微流体动力环境对磷酸钙生物陶瓷骨诱导行为的影响及机理

In this study, influence of micro-fluid dynamic environment in calcium phosphate porous bioceramics for protein adsorption/disadsorption, cell growth and distribution, osteogenic differentiation, and extracellular matrix production under stress will be researched by using experiments observation and computational fluid dynamics (CFD) modeling simulation. The mechanism of osteoinduction effected by macrostructure properties in biomaterials will be investigated based on the mechanoregulation theory. The study contents include: 1) developing 3D scaffolds models and analyzing the micro-fluid dynamic environment, that can be controlled by macrostructure parameters during the scaffold fabrication process, through a CFD model, and with the aim of predicting the protein adsorption/disadsorption and cell behavior under the hydrodynamic field; 2) evaluating the results of biomimetic mineralization and cell growth and distribution, osteogenic differentiation, and extracellular matrix production under different stress condition in bioceramics with different macrostructure properties, and comparing the experiments results to those predictive ones; 3) investigating the process of bone formation and vascularization in bioceramics with different porous structure properties when implanted at heterotopic sites in vivo. The innovation in the study is: using CFD modeling, stress condition in scaffold can be characterized and quantified as a function of parameters that can be used in optimal fabrication of calcium phosphate bioceramics with controlled osteoinduction; at the same time, the study provide a novel access to explore the interaction between scaffold pore structure, mechanical stimuli developed at the cell microscopic level, and physical conditions in vivo through micro-fluid dynamic environment effected by stress acting on scaffold systems.

本研究拟采用实验观察和模拟计算相结合的方法，研究应力作用下磷酸钙多孔生物陶瓷中的微流体动力环境对支架内的蛋白质吸附/解吸，细胞生长和分布、骨向分化，及细胞外基质生成的影响。基于力学信号转化理论,对生物材料通过自身结构特征调控骨诱导发生的机制进行探讨。研究内容：1）构建三维多孔支架模型,通过计算流体动力学（CFD）方法分析材料孔隙结构对支架内微流体动力环境的影响并预测流体场对蛋白吸附/解吸及细胞行为的作用；2）在施以应力的条件下，材料的矿化表现及细胞行为评价，并与预测结果相验证；3）在体内非骨生理环境中，材料的骨诱导行为评价。其创新点在于：CFD模型应用于生物材料骨诱导机制研究中能定性和量化应力环境，对优化设计可控骨诱导性的磷酸钙生物陶瓷具有指导意义；同时，在支架体系上施加应力，能影响支架内的微流体动力环境，为研究支架孔隙结构、细胞显微水平上的力学刺激及体内生理环境之间的相互作用提供新途径。

尽管对磷酸钙生物陶瓷骨诱导现象的生物机制进行了大量的研究，却仍缺乏对这些机制的系统性理解，这限制了生物陶瓷在临床骨再生治疗中的广泛应用。本研究从体内应力环境、支架孔隙结构和支架内细胞受到的力学刺激之间的相关性角度入手，对骨诱导发生机制进行探讨。为深入研究磷酸钙生物陶瓷与骨诱导发生的相关性机制提供新途径，并对优化设计可控骨诱导性的磷酸钙生物陶瓷提供实验和理论依据。包括以下内容：.（1）构建三维多孔支架模型,通过计算流体动力学（CFD）方法分析材料孔隙结构对支架微流体动力环境的影响。通过分析发现球粒造孔支架的贯通孔/宏孔的比值超出0.5时，支架结构将出现破损。计算结果显示，支架宏孔形状、宏孔径、宏孔径/贯通孔径比例对支架微流体动力环境有显著影响，而微流体动力环境又将影响支架内新骨形成和分布。.（2）将具有不同宏观孔隙形状和尺寸的羟基磷灰石颗粒堆积支架和羟基磷灰石颗粒造孔支架植入狗的背肌和腹腔，系统比较宏孔形状和尺寸对支架异位成骨能力的影响。结果表明宏孔径和形状对HA多孔支架骨诱导性有显著的影响，并且这种影响是两种结构变量共同作用的结果。研究提示微流体动力环境是材料的宏孔结构因素转换为骨诱导生物调控信号的重要途径。.（3）在体外模拟生理环境下，构建微振动/支架/细胞复合培养体系和灌流/支架/细胞复合培养体系。微振动培养实验结果表明微振动应力环境能提升磷酸钙支架生物矿化能力，有利于构建成骨环境；微振动应力环境通过三维多孔支架发生力学信号转换诱导BM-MSCs成骨分化；并且微振动应力环境和HA多孔支架协同提高了BM-MSCs成骨基因和成骨特征性蛋白ALP的表达。灌流实验结果表明，灌流改善了物质传输；在相同贯通性条件下，支架的孔径差异对微流体动力环境有显著影响，该结果与计算预测分析相一致。宏孔径对细胞流体灌注的剪切力刺激在一定范围内(1300–500 μm)存在临界尺寸，通过激活ERK1/2途径，将外部刺激导入细胞核内，从而引起细胞成骨基因和蛋白表达的改变。