【作者】 李瑞欣；

【导师】 张西正；

【作者基本信息】 中国人民解放军军事医学科学院 ， 军事预防医学， 2012， 博士

【摘要】 研究背景和目的作为身体的支撑框架，骨组织在整个生命过程中不断地受到各种力的作用而相应地调节其质量、密度和内部结构。成骨细胞是对应力应变信号进行感知的主要力学敏感性细胞。骨缺损的修复及组织工程化骨组织的构建，成骨细胞都是在三维支架内部贴壁生长的。支架材料作为接受外来刺激的主体，将力学刺激从外部传递到细胞，而细胞因为受到力学刺激，其增殖和分化的速率也将会发生改变。因此，研究支架在表观应变下内部的应变分布对于研究力学刺激对三维支架内细胞的增殖及分化具有重要的意义。Micro-CT技术能够非破坏性的获得支架内部结构数据，通过这些数据进行重建获得支架的模型，再用有限元分析不同的力学刺激下支架内部的应变分布。在骨移植材料和骨组织工程支架材料中，羟基磷灰石（Hydroxyapatite, HA）因能与骨形成很强的化学结合具有骨传导作用而被广泛应用，然而，单一HA的脆性及低疲劳强度限制了其进一步应用，壳聚糖（Chitosan，CS）因具有无毒性、可生物降解及良好的可加工性而被广泛应用于组织工程研究中，将HA与CS复合从而增强韧性，并充分发挥HA良好的生物相容性，有望制备出性能优异的骨支架材料。本研究拟制备微、纳米HA并与CS共混制备复合膜及多孔支架材料，考察力学刺激对MC3T3-E1细胞在微、纳米HA/CS复合膜及多孔支架上的生物学特性的影响。为骨移植材料的研制、组织工程骨的体外构建提供一定的理论依据和新方法。研究方法（1）通过将猪骨脱脂脱蛋白、煅烧再球磨的方法制备微米级HA，通过超声、搅拌的方法制备纳米级HA。将微、纳米级HA与CS共混制备复合膜，对复合膜进行理化性能、细胞相容性测试，考察MC3T3-E1细胞在微、纳米HA/CS复合膜上的增殖和凋亡，优选制备三维多孔支架材料的配比。（2）以石蜡微球为致孔剂，通过与HA/CS共混溶液混合后铸模、粒子导出方法制备复合多孔支架材料。对制备的复合支架材料进行动态力学性能测试及SEM形貌观察，将成骨前体细胞MC3T3-E1细胞接种到支架上，考察支架材料的细胞相容性；对支架进行Micro-CT扫描获得微观结构参数，并考察复合支架的体外酶解过程。（3）应用Micro-CT扫描得到的材料结构参数，有限元建模后分析材料在施加不同的表观应变时内部的应变分布，优选出适合的表观应变。对细胞/支架复合物施加波形为正弦波、频率为1Hz、表观应变3000με的动态压载荷、10min/次，1次/d，7d、14d后进行相关检测：SEM观察细胞在支架上受力后的形态变化，检测细胞增殖、凋亡，ELISA检测ALP含量，Real-time PCR检测BMP-2、Col I、OCN mRNA表达，Western blot检测BMP-2、Col I、OCN蛋白含量。实验结果（1）通过对松质骨脱脂脱蛋白再煅烧、球磨的方法，可以获得粒径较为均一的微米级天然HA粒子，中值粒径D50在1.21～1.67范围内。采用超声、搅拌的方法制备了纳米级的直径约5nm、长约50nm的棒状HA粒子。将微、纳米HA与CS共混制备HA/CS复合膜，SEM显示HA在基质CS中分布较均匀，二者结合紧密，复合膜具有较好的力学性能，在CS中加入HA可以克服HA的颗粒流动性的缺点。比较球磨工艺及原料配比对成骨前体细胞MC3T3-E1增殖及形态的影响，优选出9号复合膜的配比制备下一步的多孔支架，细胞在微、纳米HA/CS复合膜上的增殖、凋亡没有统计学差异。在复合膜材料上检测细胞相容性更加直观，为下一步制备多孔支架材料提供了理论依据。（2）以石蜡微球为致孔剂通过粒子导出方法制备了微、纳米级HA/CS复合支架材料，随着致孔剂含量的增加，支架的孔隙连通性增加。实验证实，球形致孔剂制备的支架在孔隙连通性方面较立方体致孔剂更具有优势。对复合支架分别进行动态力学性能测试，支架表现出粘弹性和滞后效应。综合力学性能和SEM结果，优选复合溶液与石蜡微球比例为50:70的支架为下一步实验用支架。经Micro-CT扫描后获得的微、纳米HA/CS复合支架的微观结构参数基本一致，将Micro-CT扫描后获得的图像在Mimics中建模，可以看出制备的支架具有良好的孔隙连通性。微、纳米HA/CS复合支架具有良好的细胞相容性，成骨前体细胞MC3T3-E1能在支架孔壁上黏附、增殖。微、纳米HA/CS复合支架在溶菌酶的PBS溶液中可以发生降解，在前4w降解速率较低，在4～8w降解速率大大提高，降解液中有还原糖生成，且其含量随降解时间的延长呈增大趋势。（3）用基于Micro-CT的有限元方法，计算了不同表观应变下复合支架内部的应变分布，存在应力集中现象，计算出应施加的最佳表观应变为3000με。对细胞/支架复合物施加动态压载荷后，SEM结果表明细胞在微、纳米HA/CS支架上可黏附、增殖，7d时细胞呈梭形、不规则状，有部分集落，14d细胞逐渐融合成片，几乎长满支架表层的孔隙。Real-time PCR和Western结果显示ColⅠmRNA及蛋白表达在14d均高于对照组，而OCN蛋白表达在7d、14d与对照组无显著性差异，表明细胞还未完全分化成熟。ALP蛋白表达在7d时明显升高，14d时又下降。实验结果显示基因mRNA达与蛋白表达二者间并没有完全的一致性。结论（1）通过球磨法可制备微米级HA粒子，通过超声、搅拌的方法可制备出纳米级HA粒子，但将二者与CS共混成膜后尺度效应不够明显，对MC3T3-E1细胞的黏附、增殖、凋亡无明显影响，可能是HA发生团聚所致。（2）应用石蜡微球做致孔剂可以得到孔隙连通性良好的微、纳米HA/CS复合支架材料，随着致孔剂含量的增加，支架的孔隙连通性增加。同法制备的微、纳米HA/CS复合支架材料的微观结构参数基本一致。微、纳米HA/CS复合支架在溶菌酶的PBS溶液中可以发生降解，在4～8w降解速率大大提高，降解液中还原糖含量随降解时间的延长呈增大趋势。（3）应用基于Micro-CT的有限元方法，可分析不同表观应变下复合支架内部的应变分布，计算出利于细胞生长的最佳表观应变。循环压载荷促进了微、纳米复合支架内MC3T3-E1细胞的BMP-2、ColⅠ的mRNA蛋白表达，但对于OCN蛋白表达无显著影响。更多还原

【Abstract】 Background and objectiveAs the frame of body, bone tissue endlessly accommodates its quality, densityand internal structure in whole vital process. Osteoblast is the main mechanicalsensitively cell to response stress and strain signal. In the process of bone defectivereparation and engineered bone tissue construction, osteoblast adherent growth. Asthe main body to receive the external irritant, scaffold impart the irritant to theosteoblast. The proliferation and differentiation of the osteoblast will be changedbecause of the mechanical stimuli. So, it is very important to investigate the straindistributions in scaffolds for studying the cell proliferation and differentiation inscaffolds under mechanical stimuli. The Micro-CT technique allows acquiring,nondestructively, images of transversal sections of the scaffold. From these data, it ispossible to reconstruct the3D geometry and obtain a virtual model that represents thereal shape of the scaffold. Stress and strain distributions in a scaffold at a microscopiclevel can be studied using the finite element method. As bone transplantationmaterials and bone tissue engineering scaffolds, hydroxyapatite (HA) has beenutilized extensively because it can form strong chemical constitution with bone, butits brittleness and low fatigue resistance restrict its further application. Chitosan (CS)is considered as an appropriate functional material for tissue engineering scaffoldsbecause of high biocompatibility, biodegradability, and favourable process properties.Incorporation of HA into the chitosan matrix improved biocompatibility and tenacity,the HA/CS suggests their potential use for bone tissue engineering applications. Weaimed to detect the effect of mechanical stimuli on the biological characterastics ofMC3T3-E1cells on micro, nanoHA/CS composite membranes and scaffolds. Perhapsit is useful for the development of bone transplantation material and engineered boneconstruction.Methods（1）MicroHA was prepared by calcinating and milling degreased, deproteinedcancellous, and nanoHA was prepared by transonic and agitation method. HA/CS composite membranes were prepared by blending, and were tested for physical andchemical properties, biocompatibility. The proliferation and apoptosis of MC3T3-E1cells on composite membrane were tested for the best content of porous scaffolds.（2）Composite scaffolds were fabricated by the technique of compressionmolding and particulate leaching method with paraffin microsphere as porogen. Thecomposite scaffolds were tested for dynamic mechanical property, SEM observationand cell compatibility with MC3T3-E1cell seeding. The microstructure parameters ofscaffolds were obtained by being scanned using Micro-CT. The enzymaticdegradation was also investigated in vitro.（3）The Micro-CT technique allows acquiring, nondestructively, images oftransversal sections of the scaffold. From these data, it is possible to reconstruct the3D geometry and obtain a virtual model that represents the real shape of the scaffold.Stress and strain distributions in scaffold at a microscopic level were analyzed usingthe finite element method through the microstructure parameters to obtain the optimalapparent strain. Dynamic loading of sinusoidal wave,1Hz frequency,3000μεapparent strain was applied to cell/scaffold composite for10min per day. Cellsmorphology was observed by SEM. The proliferation and apoptosis of MC3T3-E1cells on composite scaffolds were tested. ALP protein level was detected by ELISA,BMP-2, Col I, OCN mRNA level were detected by Real-time PCR, BMP-2, Col I,OCN protein level were detected by Western blot.Results（1）MicroHA particle with relatively homogeneous particle size was preparedby calcinating and milling degreased, deproteined cancellous, and the D50was in therange of1.21～1.67. Rod-shape nanoHA particle with5nm diameter and50nmlength was prepared by transonic and agitation method. HA/CS composite membraneswere prepared by blending micro, nanoHA and CS. HA particle was distributeduniformly in the CS matrix and combined with CS tightly, so the HA particleflowability shortcoming was overcomed. The mechanical property of the compositemembrane was good. Comparing the effect of milling parameters and mass ratio onthe proliferation and morphology of MC3T3-E1cell, we optimized the parameters ofmembrane9as the scaffolds preparation parameters. No significant difference of theproliferation and apoptosis of MC3T3-E1cell between micro, nanoHA/CS compositemembrane. （2）Composite scaffolds were fabricated by the technique of compressionmolding and particulate leaching method with paraffin microsphere as porogen. Thepore connectivity was increased with the porogen content. The results showed that itwas more effective for pore connectivity using spherical porogen than cubic porogen.Dynamic mechanical results showed that scaffolds displayed viscoelastic property andthere was a hystersis effect between strain and stress. On the whole results ofmechanical and SEM, the ratio was optimized of50:70between composite solutionand paraffin volume for the next experiment. The microstructure parameters of microand nanoHA/CS composite scaffolds with same methods were uniform basically.3Dmodel in Mimics of composite scaffolds displayed satisfactory pore connectivity.MC3T3-E1cell could adhered and proliferation on the composite scaffolds, so bothmicro and nano composite scaffolds had good cytocompatibility. Micro andnanoHA/CS composite scaffolds were experienced degradation in the PBS solution oflysozyme. The degradation rate was slow before4weeks, but increased after4weeks,and the amount of reducing sugar in the supernatant was increased with thedegradation time.（3）Stress and strain distributions in the composite scaffold at a microscopiclevel were analyzed using the finite element method based Micro-CT, and the optimalapparent strain3000με which was benefit for cell growth was calculated. The finiteelement results also showed there was stress concentration in the scaffolds. After theapplication of dynamic loading on the cell/scaffolds composite, SEM observationrevealed MC3T3-E1cell could adhered and proliferation on the composite scaffolds,and the cell change morphology from fusiform to confluence. On day14,ColⅠmRNA and protein had high level than control group, but no significantdifference of OCN protein level. This results showed that the cells were notcompletely matured. ALP protein level increased on day7, but decreased on day14.The results showed that there was consistent between mRNA and protein level.Conclusions（1）MicroHA was prepared by milling and nanoHA was prepared by transonicand agitation method, but the scale effect was not significantly. There was notsignificant influence on the adheasion, proliferation and apoptosis of MC3T3-E1cell,and the reason perhaps was the agglomeration of HA particles.（2）Micro, nanoHA/CS composite scaffolds were fabricated by the technique of compression molding and particulate leaching method with paraffin microsphere asporogen. The pore connectivity was increased with the porogen content. Themicrostructure parameters of micro and nanoHA/CS composite scaffolds with samemethods were uniform basically. Micro and nanoHA/CS composite scaffolds wereexperienced degradation in the PBS solution of lysozyme. The degradation rate wasincreased after4weeks, and the amount of reducing sugar in the supernatant wasincreased with the degradation time.（3）Stress and strain distributions in the composite scaffold at a microscopiclevel could be analyzed using the finite element method based Micro-CT, and theoptimal apparent strain benefit for cell growth was calculated. Dynamic loadingpromoted the level of BMP-2, ColⅠmRNA and protein of MC3T3-E1cells on micro,nanoHA/CS composite scaffolds, but influenced OCN protein level not significantly.更多还原