体外培养条件下骨支架形态对细胞生物力学环境影响的数值仿真研究

发布时间：2018-07-06 20:49

本文选题：支架 + 有限元法　；参考：《吉林大学》2016年博士论文

【摘要】：骨缺损是由创伤、肿瘤、先天性畸形、骨感染等原因引起的临床常见疾病,是造成肢体残障的重要原因之一。近年来,骨组织工程技术已成为骨缺损疾病治疗的重要手段。骨缺损部位的骨组织-支架复合植入物需通过体外培养获得。体外培养条件、支架材料、以及支架的形态特征是决定能否成功获取骨-支架复合物的关键因素。在体外培养过程中,支架表面黏附的种子细胞存在于复杂的力学环境下。骨组织工程支架将力学激励传递给种子细胞以促使其增殖与分化,同时也为骨的生长提供了临时的力学支撑。然而,目前的实验手段无法准确测得支架内的力学环境参数,通过有限元分析方法可以很好地解决这一问题。本研究采用Micro-CT影像与有限元技术相结合的方法,建立了具有不同形态、材料的骨组织工程支架有限元模型,定量分析了支架内的力学激励,并模拟预测骨髓间充质干细胞(BMSCs)在不同支架结构上的分化结果。本研究主要可以分为以下三个部分:第一部分对基于松质骨理想化胞元形态的骨支架模型进行细胞分化仿真分析。测量支架的三维孔形态参数,并且对体外培养条件对细胞分化结果的影响进行了预测。首先利用Rhino三维建模软件建立了5种孔隙率为65%的三维骨组织工程支架模型。支架采用线弹性PDLLA材料,在0.5-5%的压应变作用下,分析模型表面的应变分布情况,同时在孔内模拟稳态流,入口流速为0.01-1 mm/s,计算支架表面的流体剪应力(FSS)。最后,基于细胞分化定量理论对不同支架表面的BMSCs分化结果进行了预测分析。结果表明,应变和流体剪应力的分布取决于支架内孔隙的分布。进口流体速度和压应变以及支架的形态均会影响支架表面BMSCs的分化结果。当轴向压应变在0.5-5%,流体初速度在0.01-1 mm/s范围内时,所有支架的骨和软骨分化面积可达到90%以上。本研究设计的不同孔结构支架在压力和流体流动作用下可以产生不同程度激励以利于BMSCs的分化,并满足不同骨缺损部位对力学性能的需要。第二部分依据动物松质骨形态,对显微结构参数与力学参数、细胞分化参数的关系进行了分析。首先对雄鼠和牛的松质骨进行Micro-CT扫描,选取孔隙率为65%左右的松质骨结构建立1 mm3的三维立方体支架,施加进口流体速度和压应变,进行体外灌流培养条件下的有限元数值模拟。支架固体采用线弹性的聚乳酸(PDLLA)材料,对其施加0.5-5%的压缩载荷,同时模拟入口流速为0.01-1mm/s的牛顿流。根据细胞分化定量理论确定不同初始条件下,力学激励对每种结构表面BMSCs分化结果的影响。结果表明,应变和流体剪应力的分布取决于松质骨结构支架的形态。不规则的形态结构使激励分布极不均匀,并且细小孔道处出现应力集中现象。细胞分化过程中对于进口流体流速比支架所受轴向应变更加敏感。当入口流体速度在0.01-1 mm/s,整体压应变在0.5-5%范围内时,所有支架壁表面的骨分化面积可达到90%以上。与牛松质骨结构支架相比,鼠松质骨结构支架骨分化区在60-90%之间的分化激励值范围较大。相对于鼠松质骨结构支架,具有更多板状结构的牛松质骨结构支架的软骨分化情况更好,软骨分化区在60-90%之间的分化激励值范围远远大于鼠松质骨结构支架。本研究依据松质骨形态建立支架结构,提取出了能够全面反映支架形态的3个主元素,对显微结构参数与力学参数、细胞分化参数进行了相关性分析,并建立了主元素与力学参数、细胞分化参数之间的回归方程。在进行细胞体外培养时,可以为BMSCs提供更贴近于体内的力学环境,为支架形态的设计和临床治疗骨缺损提供了理论基础。第三部分针对不同工艺制备的生物材料支架,比较了支架在不同流体加载参数条件下的力学环境。基于Micro-CT影像重建三维Ti O2生物材料支架,测量支架的孔形态参数,并通过扫描电镜观察支架表观形貌。另外,将本研究得到的渗透率结果与其它研究进行了比较,验证了仿真分析的有效性。最后,对支架内的力学激励(流体剪应力和固体应变)进行定量分析,同时与其他三种商业骨支架材料(Bio-Oss,Cerabone,Maxresorb)进行了比较。结果表明Ti O2的力学性能相对较差,但壁面流体剪应力明显高于其他商业骨支架替换材料。另外,不规则的支架形态会形成不均匀的激励分布,且在四种支架内产生的流体剪应力均在BMSCs产生生物学反应的激励范围内。本研究从力学性能、生物材料、组织学等多角度评价了骨组织工程支架的性能,研究结果可以为生物反应器内体外培养条件的设计,以及临床特定部位骨缺损的修复提供重要的理论依据。所建立的三种不同形式的支架模型(理想化支架模型,动物松质骨支架模型,以及合成生物材料支架模型)可以作为研究体外灌流培养实验的基础,并为体外培养条件下支架的制备与临床应用提供更为直观的依据。
[Abstract]:Bone defect is a common clinical disease caused by trauma, tumor, congenital malformation and bone infection. It is one of the important causes of the disability of the limb. In recent years, bone tissue engineering has become an important means for the treatment of bone defects. Bone tissue scaffold composite implant in bone defect needs to be obtained through culture in vitro. The condition, scaffold material, and the morphological characteristics of the scaffold are the key factors determining whether the bone scaffold complex can be successfully obtained. In the process of culture, the adherent seed cells of the scaffold exist in the complex mechanical environment. The bone tissue engineering scaffold transfers mechanical stimulation to the seed cells to promote its proliferation and differentiation. It provides a temporary mechanical support for bone growth. However, the mechanical environment parameters in the scaffold can not be accurately measured by the present experimental methods. The finite element method can be used to solve this problem well. In this study, a combination of Micro-CT images and finite element method was used to establish a bone tissue worker with different shapes and materials. The finite element model of range stents is used to quantitatively analyze the mechanical excitation in the scaffold and to simulate and predict the differentiation of bone marrow mesenchymal stem cells (BMSCs) on different scaffolds. This study can be divided into three parts: the first part is the simulation analysis of the cell differentiation based on the idealized bone scaffold model of the cancellous bone. The three-dimensional pore shape parameters of the scaffold were measured and the effects of the culture conditions on the cell differentiation were predicted. First, 5 three-dimensional bone tissue engineering scaffolds with 65% porosity were established by using Rhino 3D modeling software. The scaffolds were linear elastic PDLLA material, and the model surface should be analyzed under the 0.5-5% pressure strain. At the same time, the steady flow is simulated in the hole, and the flow velocity of the inlet is 0.01-1 mm/s. The fluid shear stress (FSS) on the surface of the stent is calculated. Finally, the results of the BMSCs differentiation on the different scaffolds are predicted based on the cell differentiation theory. The results show that the distribution of the strain and the shear stress of the flow body depends on the distribution of the pores in the scaffold. The velocity and strain of the oral fluid and the shape of the scaffold affect the differentiation of BMSCs on the surface of the scaffold. The bone and cartilage differentiation area of all scaffolds can reach more than 90% when the axial compression strain is 0.5-5% and the initial velocity of the fluid is within the range of 0.01-1 mm/s. Different extent of stimulation was produced to help BMSCs differentiation and to meet the needs of mechanical properties of different bone defects. The second part, based on the shape of the cancellous bone, analyzed the relationship between the microstructure parameters and the mechanical parameters and the cell differentiation parameters. First, the Micro-CT scan was performed on the cancellous bone of male and cattle, and the porosity was 65%. The left and right cancellous bone structures set up a 1 mm3 three-dimensional cube scaffold, applying the inlet fluid velocity and pressure strain to simulate the finite element numerical simulation under the condition of in vitro perfusion culture. The scaffold solid adopts the linear elastic polylactic acid (PDLLA) material to apply the 0.5-5% compression load to it, and simulates the Newton flow of the inlet velocity of 0.01-1mm/s. The quantitative theory of cell differentiation determines the effect of mechanical excitation on the results of BMSCs differentiation on the surface of each structure under different initial conditions. The results show that the distribution of the strain and the shear stress of the fluid depends on the shape of the structure of the cancellous bone structure. The irregular shape and structure make the excitation distribution very uneven, and the stress concentration in the small pores. In the process of cell differentiation, the flow velocity of the inlet fluid is more sensitive than the axial stress of the stent. When the inlet fluid velocity is at 0.01-1 mm/s and the overall compressive strain is within the 0.5-5% range, the bone differentiation area of all the wall surfaces can reach more than 90%. Compared with the bovine cancellous bone structure scaffold, the bone differentiation area of the rat cancellous bone structure is between 60-90% and the bone structure. The differentiation incentive value is larger. Compared with the structure of the bone structure of the rat, the cartilage differentiation of the bovine cancellous bone structure with more plate structure is better, the differentiation incentive value of the differentiation area between 60-90% is far greater than that of the rat cancellous bone structure. The 3 main elements of the scaffold form are fully reflected. The correlation analysis of the microstructure parameters and the mechanical parameters and the cell differentiation parameters is carried out. The regression equation between the main elements and the mechanical parameters and the cell differentiation parameters is established. In the culture of the cells in vitro, the BMSCs can provide a more close to the internal mechanical environment for the scaffolding. The design of state and the clinical treatment of bone defect provide a theoretical basis. The third part compares the mechanical environment of the scaffold under the conditions of different fluid loading parameters for the scaffolds prepared by different processes. The reconstruction of the three-dimensional Ti O2 biomaterial scaffold based on the Micro-CT image is used to measure the pore shape parameters of the scaffold, and the scanning electron microscope is observed by scanning electron microscope. In addition, the results obtained from this study were compared with other studies to verify the effectiveness of the simulation analysis. Finally, the mechanical excitation (fluid shear stress and solid strain) in the support was quantitatively analyzed, and compared with the other three kinds of commercial bone scaffolds (Bio-Oss, Cerabone, Maxresorb). The results show that the mechanical properties of Ti O2 are relatively poor, but the shear stress of the wall fluid is significantly higher than that of other commercial bone scaffold replacement materials. In addition, the irregular shape of the scaffold forms an uneven excitation distribution, and the fluid shear stress produced in the four kinds of scaffolds is within the excitation range of the biologic reaction in BMSCs. The performance of bone tissue engineering scaffolds is evaluated by many angles, such as biomaterials and histology. The results can provide important theoretical basis for the design of the culture conditions in the bioreactor and the repair of bone defects in specific sites. Three different forms of scaffold models (idealized scaffold model, animal cancellous bone) have been established. The scaffolding model, as well as the synthetic biomaterial scaffold model, can be used as the basis for the study of in vitro perfusion culture, and provide a more intuitive basis for the preparation and clinical application of scaffolds in vitro culture conditions.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.04;R683

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