力学作用下微、纳米HA/CS共混体系中微观结构参数对成骨前体细胞MC3T3-E1生物学特性的影响
|
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。
Background and objective
As 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.
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。
Background and objective
As 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.
引文
[1] Karlsson MK, Johnell O, Obrant KJ. Is Bone Mineral Density Advantage MaintainedLong-term in Previous Weight Lifters. Calcif Tissue Int,1995,57(5):325-328.
[2] Burr DB, Milgrom C, Fyhrie D. In vivo measurement of human tibial strains during vigorousactivity. Bone,1996,18(5):405-410.
[3] Clara S, Sara C, Patrick JP. Simulation of angiogenesis and cell differentiation in a CaPscaffold subjected to compressive strains using a lattice modeling approach. Biomaterials,2010,31:2446-2452.
[4] Andy LO, Elia M, Josep A. Planell, Damien Lacroix. Finite element study of scaffoldarchitecture design and culture conditions for tissue engineering. Biomaterials,2009,30:6142-6149
[5] Karande TS, Ong JL, Agrawal CM. Diffusion in musculoskeletal tissue engineering scaffolds:design issues related to porosity, permeability, architecture, and nutrient mixing. Ann Biomed Eng,2004,32:1728-1743.
[6] Schriefer JL, Warden SJ, Saxon LK. Cellular accommodation and the response of bone tomechanical loading. J Biomech,2005,38:1838-1845.
[7] Martin I, Wendt D, Heberer M. The role of bioreactors in tissue engineering. TrendsBiotechnol,2004,22:80-86.
[8] Meyer U, Buchter A, Nazer N, Wiesmann HP. Design and performance of a bioreactor systemfor mechanically promoted three dimensional tissue engineering. J Oral Maxillofac Surg,2006,44:134-140.
[9] Wendt D, Marsano A, Jakob M. Oscillating perfusion of cell suspensions throughthree-dimensional scaffolds enhances cell seeding efficiency and uniformity. Biotechnology andBioengineering,2003,84:205-214.
[10] Stops A.J.F, Heraty KB, Browne M. A prediction of cell differentiation and proliferationwithin a collagen–glycosaminoglycan scaffold subjected to mechanical strain and perfusive fluidflow. Journal of Biomechanics,2010,43:618-626
[11] Jaecques SVN, Van OH, Muraru L. Individualised, micro CT-based finite element modellingas a tool for biomechanical analysis related to tissue engineering of bone. Biomaterials,2004,25:1683-1696.
[12] Damien L, Arnaud C, Maria PG. Micro-finite element models of bone tissue-engineeringscaffolds. Biomaterials,2006,27:5326-5334.
[13] Sun W, Starly B, Nam J. Bio-CAD modeling and its applications in computer-aided tissueengineering. Comput-aided Design,2005,37:1097-1114.
[14] Lacroix D, Sandino C, Villagomez M. A micro-Finite Element analysis of fluidic and solidmechanical stimuli in bone tissue engineering scaffolds. Journal of Biomechanics,2006,39(Supplement1): S415
[15] Saey T, Dietmar WH. Application of micro CT and computation modeling in bone tissueengineering. Computer-Aided Design,2005,37(11):1151-1161.
[16] Byrne DP, Lacroix D, Planell JA. Simulation of tissue differentiation in a scaffold as afunction of porosity, Young’s modulus and dissolution rate: application of mechanobiologicalmodels in tissue engineering. Biomaterials,2007,28:5544-5554.
[17] Den BFC, Wippermann BW, Blokhuis TJ. Healing of segmental bone defects with granularporous hydroxyapatite augmented with recombinant human osteogenic protein-1or autologousbone marrow. J Orthopaed Res,2003,21:521-528.
[18]许永华，施新猷，胡蕴玉.牛煅烧骨与体外培养兔骨膜成骨细胞的相容性.第四军医大学学报，2000,21(4):512-514
[19]徐小良，汤亭亭，楼觉人. β-TCP/HAP双相煅烧骨的制备及其在BMP-2基因给药中的应用.生物医学工程学杂志,2003,20(4):630-633
[20]赵巍,侯振德,任朝峰.骨细胞培养过程对羟基磷灰石力学性质影响的研究.实验力学，2009,24(1):1-7
[21] Toshiaki K, Takao Y, Takashi N. Four calcium phosphate ceramics as bone substitutes fornon-weight-bearing. Biomaterials,1993,143:216-224
[22] Kikuchi M, Matsumoto HN, Yamada T. Glutaraldehyde cross-linked hydroxyapatite/collagen self-organized nanocomposites. Biomaterials,2004,25:63-69.
[23] Manjubala I, Scheler S, Bossert J. Mineralisation of chitosan scaffolds with nano-apatiteformation by double diffusion technique. Acta Biomater,2006,2(1):75-84.
[24] Malafaya PB, Silva GA, Reis RL. Natural-origin polymers as carriers and scaffolds forbiomolecules and cell delivery in tissue engineering applications. Adv Drug Deliv Rev,2007,59(4–5):207-233.
[25] Thein-Han WW, Misra RDK. Biomimetic chitosan–nanohydroxyapatite composite scaffoldsfor bone tissue engineering. Acta Biomaterialia,2009,5:1182-1197.
[26] Hu Q, Li B, Wang M. Preparation and characterization of biodegradable chitosan/hydroxyapatite nanocomposite rods via in situ hybridization: a potential material as internalfixation of bone fracture. Biomaterials,2004,25(5):779-85.
[27] Kong L, Gao Y, Cao W. Preparation and characterization of nano-hydroxyapatite/chitosancomposite scaffolds. J Biomed Mater Res,2005,75A:275-82.
[28] Rusu VM, Ng CH, Wilke M. Size-controlled hydroxyapatite nanoparticles as self-organizedorganic–inorganic composite materials. Biomaterials,2005,26(26):5414-26.
[29] Manjubala I, Scheler S, Bossert J. Mineralisation of chitosan scaffolds with nano-apatiteformation by double diffusion technique. Acta Biomater,2006,2(1):75-84.
[30]杨菊林,周长忍,田冶.壳聚糖/羟基磷灰石膜的制备及对细胞生长的影响.生物医学工程学杂志,2009,26(3):580-584.
[31]段友容,吕万新,王朝元.在动态模拟体液中致密CaP陶瓷表面形貌对类骨磷灰石层形成的影响.生物医学工程学杂志,2002,19(2):186-190.
[32] Shel TRM, Liu Y, Cooper PR. Bone marrow cell gene expression and tissue constructassembly using octacalcium phosphate microscaffolds. Biomaterials,2006,27(14):2874-2881.
[33] Brown TD. Techniques for mechanical stimulation of cells in vitro: a review. J Biomech,2004,33:3-14.
[34]唐丽灵.王远亮,谷俐.不同应变水平拉伸对成骨细胞生理功能的影响.重庆大学学报,2003,26(3):67-70.
[35] Harter LV, H ruska KA, Duncan RL. Human osteoblast-like cells respond to mechanicalstrain with increased bone matrix protein production independent of hormonal regulation.Endocrinology,1995,136(2):528-535.
[36] Knoll BI, Mccarthy TL, Centrella M. Strain-dependent control of transforming growthfactor-beta function in osteoblasts in an vitro model: biochemical events associated withdistraction osteogenesis. Plast Reconstr Surg,2005,116(1):224-233.
[37]陈伟辉,乔鞠,罗颂椒.不同流动剪切力调节大鼠成骨样细胞增殖的体外研究.华西医大学报,2001,32(2):232-234.
[38] Vico L, Lafage–Proust MH, Alexandre C. Effect of gravitational change on bone system invitro and in vivo [J]. Bone,1998,22(5Suppl):95S
[39]毛勇,段小红,王忠义.不同应力对成骨细胞和细胞骨架影响的实验研究.牙体牙髓牙周病学杂志，2001,11(2):98-100.
[40]戚孟春，胡静,韩立赤.成骨细胞在机械力刺激下细胞骨架及细胞形态改变的体外研究.中国口腔颌面外科杂志,2004,2(3):181-184.
[41] Genge BR, Sauer GR, Wu LN. Correlation between loss of alkaline phosphatase activity andaccumulation of calcium during matrix vesicle mediated mineralization [J]. J Biol Chem,1988,263(34):18513-9.
[42] Buckley MJ, Banes AJ, Jordan RD. The effects of mechanical strain on osteoblasts in vitro. JOral Maxillofac Surg,1990;48(2):276-282.
[43]张西正,李彬,郭勇.不同应变对成骨细胞增殖活性及功能状态的影响.生物医学工程与临床,2005,9(2):69-72.
[44] Kaspar D, Seidl W, Neidlinger-Wilke. Dynamic cell stretching increases human osteoblastproliferation and CICP synthesis but decreases osteocalcin synthesis and alkaline phosphataseactivity [J]. J Biomechanics,2000,33(1):45-51.
[45]唐林,林珠,李永明.机械牵张力对成骨细胞碱性酸酶及骨钙蛋白的影响.临床口腔医学杂志,2005,21(11):648-650.
[1] Greenwald AS, Boden SD, Goldberg VM. Bone-graft substitutes: Facts, fictions, andapplications[J]. J Bone Joint Surg,2001,83A:98-103.
[2] Den BFC, Wippermann BW, Blokhuis TJ. Healing of segmental bone defects with granularporous hydroxyapatite augmented with recombinant human osteogenic protein-1or autologousbone marrow[J]. J Orthopaed Res,2003,21:521-528.
[3] Toshiaki K, Takao Y, Takashi N. Four calcium phosphate ceramics as bone substitutes fornon-weight-bearing[J]. Biomaterials,1993,143:216-224.
[4] Nelson Luis G.D. Souza, Humberto M Brandao, Luiz Fernando C. de Oliveira. Spectroscopicand thermogravimetric study of chitosan after incubation in bovine rumen[J]. Journal of MolecularStructure,2011,1005:186-191.
[5] He Q，Ao Q，Xiu B，Gong YD, Zhao NM, Zhang XF. Mechanisms for biocompatibility ofchitosan: A new viewpoint [J]. Journal of Clinical Rehabilitative Tissue Engineering Research，2007,11(35):7110-7112.
[6] Shirosaki Y, Tsuru K, Hayakawa S, Osaka A, Lopes MA, Santos JD, Fernandes MH. In vitrocytocompatibility of MG63cells on chitosan-organosiloxane hybrid membranes[J]. Biomaterials,2005,26(5):485-493.
[7] Zhang XF, Hua H, Shen XY, Yang Q. In vitro degradation and biocompatibility ofpoly(1-lactic acid)/chitosan fiber composites[J]. Polymer,2007,48(4):1005-1011.
[8] Amaral IF, Lamghari M, Sousa SR, Sampaio P, Barbosa MA. Rat bone marrow stromal cellosteogenic differentiation and fibronectin adsorbtion on chitosan membranes: the effect of thedegree of acetylation [J]. J Biomed Mater Res A,2005,75(2):387-397.
[9] Zhao F, Yin YJ, Yao KD. Preparation and histological evaluation of biomimeticthree-dimensional hydroxyapatite/chitosan-gelatin network composite scaffolds[J]. Biomaterials,2002,23:3227-3234.
[10] Kong L, Gao Y, Cao W. Preparation and characterization of nano-hydroxyapatite/chitosancomposite scaffolds[J]. J Biomed Mater Res,2005;75A:275–282.
[11] Rusu VM, Ng CH, Wilke M. Sizecontrolled hydroxyapatite nanoparticles as self-organizedorganic–inorganic composite materials[J]. Biomaterials,2005;26(26):5414–5426.
[12] Manjubala I, Scheler S, Bossert J. Mineralisation of chitosan scaffolds with nano-apatiteformation by double diffusion technique[J]. Acta Biomater,2006;2(1):75–84.
[13]张西正,侍才洪,康少华,郭勇,李瑞欣,宁波.细胞基底应变加载装置，实用新型，ZL200920250218.7.
[14]张立德,牟季美.纳米材料和纳米结构[M].北京,科学出版社,2001
[15]王嘉宁,郭宁.细胞凋亡的检测技术与方法[J].中国药理学与毒理学杂志,2005,19(6):466-470.
[16]宋海明，张宝述，孙红娟.行星式球磨机在加工蛭石粉体中的应用研究[J].矿山机械，2007，35（4）：14-16.
[17]陈彩风,陈志刚.超声场中湿法制备纳米粉末的原理和方法[J].机械工程材料,2003,27(4):30-32.
[18] Kubota N, Ohga K, Moriguchi M. Permeability properties of glycol chitosan membranemodified with thiol groups[J]. Journal of Applied Polymer Science,1991,42:495-501.
[19] Majeti NV Ravikumar. A review of chitin and chitosan applications[J]. Reactive andFunctional Polymers,2000,46:1-27.
[20]杨菊林,周长忍,田冶,田金环.壳聚糖/羟基磷灰石膜的制备及对细胞生长的影响[J].生物医学工程学杂志,2009,26(3):580-584.
[21] Altankov G, Groth T. Reorganization of substratum bound fibronectin on hydrophobicmaterials is related to biocompatibility [J]. J Mater Sci Miter Med,1994,5(9):732-737.
[22] Anselme K, Bigerell EM, Noel B. Qualitative and quantitative study of human osteoblastadhesion on materials with various surface roughness[J]. J Bio Mater Res,2000,49(2):155-166.
[23]宋辰刚，邱小忠，吴刚，余磊，赖桂华，欧阳钧. MC3T3-HA复合培养后的细胞凋亡检测[J].中国临床解剖学杂志，2007,25(5):567-570.
[1]陈际达,崔磊,刘伟,曹谊林.溶剂浇铸/颗粒沥滤技术制备组织工程支架材料[J].中国生物工程杂志,2003,23(4):32-35,42.
[2] Hutmacher DW. Scaffolds in tissue engineering bone and cartilage[J]. Biomaterials,2000,21(24):2529-2543.
[3] Agrawal CM, Ray RB. Biodegradable polymeric scaffolds for musculoskeletal tissueengineering[J]. J Biomed Mater Res,2001,55:141-150.
[4] Yang SF, Leong KF, Du ZH, Chua CK. The design of scaffolds for use in tissue engineering[J].Tissue Eng,2001,7:679-689.
[5] Chen GP, Ushida T, Tateishi T. Scaffold design for tissue engineering[J]. MacromolBiosci,2002,2(2):67-77.
[6] Liu XH, Ma PX. Polymeric scaffolds for bone tissue engineering[J]. Ann Biomed Eng,2004,32(3):477-486.
[7] Fisher JP, Holland TA, Dean D, Engel PS, Mikos AG. Synthesis and properties ofphotocross-linked poly(propylene fumarate) scaffolds[J]. J Biomater Sci Polym Ed,2001,12(6):673-87.
[8] Borden M, El-Amin SF, Attawia M, Laurencin CT. Structural and human cellular assessmentof a novel microsphere-based tissue engineered scaffold for bone repair[J]. Biomaterials,2003,24:597-609.
[9] Shen F, Cui YL, Yang LF, Yao KD, Dong XH, Jia WY, Shi HD. A study on the fabrication ofporous chitosan/gelatin network scaffold for tissue engineering[J]. Polym Int,2000,49:1596-1599.
[10] Chen G, Ushida T, Tateishi T. Preparation of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams by use of ice microparticulates[J]. Biomaterials,2001,22:2563-2567.
[11] Murphy WL, Dennis RG, Kileny JL, Mooney DJ. Salt fusion: an approach to improve poreinterconnectivity within tissue engineering scaffolds[J]. Tissue Eng,2002,8(1):43-52.
[12] Zhang RY, Ma PX. Synthetic nano-fibrillar extracellular matrices with predesignedmacroporous architectures[J]. J Biomed. Mater Res,2000,52:430-438.
[13] Liao CJ, Chen CF, Chen JH, Chiang SF, Lin YJ, Chang KY. Fabrication of porousbiodegradable polymer scaffolds using solvent merging/particulate leaching method[J]. J BiomedMater Res,2002,59:676-681.
[14] Hou QP, Grijpma DW, Feijen J. Porous polymeric structures for tissue engineering preparedby a coagulation, compression moulding and salt leaching technique[J]. Biomaterials,2003,24:1937-1947.
[15] Oh SH, Kang SG, Kim ES, Cho SH, Lee JH. Fabrication and characterization of hydrophilicpoly(lactic-co-glycolic acid)/poly(vinyl alcohol) blend cell scaffolds by melt-moldingparticulate-leaching method[J]. Biomaterials,2003,24(22):4011-4021.
[16] Sander EA, Alb AM, Nauman EA, Reed WF, Dee KC. Solvent effects on the microstructureand properties of75/25poly(D,L-lactide-coglycolide) tissue scaffolds[J]. J Biomed Mater Res A,2004,70:506-513.
[17] Cai Q, Yang JA, Bei JZ, Wang SG, A novel porous cells scaffold made of polylactide-dextranblend by combining phase-separation and particle-leaching techniques[J]. Biomaterials,2002,23:4483-4492.
[18] Shastri VP, Martin I, Langer R. Macroporous polymer foams by hydrocarbon templating[J].Proc Natl Acad Sci USA,2000,97(5):1970-1975.
[19] Lin HR, Kuo CJ, Yang CY, Shaw SY, Wu YJ. Preparation of Macroporous BiodegradablePLGA Scaffolds for Cell Attachment with the Use of Mixed Salts as Porogen Additives[J]. JBiomed Mater Res,2002,63:271-279.
[20] Nam YS, Yoon JJ, Park TG. A novel fabrication method of macroporous biodegradablepolymer scaffolds using gas foaming salt as a porogen additive[J]. J. Biomed. Mater. Res.(ApplBiomater),2000,53:1-7.
[21] Lin ASP, Barrows TH, Cartmell SH, Guldberg RE. Microarchitectural and mechanicalcharacterization of oriented porous polymer scaffolds[J]. Biomaterials,2003,24:481-489.
[22] Ma ZW, Gao CY, Gong YH, Shen JC. Paraffin spheres as porogen to fabricate poly(L-lacticacid) scaffolds with improved cytocom-patibility for Cartilage Tissue Engineering[J]. J BiomedMater Res Part B,2003,67B(1):610-617.
[23] Chen VJ, Ma PX. Nano-fibrous poly(L-lactic acid) scaffolds with interconnected sphericalmacropores[J]. Biomaterials,2004,25(11):2065-2073.
[24] Zhang JC, Wu LB, Jing DY, Ding JD. A comparative study of porous scaffolds with cubic andspherical macropores[J]. Polymer,2005,46:4979-4985
[25]曹谊林，陈际达，崔磊，刘伟.水溶性粘结剂及其在内部结构可控的大体积细胞支架制备中的应用. CN200410016133.4
[26] Ma PX, Choi JW. Biodegradable polymer scaffolds with well-defined interconnectedspherical pore network[J]. Tissue Eng,2001,7(1):23-33.
[27] Wu LB, Ding JD. In vitro degradation of three dimensional porous poly(D,L-lactide-co-glycolide) scaffolds for tissue engineering[J]. Biomaterials2004;25:5821-5830.
[28] Babense JE, Anderson JM, Mcintire LV, Mikos AG. Host response to tissue engineereddevices[J]. Adv. Drug. Deliv. Rev.,1998,33,111–139.
[29] Heggset E.B., Hoell1.A.，Kristoffersen M.，Eijsink V.G.H.，VarumK.M. Degradation ofChitosans with Chitinase G from Streptomyces coelicolor A3(2): Production ofChito-oligosaccharides and Insight into Subsite Specificities[J]. Biomacromolecules，2009,10(4):892-899.
[30] Heggset E.B.，Dybvik A.I.，HoellI.A.，Norberg A.L.，Sorlie M., Eijsink V.G.H.,Varum K.M.Degradation of Chitosans with a Family46Chitosanase from Streptomyces coelicolor A3(2)[J].Biomacromolecules,2010,11(9):2487-2497.
[31] Lee D.X.，Xia W.S.，Zhang J.L. Enzymatie preparation of chitooligosaccharides bycommercial lipase[J]. Food Chem,2008,111(2):291-295.
[32] Xie Y.，WeiY，HuJ.G.Depolymerization of Chitosan with a Crude Cellulase Preparation fromAspergillus Niger[J]. Appl Biochem Biotech,2010,160(4):1074-1083.
[33] Sikorski P, Sorbotten A, Horn SJ, Eijsink VGH, Varum KM. Serratia marcescens chitinaseswith tunnel-shaped substrate-binding grooves show endo activity and different degrees ofprocessivity during enzymatic hydrolysis of chitosan[J]. Biochemistry-Us,2006,45(31):9566-9574.
[34] Henry, J. B. Clinical Diagnosis and Management by Laboratory Methods,18th ed.;Saunerders: Philadelphia,1991,1371.
[35] Temel A.，Kazokoglu H.，Taga Y. Tear Lysozyme Levels in Contact-Lens Wearers[J]. AnnOphthalmol,1991,23(5)：191-194.
[36] Nordtceit RJ, Varum KM, Smidsrod O. Degradation of partially N-acetylated chitosans withhen egg white and human lysozyme[J]. Carbohydr Polym,1996,29:163-167
[37]马如,黄明智.壳聚糖的溶菌酶降解[J].北京化工大学学报,2002,29(6):40-43.
[38] Yang B, Li XY, Shi S, Kong XY, Guo G, Huang MJ, Luo F, Wei YQ, Zhao X, Qian ZY.Preparation and characterization of a novel chitosan scaffold[J]. Carbohydrate Polymers,2010,80:860-865
[39] Imoto T, Yagshita K. A simple activity measurement of lysozyme [J]. Agri Bio Chem,1971,35:1154-1156
[40]陈学忠，李志宏，李瑞欣，郭勇，刘璐，王亮，张西正.胶原多糖基纳米羟基磷灰石仿生骨支架材料的研制[J].国际生物医学工程杂志,2011,34(2):65-70
[41] Cowin SC. The relationship between the elasticity tensor and the fabric tensor[J]. MechMater,1985,4:137-147.
[42] Gundersen HJG, Jensen TB, Osterby R. Distribution of membrane thickness determined bylineal analysis[J]. J Microsc,1978,113:27-43.
[43] Harrigan TP, Mann RW. Characterization of microstructural anisotropy in orthotropicmaterials using a second rank tensor[J]. J Mater Sci,1984,19:761-767
[44] Zhang Y, Li RX, Fan YB, Liu H, Guo Y, Wang L, Shi CH, Zhu D, Zhang XZ.Microarchitectural and mechanical characterization of chitosan/hydroxyapatite/demineralizedbone matrix composite scaffold[J]. Journal of porous material,2011:1-9.
[45] Ren DW, Yi HF, Wang W, Ma XJ. The enzymatic degradation and swelling properties ofchitosan matrices with different degrees of N-acetylation[J]. Carbohydrate Research,2005,340:2403-2410.
[46] Murphy WL, Dennis RG, Kileny JL, Mooney DJ. Salt fusion: an approach to improve poreinterconnectivity within tissue engineering scaffolds[J]. Tissue Engineering,2002,8(1):43-52.
[47] Nam YS, Yoon JJ, Park TG. A novel fabrication method of macroporous biodegradablepolymer scaffolds using gas foaming salt as a porogen additive[J]. J Biomed MaterRes,2000,53:1-7.
[48] Muller B, Beckmann F, Huser M, Maspero F, Szekely G, Ruffieux K, Thurner P,Wintermantel E. Non-destructive three-dimensional evaluation of a polymer sponge bymicro-tomography using synchrotron radiation[J]. Biomol Eng,2002,19:73-78.
[49] Lin AS, Barrows TH, Cartmell SH, Guldberg RE. Microarchitectural and mechanicalcharacterization of oriented porous polymer scaffolds[J]. Biomaterials,2003,24:481-489.
[50] Ho ST, Hutmacher DW. A comparison of micro CT with other techniques used in thecharacterization of scaffolds[J]. Biomaterials,2006,27:1362-1376.
[51] Wan Y, Yu AX，Wu H，Wang ZX，Wen DJ. Porous-conductive chitosan scaffolds for tissueengineering II. in vitro and in vivo degradation[J]. J Mater Sci-Mater M,2005，16(11):1017-1028.
[1] Cheng B, Kato Y, Zhao S, Luo J, Sprague E, Bonewald LF, Jiang JX. PGE(2) is essential forgap junction-mediated intercellular communication between osteocyte-like MLO-Y4cells inresponse to mechanical strain. Endocrinology,2001,142,3464-3473.
[2] Tanaka SM. A new mechanical stimulator for cultured bone cells using piezoelectric actuator.Journal of Biomechanics,1999,32:427-430.
[3] Meyer U, Meyer T, Wiesmann HP, Stratmann U, Kruse-Losler B, Maas H, Joos U. The effectof magnitude and frequency of interfragmentary strain on the tissue response to distractionosteogenesis. Journal of Oral and Maxillofaciale Surgery,1999,57:1331-1339.
[4] Peake MA, Cooling LM, Magnay JL, Thomas PB, ElHaj AJ. Selected contribution: regulatorypathways involved in mechanical induction of c-fos gene expression in bone cells. Journal ofApplied Physiology,2000,89:2498-2507.
[5] Saunders MM, Taylor AF, Du C, Zhou Z, PellegriniJr VD, Donahue HJ. Mechanicalstimulation effects on functional end effectors in osteoblastic MG-63cells. Journal ofBiomechanics,2006,39:1419-1427
[6] Visconti LA, Yen EHK, Johnson RB. Effect of strain on bone nodule formation by ratosteogenic cells in vitro. Archives of Oral Biology,2004,49:485-492.
[7] Kaspar D, Seidl W, Neidlinger-Wilke C, Ignatius A, Claes L. Dynamic cell stretching increaseshuman osteoblast proliferation and CICP synthesis but decreases osteocalcin synthesis andalkaline phosphatase activity. Journal of Biomechanics,2000,33:45-51.
[8] Mauney JR, Sjostorm S, Blumberg J, Horan R, O’Leary JP, Vunjak-Novakovic G, Volloch V,Kaplan DL,. Mechanical stimulation promotes osteogenic differentiation of human bone marrowstromal cells on3-D partially demineralized bone scaffolds in vitro. Calcified Tissue International2004,74:458-468.
[9] Yang Y, Wood MA, ElHaj AJ. Enhancement of mechanical signals for tissue engineering boneby mechano-active scaffolds. Abstract of conference: ECM V The cell biomaterials reaction,DAVOS, Switzerland, European Cells and Materials,2004,7, supplement1:35
[10] Wood MA, Yang Y, Thomas PBM, ElHaj AJ. Utilising dihydropyridine-release strategies toenhance load effects in engineered human bone constructs. Tissue Engineering,2006,12:2489-2497.
[11] Gemmiti CV, Guldberg RE. Fluid flow increases type II collagen deposit and tensilemechanical properties in bioreactor-grown tissueengineered cartilage. Tissue Engineering,2006，12,469-479.
[12] Martin I, Wendt D, Heberer M. The role of bioreactors in tissue engineering. Trends inBiotechnology,2004,22,80-86.
[13] Meyer U, Buchter A, Nazer N, Wiesmann HP. Design and performance of a bioreactorsystem for mechanically promoted three dimensional tissue engineering. British Journal of Oraland Maxillofacial Surgery,2006,44,134-140.
[14] Baas E, Kuiper JH, Yang Y, Wood MA, ElHaj AJ. In vitro bone growth responds to localmechanical strain in three-dimensional polymer scaffolds. Journal of Biomechanics,2010,43:733-739
[15] Lacroix D, Sandino C, Villagomez M. A micro-finite element analysis of fluidic and solidmechanical stimuli in bone tissue engineering scaffolds. Journal of Biomechanics,2006,39(Supplement1):415
[16] Gibson LJ, Ashby MF. Cellular Solids: Structure and Properties.1988. Pergamon Press, NewYork, USA.
[17]宫元伟,闫玉仙,张媛,张西正.基底拉伸应变对小鼠骨细胞Rnnx2表达的影响.中国骨质疏松杂志.2011,17(3):185-188
[18] Wang L, Zhang XZ, Guo Y, Chen XZ, Li RX, Liu L, Shi CH, Guo C, Zhang Y. Involvementof BMPs/Smad Signaling Pathway in Mechanical Response in Osteoblasts. Cell Physiol Biochem2010;26:1093-1102.
[19]闫玉仙,宋梅,郭春,郭勇,宫元伟,李瑞欣,张西正.基底拉伸应变对小鼠三种骨组织细胞BMP-2mRNA表达的影响.中国老年学杂志,2010,30(21):3092-3095
[20] Chen XY，Zhang XZ，Guo Y, Li RX, Lin JJ, Wei Y. The establishment of a mechanobiologymodel of bone and functional adaptation in response to mechanical loading. ClinicalBiomechanics,2008, s88-95
[21]赵红斌,吕同德,马敬,马慧,张西正.机械力对间充质干细胞向成骨细胞分化的力学响应机制.生物化学与生物物理进展.2007,34(7):718-723
[22] Cɑrɑno A, Siciliɑni G. Effects of continuous ɑnd intermittent forces on humɑn fibroblɑsts invitro. Eur J Orthod,1996,18:19-26.
[23]黎小坚, Frost HM,朱绍舜,柯华珠.基础骨生物学新观.中国骨质疏松杂志,2001,7(2):152-174
[24]李广州,蒋电明.纳米羟基磷灰石生物安全性评价与研究进展.生物骨科材料与临床研究,2009,6(1):31-33
[25] Genge BR, Sauer GR, Wu LN, McleanFM, WuthierRE. Correlation between loss of alkalinephosphatase activity and accumulation of calcium during matrix vesicle-mediated mineralization.J Biol. Chem.,1988,263(34):18513-18519.
[26] Guicheux J, Lemonnier J, Ghayor C, Suzuki A, Palmer G, Caverzasio J. Activation of p38mitogen activated protein kinase and c-jun NH2terminal kinase by BMP-2and their implicationin the stimulation of osteoblastic cell differentiation. Bone Miner Res,2003,18(11):2060-2068.
[27] Islam AA, Rasubala L, Yoshikawa H, Shiratsuchi Y, Ohishi M. Healing of fractures inosteoporotic rat mandible shown by the expression of bone morphogenetic protein-2and tumournecrosis factor-alpha. Br J Oral Maxillofac Surg,2005,43(5):383-391.
[28] Rauch F, Lauzier D, Croteau S, Travers R, Glorieux FH, Hamdy R. Temporal and spatialexpression of bone morphogenetic protein-2,4and7during distraction osteogenesis.Bone,2000,26:611-617.
[29] Lai CF, Cheng SL. Signal transductions induced by bone morphogenetic protein-2andtransforming growth factor-beta in normal human osteoblastic cells. J Biol Chem.,2002,277(18):15514-15522.
[30] Chai F, Jin Y, Zhao YM. Expression of osteopontin and osteocalcin mRNA inectomesenchymal stem cells under induction of EMPs&EMD in vitro. Journal of Oral ScienceResearch,2004,20(3):243-245.
[1] Li Yubao, Klein C, Wijin J, Meer S, Groot K. Shape change and phase transition of needle-likenonstichiometic apatite crystals [J]. J Mater Sci: Mater Med,1994,5:263-267.
[2]许永华,施新猷,胡蕴玉.牛煅烧骨与体外培养兔骨膜成骨细胞的相容性[J].第四军医大学学报,2000,21(4)：512-514.
[3] Den BFC, Wippermann BW, Blokhuis TJ. Healing of segmental bone defects with granularporous hydroxyapatite augmented with recombinant human osteogenic protein-1or autologousbone marrow[J]. J Orthopaed Res,2003,21:521-528.
[4] Webster TJ. Nanophase ceramies: The future orthopedic and dental implant material.Advances in Chemical Engineering. Academic Press New York,2001:125-166.
[5] Yoshikawa T, Ohgushi H,Tamai S. Immediate bone forming capability of prefabrieatedosteogenic hydroxyapatite[J]. Biomed Mater Res,1996,32(3):481-492.
[6] Rusu VM, Ng CH, Wilke M, Tiersch B, Fratzl P, Peter MG. Sizecontrolled hydroxyapatitenanoparticles as self-organized organic–inorganic composite materials[J]. Biomaterials2005;26(26):5414-26.
[7] Zhao F, Yin YJ, Lu WW, Leong JC, Zhang WJ, Zhang JY, Zhang MF, Yao KD. Preparationand histological evaluation of biomimetic three-dimensional hydroxyapatite/chitosan-gelatinnetwork composite scaffolds [J]. Biomaterials,2002,23:3227-3234.
[8] Thein-Han WW, Kitiyanant Y, Misra RDK. Chitosan as a scaffold matrix for tissueengineering. Mater Sci Technol2008;24:1062-1075.
[9] Martin HJ, Sehulz KH, Bumgardner JD, Schneider JA. Enhanced bonding of chitosan toimplant qualitytitanium via four treatment combinations[J]. Thin Solid Films,2008,516(18):6277-6286.
[10] Lahiji A, Sohrabi A, Hungerford DS, Frondoza CG. Chitosan surports the expression of theextracellular matrix proteins in human osteoblasts and ehondroeytes[J]. Biomed MaterRes.2000.51(4):586-595.
[11] Thein-Han WW, Misra RDK. Biomimetic chitosan–nanohydroxyapatite composite scaffoldsfor bone tissue engineering[J]. Acta Biomaterialia2009;5:1182-1197.
[12] Ito M, Hidaka Y, Nakajima M, Yagasaki H, Kafrawy AH. Effect of hydroxyapatite contenton physical properties and connective tissue reactions to a chitosan／hydroxyapatite compositemembrane[J]．Journal of Biomedical Materials Research,1999,45(3)：204-208．
[13]赵峰,尹玉姬,宋雪峰,姚康德,郭善一,郭若霖,张镜宇.壳聚糖-明胶网络／羟基磷灰石复合材料支架的研究-制备及形貌[J].中国修复重建外科杂志,2001,15(5)：276-279.
[14] Mukherjee DP, Tunkle AS, Roberts RA, Clavenna A, Rogers S, Smith D. An animalevaluation of a paste of chitosan glutamate and hydroxyapatite as a synthetic bone graftmateria1[J]．Journal of Biomedical Materials Research Part B-Applied Biomaterials,2006,67B(1)：603-609．
[15]郑雄飞,翟文杰,梁迎春,孙涛.低温沉积制造壳聚糖-纳米羟基磷灰石支架[J].无机材料学报,2011,26(1)：12-16.
[16]吴芳,戴伯川,李为祖.纳米羟基磷灰石/壳聚糖-海藻酸钠复合材料的制备及性能研究[J].海峡药学,2009,21(3):21-23.
[17]张利,李玉宝,魏杰.纳米羟基磷灰石/壳聚糖复合骨修复材料的共沉淀法制备及其性能表征[J].功能材料,2005,36(3):441-444.
[18] Chen F, Wang Z C, Lin C J. Preparation and characterization of nano-sized hydroxyapatiteparticles and hydroxy apatite/chitosan nano-composite for use in biomedical materials[J]. Materials Letters,2002,57:858-861.
[19]刘敬肖,史非,于玲,王井,牛丽婷,李佳颖,胡志强.模拟体液中纳米羟基磷灰石/壳聚糖的制备及表征[J].大连轻工业学院学报,2007,26(4):332-336.
[20]薛博,施云峰,何淑梅,李兰英,郭振招,张兰兰,李红.常温环境下壳聚糖诱导合成单斜羟基磷灰石纳米晶体及其结构表征[J].高等学校化学学报,2010,31(12)：2339-2343.
[21]杨辉,张园园.壳聚糖分子对共沉淀法合成羟基磷灰石/壳聚糖粉体材料晶体形貌的影响[J].人工晶体学报,2012,41(1):209-214.
[22] Viorel Matin Rusu, Chuen-How Ng, Max Wilke, Brigitte Tiersch, Peter Fratzl, Matin G Peter.Size-controlled hydroxyapatite nanoparticles as self organized organic-inorganic compositematerials[J]. Biomaterials,2005,26:5414-5426.
[23]何丹,许素华,游青青,张明霞,肖秀峰,刘榕芳.壳聚糖模板诱导梭状纳米羟基磷灰石的仿生合成[J].化工时刊,2007,21(7):7-10.
[24] Yang F, Wolke J G C, Jansen J A. Biomimetic calcium phosphate coating on electrospun poly(ε-caprolactone) scaffolds for for bone tissue engineering[J]. Chem Eng J,2008,137(1):154-161.
[25] Beppu MM,Santana CC．PAA influence on chitosan membrane calcification[J]．MaterialsScience and Engineering C,2003,23(5)：651-658．
[26] Tachaboonyakiat W, Serizawa T, Akashi M. Hydroxyapatite formation on/in biodegradablechitosan hydrogels by an alternate soaking process[J].Polymer Journal,2001,33(2):177-181.
[27]李红,朱敏鹰,李立华,周长忍.原位沉析羟基磷灰石-壳聚糖骨组织工程支架材料的研制[J].功能材料,2006,37(6):909-911.
[28]王新,刘玲蓉,张其清.纳米羟基磷灰石-壳聚糖骨组织工程支架的研究[J].中国修复重建外科杂志,2007,21(2):120-124.
[29]李波,李立华,赵名艳,周长忍. Sol-gel原位相转变矿化制备纳米羟基磷灰石/壳聚糖复合支架材料.复合材料学报,2011,28(4)：83-88.
[30]卢晓英,王秀红,刘治,翁杰.反应pH值对原位水热沉积法制备纳米羟基磷灰石/壳聚糖复合材料的影响[J].复合材料学报,2009,26(4):53-58.
[31] Li BQ, Hu QL, Qian XZ, Fang ZP, Shen JC. Bioabsorbable chitosan/hydroxyapatitecomposite rod prepared by in situ precipitation for internal fixation of bone fracture[J].Acta.Polymerica.Sinica.,2002,36(6):828-833.
[32] Manjubala I, Scheler S, Bossert J. Mineralisation of chitosan scaffolds with nano-apatiteformation by double diffusion technique[J]. Acta Biomater2006;2(1):75-84.
[33]黄志海,李纪宏,林萍华,董寅生,郭超,盛晓波,储成林.壳聚糖多孔支架电化学辅助沉积羟基磷灰石涂层研究[J].高分子学报,2009,(3):273-277.
[34]胡仁,胡皓冰,林吕健．CaP／壳聚糖复合膜层的电化学共沉积研究[J]．高等学校化学学报,2002,23(11)：2142-2146.
[35] Huang LY, Xu KW, Lu J．A study of the process and kinetics of electrochemical depositionand the hydrothermal synthesis of hydroxyapatite coatings[J]．Journal of Materials Science-Materials in Medicine,2000,11(11)：667-673．
[36] Huang ZH, Dong YS, Chu CL. Electrochemistry assisted reacting deposition ofhydroxyapatite in porous chitosan scaffolds [J]. Materials Letters,2008,62(19):3376-3378.
[37] Hu Q, Li B, Wang M, Shen J. Preparation and characterization of biodegradablechitosan/hydroxyapatite nanocomposite rods via in situ hybridization: a potential material asinternal fixation of bone fracture[J]. Biomaterials2004;25(5):779-785.
[38] Kong L, Gao Y, Cao W, Gong Y, Zhao N, Zhang X. Preparation and characterization ofnano-hydroxyapatite/chitosan composite scaffolds[J]. J Biomed Mater Res2005;75A:275-282.
[39] Yamaguchi I,Iizuka S,Osaka A, Monma H, Tanaka J. The effect of citric acid addition onchitosan/hydroxyapatite composites [J]. Colloids and Surfaces A: Physicochem Eng Aspects,2003,214:111-118.
[40] Yamaguchi I, Tokuchi K, Fukuzaki H, Koyama Y, Takakuda K, Monma H. Preparation andmicrostructure analysis of chitosan/hydroxyapatite nanocomposites. Journal of BiomedicalMaterials Research,2001,55:20-27.
[41]陈景帝,王迎军,陈晓峰,尹诗衡.利用冷冻干燥原位构筑仿生型纳米羟基磷灰石、壳聚糖多孔支架材料[J].稀有金属材料与工程,2009,38：271-273.
[42]李保强,胡巧玲,汪茫,沈家骢.原位复合法制备层状结构的壳聚糖/羟基磷灰石纳米材料[J].高等学校化学学报,2004,25(10)：1949-1952.
[43]赵巍,侯振德,任朝峰.骨细胞培养过程对羟基磷灰石力学性质影响的研究[J].实验力学,2009,24(1):1-7.
[44]汪皖,徐培,吴海珍.壳聚糖-纳米羟基磷灰石复合材料修复兔骨缺损过程中BMp-2的表达[J].安徽医科大学学报,20l0,45(4):491-493.
[45] Yuan H, Chen N, Lu XY, Zheng BZ. Experimental study of natural hydroxyapatite/chitosancomposite on reconstructing bone defects[J]. Journal of Nanjing Medical University,2008,6(22):372-375.
[46] Kong LJ, Ao Q, Xi J, Zhang L, Gong YD, Zhao NM, Zhang XF. Proliferation anddifferentiation of MC3T3-E1cells cultured on nano-hydroxyapatite/chitosan compositescaffolds[J]. Joumal of Biomedical Materials Research,2007,2(75):275-282.
[47]袁华,陈宁,吕晓迎,郑步中,宋晓陵.天然羟基磷灰石-壳聚糖复合材料修复骨缺损的实验研究[J].口腔医学,2005,25(2):65-67.
[48] Aylin S U, Russell D. The addition of biphasic calcium phosphate to porous chitosanscaffolds enhances bone tissue development in vitro[J]. J. Biomed. Mater. Res. A,2007,81A(3):624-633.
[49]杨菊林,周长忍,田冶.壳聚糖/羟基磷灰石膜的制备及对细胞生长的影响[J].生物医学工程学杂志,2009,26(3):580-584.
[50] Shel TRM, Liu Y, Cooper PR. Bone marrow cell gene expression and tissue constructassembly using octacalcium phosphate microscaffolds[J]. Biomaterials2006;27(14):2874-2881.
[51] Oberdors ter G, Sharp Z, Atudorei V, et al. Extrapulmonary translocation of ultrafine carbonparticles following whole-body inhalation exposure of rats[J]. J Toxicol Environ Health A2002;65(20):1531-1543.
[52]李广州,蒋电明.纳米羟基磷灰石生物安全性评价与研究进展[J].生物骨科材料与临床研究,2009,6(1):31-33.
[2] Burr DB, Milgrom C, Fyhrie D. In vivo measurement of human tibial strains during vigorousactivity. Bone,1996,18(5):405-410.
[3] Clara S, Sara C, Patrick JP. Simulation of angiogenesis and cell differentiation in a CaPscaffold subjected to compressive strains using a lattice modeling approach. Biomaterials,2010,31:2446-2452.
[4] Andy LO, Elia M, Josep A. Planell, Damien Lacroix. Finite element study of scaffoldarchitecture design and culture conditions for tissue engineering. Biomaterials,2009,30:6142-6149
[5] Karande TS, Ong JL, Agrawal CM. Diffusion in musculoskeletal tissue engineering scaffolds:design issues related to porosity, permeability, architecture, and nutrient mixing. Ann Biomed Eng,2004,32:1728-1743.
[6] Schriefer JL, Warden SJ, Saxon LK. Cellular accommodation and the response of bone tomechanical loading. J Biomech,2005,38:1838-1845.
[7] Martin I, Wendt D, Heberer M. The role of bioreactors in tissue engineering. TrendsBiotechnol,2004,22:80-86.
[8] Meyer U, Buchter A, Nazer N, Wiesmann HP. Design and performance of a bioreactor systemfor mechanically promoted three dimensional tissue engineering. J Oral Maxillofac Surg,2006,44:134-140.
[9] Wendt D, Marsano A, Jakob M. Oscillating perfusion of cell suspensions throughthree-dimensional scaffolds enhances cell seeding efficiency and uniformity. Biotechnology andBioengineering,2003,84:205-214.
[10] Stops A.J.F, Heraty KB, Browne M. A prediction of cell differentiation and proliferationwithin a collagen–glycosaminoglycan scaffold subjected to mechanical strain and perfusive fluidflow. Journal of Biomechanics,2010,43:618-626
[11] Jaecques SVN, Van OH, Muraru L. Individualised, micro CT-based finite element modellingas a tool for biomechanical analysis related to tissue engineering of bone. Biomaterials,2004,25:1683-1696.
[12] Damien L, Arnaud C, Maria PG. Micro-finite element models of bone tissue-engineeringscaffolds. Biomaterials,2006,27:5326-5334.
[13] Sun W, Starly B, Nam J. Bio-CAD modeling and its applications in computer-aided tissueengineering. Comput-aided Design,2005,37:1097-1114.
[14] Lacroix D, Sandino C, Villagomez M. A micro-Finite Element analysis of fluidic and solidmechanical stimuli in bone tissue engineering scaffolds. Journal of Biomechanics,2006,39(Supplement1): S415
[15] Saey T, Dietmar WH. Application of micro CT and computation modeling in bone tissueengineering. Computer-Aided Design,2005,37(11):1151-1161.
[16] Byrne DP, Lacroix D, Planell JA. Simulation of tissue differentiation in a scaffold as afunction of porosity, Young’s modulus and dissolution rate: application of mechanobiologicalmodels in tissue engineering. Biomaterials,2007,28:5544-5554.
[17] Den BFC, Wippermann BW, Blokhuis TJ. Healing of segmental bone defects with granularporous hydroxyapatite augmented with recombinant human osteogenic protein-1or autologousbone marrow. J Orthopaed Res,2003,21:521-528.
[18]许永华，施新猷，胡蕴玉.牛煅烧骨与体外培养兔骨膜成骨细胞的相容性.第四军医大学学报，2000,21(4):512-514
[19]徐小良，汤亭亭，楼觉人. β-TCP/HAP双相煅烧骨的制备及其在BMP-2基因给药中的应用.生物医学工程学杂志,2003,20(4):630-633
[20]赵巍,侯振德,任朝峰.骨细胞培养过程对羟基磷灰石力学性质影响的研究.实验力学，2009,24(1):1-7
[21] Toshiaki K, Takao Y, Takashi N. Four calcium phosphate ceramics as bone substitutes fornon-weight-bearing. Biomaterials,1993,143:216-224
[22] Kikuchi M, Matsumoto HN, Yamada T. Glutaraldehyde cross-linked hydroxyapatite/collagen self-organized nanocomposites. Biomaterials,2004,25:63-69.
[23] Manjubala I, Scheler S, Bossert J. Mineralisation of chitosan scaffolds with nano-apatiteformation by double diffusion technique. Acta Biomater,2006,2(1):75-84.
[24] Malafaya PB, Silva GA, Reis RL. Natural-origin polymers as carriers and scaffolds forbiomolecules and cell delivery in tissue engineering applications. Adv Drug Deliv Rev,2007,59(4–5):207-233.
[25] Thein-Han WW, Misra RDK. Biomimetic chitosan–nanohydroxyapatite composite scaffoldsfor bone tissue engineering. Acta Biomaterialia,2009,5:1182-1197.
[26] Hu Q, Li B, Wang M. Preparation and characterization of biodegradable chitosan/hydroxyapatite nanocomposite rods via in situ hybridization: a potential material as internalfixation of bone fracture. Biomaterials,2004,25(5):779-85.
[27] Kong L, Gao Y, Cao W. Preparation and characterization of nano-hydroxyapatite/chitosancomposite scaffolds. J Biomed Mater Res,2005,75A:275-82.
[28] Rusu VM, Ng CH, Wilke M. Size-controlled hydroxyapatite nanoparticles as self-organizedorganic–inorganic composite materials. Biomaterials,2005,26(26):5414-26.
[29] Manjubala I, Scheler S, Bossert J. Mineralisation of chitosan scaffolds with nano-apatiteformation by double diffusion technique. Acta Biomater,2006,2(1):75-84.
[30]杨菊林,周长忍,田冶.壳聚糖/羟基磷灰石膜的制备及对细胞生长的影响.生物医学工程学杂志,2009,26(3):580-584.
[31]段友容,吕万新,王朝元.在动态模拟体液中致密CaP陶瓷表面形貌对类骨磷灰石层形成的影响.生物医学工程学杂志,2002,19(2):186-190.
[32] Shel TRM, Liu Y, Cooper PR. Bone marrow cell gene expression and tissue constructassembly using octacalcium phosphate microscaffolds. Biomaterials,2006,27(14):2874-2881.
[33] Brown TD. Techniques for mechanical stimulation of cells in vitro: a review. J Biomech,2004,33:3-14.
[34]唐丽灵.王远亮,谷俐.不同应变水平拉伸对成骨细胞生理功能的影响.重庆大学学报,2003,26(3):67-70.
[35] Harter LV, H ruska KA, Duncan RL. Human osteoblast-like cells respond to mechanicalstrain with increased bone matrix protein production independent of hormonal regulation.Endocrinology,1995,136(2):528-535.
[36] Knoll BI, Mccarthy TL, Centrella M. Strain-dependent control of transforming growthfactor-beta function in osteoblasts in an vitro model: biochemical events associated withdistraction osteogenesis. Plast Reconstr Surg,2005,116(1):224-233.
[37]陈伟辉,乔鞠,罗颂椒.不同流动剪切力调节大鼠成骨样细胞增殖的体外研究.华西医大学报,2001,32(2):232-234.
[38] Vico L, Lafage–Proust MH, Alexandre C. Effect of gravitational change on bone system invitro and in vivo [J]. Bone,1998,22(5Suppl):95S
[39]毛勇,段小红,王忠义.不同应力对成骨细胞和细胞骨架影响的实验研究.牙体牙髓牙周病学杂志，2001,11(2):98-100.
[40]戚孟春，胡静,韩立赤.成骨细胞在机械力刺激下细胞骨架及细胞形态改变的体外研究.中国口腔颌面外科杂志,2004,2(3):181-184.
[41] Genge BR, Sauer GR, Wu LN. Correlation between loss of alkaline phosphatase activity andaccumulation of calcium during matrix vesicle mediated mineralization [J]. J Biol Chem,1988,263(34):18513-9.
[42] Buckley MJ, Banes AJ, Jordan RD. The effects of mechanical strain on osteoblasts in vitro. JOral Maxillofac Surg,1990;48(2):276-282.
[43]张西正,李彬,郭勇.不同应变对成骨细胞增殖活性及功能状态的影响.生物医学工程与临床,2005,9(2):69-72.
[44] Kaspar D, Seidl W, Neidlinger-Wilke. Dynamic cell stretching increases human osteoblastproliferation and CICP synthesis but decreases osteocalcin synthesis and alkaline phosphataseactivity [J]. J Biomechanics,2000,33(1):45-51.
[45]唐林,林珠,李永明.机械牵张力对成骨细胞碱性酸酶及骨钙蛋白的影响.临床口腔医学杂志,2005,21(11):648-650.
[1] Greenwald AS, Boden SD, Goldberg VM. Bone-graft substitutes: Facts, fictions, andapplications[J]. J Bone Joint Surg,2001,83A:98-103.
[2] Den BFC, Wippermann BW, Blokhuis TJ. Healing of segmental bone defects with granularporous hydroxyapatite augmented with recombinant human osteogenic protein-1or autologousbone marrow[J]. J Orthopaed Res,2003,21:521-528.
[3] Toshiaki K, Takao Y, Takashi N. Four calcium phosphate ceramics as bone substitutes fornon-weight-bearing[J]. Biomaterials,1993,143:216-224.
[4] Nelson Luis G.D. Souza, Humberto M Brandao, Luiz Fernando C. de Oliveira. Spectroscopicand thermogravimetric study of chitosan after incubation in bovine rumen[J]. Journal of MolecularStructure,2011,1005:186-191.
[5] He Q，Ao Q，Xiu B，Gong YD, Zhao NM, Zhang XF. Mechanisms for biocompatibility ofchitosan: A new viewpoint [J]. Journal of Clinical Rehabilitative Tissue Engineering Research，2007,11(35):7110-7112.
[6] Shirosaki Y, Tsuru K, Hayakawa S, Osaka A, Lopes MA, Santos JD, Fernandes MH. In vitrocytocompatibility of MG63cells on chitosan-organosiloxane hybrid membranes[J]. Biomaterials,2005,26(5):485-493.
[7] Zhang XF, Hua H, Shen XY, Yang Q. In vitro degradation and biocompatibility ofpoly(1-lactic acid)/chitosan fiber composites[J]. Polymer,2007,48(4):1005-1011.
[8] Amaral IF, Lamghari M, Sousa SR, Sampaio P, Barbosa MA. Rat bone marrow stromal cellosteogenic differentiation and fibronectin adsorbtion on chitosan membranes: the effect of thedegree of acetylation [J]. J Biomed Mater Res A,2005,75(2):387-397.
[9] Zhao F, Yin YJ, Yao KD. Preparation and histological evaluation of biomimeticthree-dimensional hydroxyapatite/chitosan-gelatin network composite scaffolds[J]. Biomaterials,2002,23:3227-3234.
[10] Kong L, Gao Y, Cao W. Preparation and characterization of nano-hydroxyapatite/chitosancomposite scaffolds[J]. J Biomed Mater Res,2005;75A:275–282.
[11] Rusu VM, Ng CH, Wilke M. Sizecontrolled hydroxyapatite nanoparticles as self-organizedorganic–inorganic composite materials[J]. Biomaterials,2005;26(26):5414–5426.
[12] Manjubala I, Scheler S, Bossert J. Mineralisation of chitosan scaffolds with nano-apatiteformation by double diffusion technique[J]. Acta Biomater,2006;2(1):75–84.
[13]张西正,侍才洪,康少华,郭勇,李瑞欣,宁波.细胞基底应变加载装置，实用新型，ZL200920250218.7.
[14]张立德,牟季美.纳米材料和纳米结构[M].北京,科学出版社,2001
[15]王嘉宁,郭宁.细胞凋亡的检测技术与方法[J].中国药理学与毒理学杂志,2005,19(6):466-470.
[16]宋海明，张宝述，孙红娟.行星式球磨机在加工蛭石粉体中的应用研究[J].矿山机械，2007，35（4）：14-16.
[17]陈彩风,陈志刚.超声场中湿法制备纳米粉末的原理和方法[J].机械工程材料,2003,27(4):30-32.
[18] Kubota N, Ohga K, Moriguchi M. Permeability properties of glycol chitosan membranemodified with thiol groups[J]. Journal of Applied Polymer Science,1991,42:495-501.
[19] Majeti NV Ravikumar. A review of chitin and chitosan applications[J]. Reactive andFunctional Polymers,2000,46:1-27.
[20]杨菊林,周长忍,田冶,田金环.壳聚糖/羟基磷灰石膜的制备及对细胞生长的影响[J].生物医学工程学杂志,2009,26(3):580-584.
[21] Altankov G, Groth T. Reorganization of substratum bound fibronectin on hydrophobicmaterials is related to biocompatibility [J]. J Mater Sci Miter Med,1994,5(9):732-737.
[22] Anselme K, Bigerell EM, Noel B. Qualitative and quantitative study of human osteoblastadhesion on materials with various surface roughness[J]. J Bio Mater Res,2000,49(2):155-166.
[23]宋辰刚，邱小忠，吴刚，余磊，赖桂华，欧阳钧. MC3T3-HA复合培养后的细胞凋亡检测[J].中国临床解剖学杂志，2007,25(5):567-570.
[1]陈际达,崔磊,刘伟,曹谊林.溶剂浇铸/颗粒沥滤技术制备组织工程支架材料[J].中国生物工程杂志,2003,23(4):32-35,42.
[2] Hutmacher DW. Scaffolds in tissue engineering bone and cartilage[J]. Biomaterials,2000,21(24):2529-2543.
[3] Agrawal CM, Ray RB. Biodegradable polymeric scaffolds for musculoskeletal tissueengineering[J]. J Biomed Mater Res,2001,55:141-150.
[4] Yang SF, Leong KF, Du ZH, Chua CK. The design of scaffolds for use in tissue engineering[J].Tissue Eng,2001,7:679-689.
[5] Chen GP, Ushida T, Tateishi T. Scaffold design for tissue engineering[J]. MacromolBiosci,2002,2(2):67-77.
[6] Liu XH, Ma PX. Polymeric scaffolds for bone tissue engineering[J]. Ann Biomed Eng,2004,32(3):477-486.
[7] Fisher JP, Holland TA, Dean D, Engel PS, Mikos AG. Synthesis and properties ofphotocross-linked poly(propylene fumarate) scaffolds[J]. J Biomater Sci Polym Ed,2001,12(6):673-87.
[8] Borden M, El-Amin SF, Attawia M, Laurencin CT. Structural and human cellular assessmentof a novel microsphere-based tissue engineered scaffold for bone repair[J]. Biomaterials,2003,24:597-609.
[9] Shen F, Cui YL, Yang LF, Yao KD, Dong XH, Jia WY, Shi HD. A study on the fabrication ofporous chitosan/gelatin network scaffold for tissue engineering[J]. Polym Int,2000,49:1596-1599.
[10] Chen G, Ushida T, Tateishi T. Preparation of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams by use of ice microparticulates[J]. Biomaterials,2001,22:2563-2567.
[11] Murphy WL, Dennis RG, Kileny JL, Mooney DJ. Salt fusion: an approach to improve poreinterconnectivity within tissue engineering scaffolds[J]. Tissue Eng,2002,8(1):43-52.
[12] Zhang RY, Ma PX. Synthetic nano-fibrillar extracellular matrices with predesignedmacroporous architectures[J]. J Biomed. Mater Res,2000,52:430-438.
[13] Liao CJ, Chen CF, Chen JH, Chiang SF, Lin YJ, Chang KY. Fabrication of porousbiodegradable polymer scaffolds using solvent merging/particulate leaching method[J]. J BiomedMater Res,2002,59:676-681.
[14] Hou QP, Grijpma DW, Feijen J. Porous polymeric structures for tissue engineering preparedby a coagulation, compression moulding and salt leaching technique[J]. Biomaterials,2003,24:1937-1947.
[15] Oh SH, Kang SG, Kim ES, Cho SH, Lee JH. Fabrication and characterization of hydrophilicpoly(lactic-co-glycolic acid)/poly(vinyl alcohol) blend cell scaffolds by melt-moldingparticulate-leaching method[J]. Biomaterials,2003,24(22):4011-4021.
[16] Sander EA, Alb AM, Nauman EA, Reed WF, Dee KC. Solvent effects on the microstructureand properties of75/25poly(D,L-lactide-coglycolide) tissue scaffolds[J]. J Biomed Mater Res A,2004,70:506-513.
[17] Cai Q, Yang JA, Bei JZ, Wang SG, A novel porous cells scaffold made of polylactide-dextranblend by combining phase-separation and particle-leaching techniques[J]. Biomaterials,2002,23:4483-4492.
[18] Shastri VP, Martin I, Langer R. Macroporous polymer foams by hydrocarbon templating[J].Proc Natl Acad Sci USA,2000,97(5):1970-1975.
[19] Lin HR, Kuo CJ, Yang CY, Shaw SY, Wu YJ. Preparation of Macroporous BiodegradablePLGA Scaffolds for Cell Attachment with the Use of Mixed Salts as Porogen Additives[J]. JBiomed Mater Res,2002,63:271-279.
[20] Nam YS, Yoon JJ, Park TG. A novel fabrication method of macroporous biodegradablepolymer scaffolds using gas foaming salt as a porogen additive[J]. J. Biomed. Mater. Res.(ApplBiomater),2000,53:1-7.
[21] Lin ASP, Barrows TH, Cartmell SH, Guldberg RE. Microarchitectural and mechanicalcharacterization of oriented porous polymer scaffolds[J]. Biomaterials,2003,24:481-489.
[22] Ma ZW, Gao CY, Gong YH, Shen JC. Paraffin spheres as porogen to fabricate poly(L-lacticacid) scaffolds with improved cytocom-patibility for Cartilage Tissue Engineering[J]. J BiomedMater Res Part B,2003,67B(1):610-617.
[23] Chen VJ, Ma PX. Nano-fibrous poly(L-lactic acid) scaffolds with interconnected sphericalmacropores[J]. Biomaterials,2004,25(11):2065-2073.
[24] Zhang JC, Wu LB, Jing DY, Ding JD. A comparative study of porous scaffolds with cubic andspherical macropores[J]. Polymer,2005,46:4979-4985
[25]曹谊林，陈际达，崔磊，刘伟.水溶性粘结剂及其在内部结构可控的大体积细胞支架制备中的应用. CN200410016133.4
[26] Ma PX, Choi JW. Biodegradable polymer scaffolds with well-defined interconnectedspherical pore network[J]. Tissue Eng,2001,7(1):23-33.
[27] Wu LB, Ding JD. In vitro degradation of three dimensional porous poly(D,L-lactide-co-glycolide) scaffolds for tissue engineering[J]. Biomaterials2004;25:5821-5830.
[28] Babense JE, Anderson JM, Mcintire LV, Mikos AG. Host response to tissue engineereddevices[J]. Adv. Drug. Deliv. Rev.,1998,33,111–139.
[29] Heggset E.B., Hoell1.A.，Kristoffersen M.，Eijsink V.G.H.，VarumK.M. Degradation ofChitosans with Chitinase G from Streptomyces coelicolor A3(2): Production ofChito-oligosaccharides and Insight into Subsite Specificities[J]. Biomacromolecules，2009,10(4):892-899.
[30] Heggset E.B.，Dybvik A.I.，HoellI.A.，Norberg A.L.，Sorlie M., Eijsink V.G.H.,Varum K.M.Degradation of Chitosans with a Family46Chitosanase from Streptomyces coelicolor A3(2)[J].Biomacromolecules,2010,11(9):2487-2497.
[31] Lee D.X.，Xia W.S.，Zhang J.L. Enzymatie preparation of chitooligosaccharides bycommercial lipase[J]. Food Chem,2008,111(2):291-295.
[32] Xie Y.，WeiY，HuJ.G.Depolymerization of Chitosan with a Crude Cellulase Preparation fromAspergillus Niger[J]. Appl Biochem Biotech,2010,160(4):1074-1083.
[33] Sikorski P, Sorbotten A, Horn SJ, Eijsink VGH, Varum KM. Serratia marcescens chitinaseswith tunnel-shaped substrate-binding grooves show endo activity and different degrees ofprocessivity during enzymatic hydrolysis of chitosan[J]. Biochemistry-Us,2006,45(31):9566-9574.
[34] Henry, J. B. Clinical Diagnosis and Management by Laboratory Methods,18th ed.;Saunerders: Philadelphia,1991,1371.
[35] Temel A.，Kazokoglu H.，Taga Y. Tear Lysozyme Levels in Contact-Lens Wearers[J]. AnnOphthalmol,1991,23(5)：191-194.
[36] Nordtceit RJ, Varum KM, Smidsrod O. Degradation of partially N-acetylated chitosans withhen egg white and human lysozyme[J]. Carbohydr Polym,1996,29:163-167
[37]马如,黄明智.壳聚糖的溶菌酶降解[J].北京化工大学学报,2002,29(6):40-43.
[38] Yang B, Li XY, Shi S, Kong XY, Guo G, Huang MJ, Luo F, Wei YQ, Zhao X, Qian ZY.Preparation and characterization of a novel chitosan scaffold[J]. Carbohydrate Polymers,2010,80:860-865
[39] Imoto T, Yagshita K. A simple activity measurement of lysozyme [J]. Agri Bio Chem,1971,35:1154-1156
[40]陈学忠，李志宏，李瑞欣，郭勇，刘璐，王亮，张西正.胶原多糖基纳米羟基磷灰石仿生骨支架材料的研制[J].国际生物医学工程杂志,2011,34(2):65-70
[41] Cowin SC. The relationship between the elasticity tensor and the fabric tensor[J]. MechMater,1985,4:137-147.
[42] Gundersen HJG, Jensen TB, Osterby R. Distribution of membrane thickness determined bylineal analysis[J]. J Microsc,1978,113:27-43.
[43] Harrigan TP, Mann RW. Characterization of microstructural anisotropy in orthotropicmaterials using a second rank tensor[J]. J Mater Sci,1984,19:761-767
[44] Zhang Y, Li RX, Fan YB, Liu H, Guo Y, Wang L, Shi CH, Zhu D, Zhang XZ.Microarchitectural and mechanical characterization of chitosan/hydroxyapatite/demineralizedbone matrix composite scaffold[J]. Journal of porous material,2011:1-9.
[45] Ren DW, Yi HF, Wang W, Ma XJ. The enzymatic degradation and swelling properties ofchitosan matrices with different degrees of N-acetylation[J]. Carbohydrate Research,2005,340:2403-2410.
[46] Murphy WL, Dennis RG, Kileny JL, Mooney DJ. Salt fusion: an approach to improve poreinterconnectivity within tissue engineering scaffolds[J]. Tissue Engineering,2002,8(1):43-52.
[47] Nam YS, Yoon JJ, Park TG. A novel fabrication method of macroporous biodegradablepolymer scaffolds using gas foaming salt as a porogen additive[J]. J Biomed MaterRes,2000,53:1-7.
[48] Muller B, Beckmann F, Huser M, Maspero F, Szekely G, Ruffieux K, Thurner P,Wintermantel E. Non-destructive three-dimensional evaluation of a polymer sponge bymicro-tomography using synchrotron radiation[J]. Biomol Eng,2002,19:73-78.
[49] Lin AS, Barrows TH, Cartmell SH, Guldberg RE. Microarchitectural and mechanicalcharacterization of oriented porous polymer scaffolds[J]. Biomaterials,2003,24:481-489.
[50] Ho ST, Hutmacher DW. A comparison of micro CT with other techniques used in thecharacterization of scaffolds[J]. Biomaterials,2006,27:1362-1376.
[51] Wan Y, Yu AX，Wu H，Wang ZX，Wen DJ. Porous-conductive chitosan scaffolds for tissueengineering II. in vitro and in vivo degradation[J]. J Mater Sci-Mater M,2005，16(11):1017-1028.
[1] Cheng B, Kato Y, Zhao S, Luo J, Sprague E, Bonewald LF, Jiang JX. PGE(2) is essential forgap junction-mediated intercellular communication between osteocyte-like MLO-Y4cells inresponse to mechanical strain. Endocrinology,2001,142,3464-3473.
[2] Tanaka SM. A new mechanical stimulator for cultured bone cells using piezoelectric actuator.Journal of Biomechanics,1999,32:427-430.
[3] Meyer U, Meyer T, Wiesmann HP, Stratmann U, Kruse-Losler B, Maas H, Joos U. The effectof magnitude and frequency of interfragmentary strain on the tissue response to distractionosteogenesis. Journal of Oral and Maxillofaciale Surgery,1999,57:1331-1339.
[4] Peake MA, Cooling LM, Magnay JL, Thomas PB, ElHaj AJ. Selected contribution: regulatorypathways involved in mechanical induction of c-fos gene expression in bone cells. Journal ofApplied Physiology,2000,89:2498-2507.
[5] Saunders MM, Taylor AF, Du C, Zhou Z, PellegriniJr VD, Donahue HJ. Mechanicalstimulation effects on functional end effectors in osteoblastic MG-63cells. Journal ofBiomechanics,2006,39:1419-1427
[6] Visconti LA, Yen EHK, Johnson RB. Effect of strain on bone nodule formation by ratosteogenic cells in vitro. Archives of Oral Biology,2004,49:485-492.
[7] Kaspar D, Seidl W, Neidlinger-Wilke C, Ignatius A, Claes L. Dynamic cell stretching increaseshuman osteoblast proliferation and CICP synthesis but decreases osteocalcin synthesis andalkaline phosphatase activity. Journal of Biomechanics,2000,33:45-51.
[8] Mauney JR, Sjostorm S, Blumberg J, Horan R, O’Leary JP, Vunjak-Novakovic G, Volloch V,Kaplan DL,. Mechanical stimulation promotes osteogenic differentiation of human bone marrowstromal cells on3-D partially demineralized bone scaffolds in vitro. Calcified Tissue International2004,74:458-468.
[9] Yang Y, Wood MA, ElHaj AJ. Enhancement of mechanical signals for tissue engineering boneby mechano-active scaffolds. Abstract of conference: ECM V The cell biomaterials reaction,DAVOS, Switzerland, European Cells and Materials,2004,7, supplement1:35
[10] Wood MA, Yang Y, Thomas PBM, ElHaj AJ. Utilising dihydropyridine-release strategies toenhance load effects in engineered human bone constructs. Tissue Engineering,2006,12:2489-2497.
[11] Gemmiti CV, Guldberg RE. Fluid flow increases type II collagen deposit and tensilemechanical properties in bioreactor-grown tissueengineered cartilage. Tissue Engineering,2006，12,469-479.
[12] Martin I, Wendt D, Heberer M. The role of bioreactors in tissue engineering. Trends inBiotechnology,2004,22,80-86.
[13] Meyer U, Buchter A, Nazer N, Wiesmann HP. Design and performance of a bioreactorsystem for mechanically promoted three dimensional tissue engineering. British Journal of Oraland Maxillofacial Surgery,2006,44,134-140.
[14] Baas E, Kuiper JH, Yang Y, Wood MA, ElHaj AJ. In vitro bone growth responds to localmechanical strain in three-dimensional polymer scaffolds. Journal of Biomechanics,2010,43:733-739
[15] Lacroix D, Sandino C, Villagomez M. A micro-finite element analysis of fluidic and solidmechanical stimuli in bone tissue engineering scaffolds. Journal of Biomechanics,2006,39(Supplement1):415
[16] Gibson LJ, Ashby MF. Cellular Solids: Structure and Properties.1988. Pergamon Press, NewYork, USA.
[17]宫元伟,闫玉仙,张媛,张西正.基底拉伸应变对小鼠骨细胞Rnnx2表达的影响.中国骨质疏松杂志.2011,17(3):185-188
[18] Wang L, Zhang XZ, Guo Y, Chen XZ, Li RX, Liu L, Shi CH, Guo C, Zhang Y. Involvementof BMPs/Smad Signaling Pathway in Mechanical Response in Osteoblasts. Cell Physiol Biochem2010;26:1093-1102.
[19]闫玉仙,宋梅,郭春,郭勇,宫元伟,李瑞欣,张西正.基底拉伸应变对小鼠三种骨组织细胞BMP-2mRNA表达的影响.中国老年学杂志,2010,30(21):3092-3095
[20] Chen XY，Zhang XZ，Guo Y, Li RX, Lin JJ, Wei Y. The establishment of a mechanobiologymodel of bone and functional adaptation in response to mechanical loading. ClinicalBiomechanics,2008, s88-95
[21]赵红斌,吕同德,马敬,马慧,张西正.机械力对间充质干细胞向成骨细胞分化的力学响应机制.生物化学与生物物理进展.2007,34(7):718-723
[22] Cɑrɑno A, Siciliɑni G. Effects of continuous ɑnd intermittent forces on humɑn fibroblɑsts invitro. Eur J Orthod,1996,18:19-26.
[23]黎小坚, Frost HM,朱绍舜,柯华珠.基础骨生物学新观.中国骨质疏松杂志,2001,7(2):152-174
[24]李广州,蒋电明.纳米羟基磷灰石生物安全性评价与研究进展.生物骨科材料与临床研究,2009,6(1):31-33
[25] Genge BR, Sauer GR, Wu LN, McleanFM, WuthierRE. Correlation between loss of alkalinephosphatase activity and accumulation of calcium during matrix vesicle-mediated mineralization.J Biol. Chem.,1988,263(34):18513-18519.
[26] Guicheux J, Lemonnier J, Ghayor C, Suzuki A, Palmer G, Caverzasio J. Activation of p38mitogen activated protein kinase and c-jun NH2terminal kinase by BMP-2and their implicationin the stimulation of osteoblastic cell differentiation. Bone Miner Res,2003,18(11):2060-2068.
[27] Islam AA, Rasubala L, Yoshikawa H, Shiratsuchi Y, Ohishi M. Healing of fractures inosteoporotic rat mandible shown by the expression of bone morphogenetic protein-2and tumournecrosis factor-alpha. Br J Oral Maxillofac Surg,2005,43(5):383-391.
[28] Rauch F, Lauzier D, Croteau S, Travers R, Glorieux FH, Hamdy R. Temporal and spatialexpression of bone morphogenetic protein-2,4and7during distraction osteogenesis.Bone,2000,26:611-617.
[29] Lai CF, Cheng SL. Signal transductions induced by bone morphogenetic protein-2andtransforming growth factor-beta in normal human osteoblastic cells. J Biol Chem.,2002,277(18):15514-15522.
[30] Chai F, Jin Y, Zhao YM. Expression of osteopontin and osteocalcin mRNA inectomesenchymal stem cells under induction of EMPs&EMD in vitro. Journal of Oral ScienceResearch,2004,20(3):243-245.
[1] Li Yubao, Klein C, Wijin J, Meer S, Groot K. Shape change and phase transition of needle-likenonstichiometic apatite crystals [J]. J Mater Sci: Mater Med,1994,5:263-267.
[2]许永华,施新猷,胡蕴玉.牛煅烧骨与体外培养兔骨膜成骨细胞的相容性[J].第四军医大学学报,2000,21(4)：512-514.
[3] Den BFC, Wippermann BW, Blokhuis TJ. Healing of segmental bone defects with granularporous hydroxyapatite augmented with recombinant human osteogenic protein-1or autologousbone marrow[J]. J Orthopaed Res,2003,21:521-528.
[4] Webster TJ. Nanophase ceramies: The future orthopedic and dental implant material.Advances in Chemical Engineering. Academic Press New York,2001:125-166.
[5] Yoshikawa T, Ohgushi H,Tamai S. Immediate bone forming capability of prefabrieatedosteogenic hydroxyapatite[J]. Biomed Mater Res,1996,32(3):481-492.
[6] Rusu VM, Ng CH, Wilke M, Tiersch B, Fratzl P, Peter MG. Sizecontrolled hydroxyapatitenanoparticles as self-organized organic–inorganic composite materials[J]. Biomaterials2005;26(26):5414-26.
[7] Zhao F, Yin YJ, Lu WW, Leong JC, Zhang WJ, Zhang JY, Zhang MF, Yao KD. Preparationand histological evaluation of biomimetic three-dimensional hydroxyapatite/chitosan-gelatinnetwork composite scaffolds [J]. Biomaterials,2002,23:3227-3234.
[8] Thein-Han WW, Kitiyanant Y, Misra RDK. Chitosan as a scaffold matrix for tissueengineering. Mater Sci Technol2008;24:1062-1075.
[9] Martin HJ, Sehulz KH, Bumgardner JD, Schneider JA. Enhanced bonding of chitosan toimplant qualitytitanium via four treatment combinations[J]. Thin Solid Films,2008,516(18):6277-6286.
[10] Lahiji A, Sohrabi A, Hungerford DS, Frondoza CG. Chitosan surports the expression of theextracellular matrix proteins in human osteoblasts and ehondroeytes[J]. Biomed MaterRes.2000.51(4):586-595.
[11] Thein-Han WW, Misra RDK. Biomimetic chitosan–nanohydroxyapatite composite scaffoldsfor bone tissue engineering[J]. Acta Biomaterialia2009;5:1182-1197.
[12] Ito M, Hidaka Y, Nakajima M, Yagasaki H, Kafrawy AH. Effect of hydroxyapatite contenton physical properties and connective tissue reactions to a chitosan／hydroxyapatite compositemembrane[J]．Journal of Biomedical Materials Research,1999,45(3)：204-208．
[13]赵峰,尹玉姬,宋雪峰,姚康德,郭善一,郭若霖,张镜宇.壳聚糖-明胶网络／羟基磷灰石复合材料支架的研究-制备及形貌[J].中国修复重建外科杂志,2001,15(5)：276-279.
[14] Mukherjee DP, Tunkle AS, Roberts RA, Clavenna A, Rogers S, Smith D. An animalevaluation of a paste of chitosan glutamate and hydroxyapatite as a synthetic bone graftmateria1[J]．Journal of Biomedical Materials Research Part B-Applied Biomaterials,2006,67B(1)：603-609．
[15]郑雄飞,翟文杰,梁迎春,孙涛.低温沉积制造壳聚糖-纳米羟基磷灰石支架[J].无机材料学报,2011,26(1)：12-16.
[16]吴芳,戴伯川,李为祖.纳米羟基磷灰石/壳聚糖-海藻酸钠复合材料的制备及性能研究[J].海峡药学,2009,21(3):21-23.
[17]张利,李玉宝,魏杰.纳米羟基磷灰石/壳聚糖复合骨修复材料的共沉淀法制备及其性能表征[J].功能材料,2005,36(3):441-444.
[18] Chen F, Wang Z C, Lin C J. Preparation and characterization of nano-sized hydroxyapatiteparticles and hydroxy apatite/chitosan nano-composite for use in biomedical materials[J]. Materials Letters,2002,57:858-861.
[19]刘敬肖,史非,于玲,王井,牛丽婷,李佳颖,胡志强.模拟体液中纳米羟基磷灰石/壳聚糖的制备及表征[J].大连轻工业学院学报,2007,26(4):332-336.
[20]薛博,施云峰,何淑梅,李兰英,郭振招,张兰兰,李红.常温环境下壳聚糖诱导合成单斜羟基磷灰石纳米晶体及其结构表征[J].高等学校化学学报,2010,31(12)：2339-2343.
[21]杨辉,张园园.壳聚糖分子对共沉淀法合成羟基磷灰石/壳聚糖粉体材料晶体形貌的影响[J].人工晶体学报,2012,41(1):209-214.
[22] Viorel Matin Rusu, Chuen-How Ng, Max Wilke, Brigitte Tiersch, Peter Fratzl, Matin G Peter.Size-controlled hydroxyapatite nanoparticles as self organized organic-inorganic compositematerials[J]. Biomaterials,2005,26:5414-5426.
[23]何丹,许素华,游青青,张明霞,肖秀峰,刘榕芳.壳聚糖模板诱导梭状纳米羟基磷灰石的仿生合成[J].化工时刊,2007,21(7):7-10.
[24] Yang F, Wolke J G C, Jansen J A. Biomimetic calcium phosphate coating on electrospun poly(ε-caprolactone) scaffolds for for bone tissue engineering[J]. Chem Eng J,2008,137(1):154-161.
[25] Beppu MM,Santana CC．PAA influence on chitosan membrane calcification[J]．MaterialsScience and Engineering C,2003,23(5)：651-658．
[26] Tachaboonyakiat W, Serizawa T, Akashi M. Hydroxyapatite formation on/in biodegradablechitosan hydrogels by an alternate soaking process[J].Polymer Journal,2001,33(2):177-181.
[27]李红,朱敏鹰,李立华,周长忍.原位沉析羟基磷灰石-壳聚糖骨组织工程支架材料的研制[J].功能材料,2006,37(6):909-911.
[28]王新,刘玲蓉,张其清.纳米羟基磷灰石-壳聚糖骨组织工程支架的研究[J].中国修复重建外科杂志,2007,21(2):120-124.
[29]李波,李立华,赵名艳,周长忍. Sol-gel原位相转变矿化制备纳米羟基磷灰石/壳聚糖复合支架材料.复合材料学报,2011,28(4)：83-88.
[30]卢晓英,王秀红,刘治,翁杰.反应pH值对原位水热沉积法制备纳米羟基磷灰石/壳聚糖复合材料的影响[J].复合材料学报,2009,26(4):53-58.
[31] Li BQ, Hu QL, Qian XZ, Fang ZP, Shen JC. Bioabsorbable chitosan/hydroxyapatitecomposite rod prepared by in situ precipitation for internal fixation of bone fracture[J].Acta.Polymerica.Sinica.,2002,36(6):828-833.
[32] Manjubala I, Scheler S, Bossert J. Mineralisation of chitosan scaffolds with nano-apatiteformation by double diffusion technique[J]. Acta Biomater2006;2(1):75-84.
[33]黄志海,李纪宏,林萍华,董寅生,郭超,盛晓波,储成林.壳聚糖多孔支架电化学辅助沉积羟基磷灰石涂层研究[J].高分子学报,2009,(3):273-277.
[34]胡仁,胡皓冰,林吕健．CaP／壳聚糖复合膜层的电化学共沉积研究[J]．高等学校化学学报,2002,23(11)：2142-2146.
[35] Huang LY, Xu KW, Lu J．A study of the process and kinetics of electrochemical depositionand the hydrothermal synthesis of hydroxyapatite coatings[J]．Journal of Materials Science-Materials in Medicine,2000,11(11)：667-673．
[36] Huang ZH, Dong YS, Chu CL. Electrochemistry assisted reacting deposition ofhydroxyapatite in porous chitosan scaffolds [J]. Materials Letters,2008,62(19):3376-3378.
[37] Hu Q, Li B, Wang M, Shen J. Preparation and characterization of biodegradablechitosan/hydroxyapatite nanocomposite rods via in situ hybridization: a potential material asinternal fixation of bone fracture[J]. Biomaterials2004;25(5):779-785.
[38] Kong L, Gao Y, Cao W, Gong Y, Zhao N, Zhang X. Preparation and characterization ofnano-hydroxyapatite/chitosan composite scaffolds[J]. J Biomed Mater Res2005;75A:275-282.
[39] Yamaguchi I,Iizuka S,Osaka A, Monma H, Tanaka J. The effect of citric acid addition onchitosan/hydroxyapatite composites [J]. Colloids and Surfaces A: Physicochem Eng Aspects,2003,214:111-118.
[40] Yamaguchi I, Tokuchi K, Fukuzaki H, Koyama Y, Takakuda K, Monma H. Preparation andmicrostructure analysis of chitosan/hydroxyapatite nanocomposites. Journal of BiomedicalMaterials Research,2001,55:20-27.
[41]陈景帝,王迎军,陈晓峰,尹诗衡.利用冷冻干燥原位构筑仿生型纳米羟基磷灰石、壳聚糖多孔支架材料[J].稀有金属材料与工程,2009,38：271-273.
[42]李保强,胡巧玲,汪茫,沈家骢.原位复合法制备层状结构的壳聚糖/羟基磷灰石纳米材料[J].高等学校化学学报,2004,25(10)：1949-1952.
[43]赵巍,侯振德,任朝峰.骨细胞培养过程对羟基磷灰石力学性质影响的研究[J].实验力学,2009,24(1):1-7.
[44]汪皖,徐培,吴海珍.壳聚糖-纳米羟基磷灰石复合材料修复兔骨缺损过程中BMp-2的表达[J].安徽医科大学学报,20l0,45(4):491-493.
[45] Yuan H, Chen N, Lu XY, Zheng BZ. Experimental study of natural hydroxyapatite/chitosancomposite on reconstructing bone defects[J]. Journal of Nanjing Medical University,2008,6(22):372-375.
[46] Kong LJ, Ao Q, Xi J, Zhang L, Gong YD, Zhao NM, Zhang XF. Proliferation anddifferentiation of MC3T3-E1cells cultured on nano-hydroxyapatite/chitosan compositescaffolds[J]. Joumal of Biomedical Materials Research,2007,2(75):275-282.
[47]袁华,陈宁,吕晓迎,郑步中,宋晓陵.天然羟基磷灰石-壳聚糖复合材料修复骨缺损的实验研究[J].口腔医学,2005,25(2):65-67.
[48] Aylin S U, Russell D. The addition of biphasic calcium phosphate to porous chitosanscaffolds enhances bone tissue development in vitro[J]. J. Biomed. Mater. Res. A,2007,81A(3):624-633.
[49]杨菊林,周长忍,田冶.壳聚糖/羟基磷灰石膜的制备及对细胞生长的影响[J].生物医学工程学杂志,2009,26(3):580-584.
[50] Shel TRM, Liu Y, Cooper PR. Bone marrow cell gene expression and tissue constructassembly using octacalcium phosphate microscaffolds[J]. Biomaterials2006;27(14):2874-2881.
[51] Oberdors ter G, Sharp Z, Atudorei V, et al. Extrapulmonary translocation of ultrafine carbonparticles following whole-body inhalation exposure of rats[J]. J Toxicol Environ Health A2002;65(20):1531-1543.
[52]李广州,蒋电明.纳米羟基磷灰石生物安全性评价与研究进展[J].生物骨科材料与临床研究,2009,6(1):31-33.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |