also optimized. Porous HA scaffolds with different outer shapes like cubic, cylinder and tibia bone, and different pore structures like triangle, square and hexagon were fabricated. The structure features of scaffolds are controllable, and their porosities are adjustable as well. The scaffolds have good connectivity. The microwave sintered scaffolds (1200℃,40℃/min,30min) gain uniform shrinkage with an average shrinkage rate about31.8%. They also gain high density and fine grain size (1.12±0.23μm) in the rods. The compressive strength test shows that microwave sintering is conducive to the improvement of mechanical strength of porous scaffolds. The average compressive strength of microwave sintered scaffolds with porosity of50%is45.57MPa, much higher than that of the conventional sintered ones.
Finite element analysis and experimental results show that scaffold structure affects the load capacity by changing their stress intensity and distribution. The weight relationship of the impact of structure features on mechanical property is porosity> pore size> pore shape. For a uniform structure scaffold, the quantitative relationship between compressive strength and porosity inσ=ln432.3-4.825p.In order to further improve the mechanical strength, porous scaffold strengthened with micro-ribs was also designed and controllably fabricated. The average compressive strength is84.2MPa (porosity about50%), about80%higher than the scaffolds without micro-rib with the same porosity, and close to the strength of the cortical bone. Moreover, it also exhibits more stable mechanical strength during degradation in vitro, which could provide longer support for cell proliferation and tissue formation. The fracture mechanism of porous ceramic scaffold is cleavage. Scaffold will crack from the rod joint where the tensile stress concentrated.
In vitro degradation and cell scaffold co-culturing experiments results show that the porous scaffold could slowly dissolve and release Ca2+in saline. It also could generate bone-like apatite layer in SBF. The fabricated scaffold has good biocompatibility. Cells could adhere to the outer surface, side walls and interal surface quickly and form a tightly biological adhesion to promote proliferation and growth. The microwave sintered scaffold possesses finer grains and better degradation ability than conventional sintering scaffold due to short sintering time and high heating rate, which could promote the adhesion and proliferation of osteoblast.
As a conclusion, extrusion deposition technique, a representative of direct writing, combined with microwave sintering could achieve personalized fabrication of scaffold with improved mechanical and biological properties, which could solve the clinical application problems of calcium phosphate ceramic and enrich the biofabrication methods.
引文
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