颅骨组织工程复合支架动态灌注装置的设计
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摘要
为了解决颅骨组织工程支架中存在的中心处细胞存活率低的问题,针对具有血管嵌入的颅骨复合支架研制动态灌注培养装置,为多孔支架内部细胞提供营养物质以提高细胞存活率,使复合支架达到体内整合的要求.基于颅骨复合支架结构特点,设计双通道循环分别对多孔支架和血管进行灌注,为颅骨复合支架中成骨细胞和内皮细胞提供物质传输,气体循环为循环培养液提供氧气、二氧化碳,保证了培养液氧气充足和pH稳定;模拟人体自然颅骨组织中机械刺激,建立多孔支架、血管流体模型,根据灌注室和血管入口流量计算流体所需的剪切应力,据此确定流体流量和压力的控制范围与精度,为复合支架提供合适的流量;设计三因素三水平正交试验对灌注室的结构尺寸进行优选,确定关键部位灌注室的结构,通过硅胶管连接灌注室、储液瓶、蠕动泵形成循环系统,实现动态灌注装置的总体结构安装;建立流体仿真分析有限元模型,分析不同流速的流体培养液分别对颅骨多孔支架和血管结构的影响,结果表明灌注室和血管结构内流体速度、剪切应力、压力分布均匀,满足骨组织和内皮细胞的培养生理环境,验证了装置设计的合理性.该装置可用于骨组织体外细胞的动态培养,为骨组织工程支架体外构建及血管化奠定了基础.
To solve the problem of low survival rate of cells at the center of a scaffold,a dynamic perfusion culture device is developed for skull composite scaffold with embedded vessels. The device improves cell survival rate and makes composite scaffolds meet the integration requirement in vivo. Based on the structural features of the skull composite scaffold,a double-channel perfusion circulation is designed to separately perfuse porous scaffold and vessels to provide material transmission for osteoblasts and endothelial cells.Moreover,gas circulation provides oxygen and carbon dioxide for circulating culture medium,which ensures sufficient oxygen and stable pH. To simulate the mechanical environment in a natural human skull,a porous scaffold and a vascular fluid model are established,and the shear stress is calculated according to the inlet flow rate of perfusion chamber and vessel. Therefore,the control range and accuracy of fluid flow and pressure are determined,and the suitable flow rate for composite scaffolds are obtained. An orthogonal test with three factors and three levels is designed to optimize the size and determine the key parts of the perfusion chamber structure. The whole structure of the dynamic perfusion device is installed by connecting the perfusion chamber,the liquid storage bottle,and the peristaltic pump with a silica gel tube to form a circulating system. A finite element fluid analysis model is established to analyze the effect of flow with different velocities oncomposite scaffold. The results show that the fluid velocity,shear stress,and pressure are uniformly distributed in the perfusion chamber and vessel,and the physiological environment of bone tissue and endothelial cells is satisfactorily simulated;therefore,the rationality of the device is verified. The device can be used for dynamic cell culture of bone tissue in vitro. It lays a foundation for the construction and vascularization of bone tissue-engineered scaffolds.
To solve the problem of low survival rate of cells at the center of a scaffold,a dynamic perfusion culture device is developed for skull composite scaffold with embedded vessels. The device improves cell survival rate and makes composite scaffolds meet the integration requirement in vivo. Based on the structural features of the skull composite scaffold,a double-channel perfusion circulation is designed to separately perfuse porous scaffold and vessels to provide material transmission for osteoblasts and endothelial cells.Moreover,gas circulation provides oxygen and carbon dioxide for circulating culture medium,which ensures sufficient oxygen and stable pH. To simulate the mechanical environment in a natural human skull,a porous scaffold and a vascular fluid model are established,and the shear stress is calculated according to the inlet flow rate of perfusion chamber and vessel. Therefore,the control range and accuracy of fluid flow and pressure are determined,and the suitable flow rate for composite scaffolds are obtained. An orthogonal test with three factors and three levels is designed to optimize the size and determine the key parts of the perfusion chamber structure. The whole structure of the dynamic perfusion device is installed by connecting the perfusion chamber,the liquid storage bottle,and the peristaltic pump with a silica gel tube to form a circulating system. A finite element fluid analysis model is established to analyze the effect of flow with different velocities oncomposite scaffold. The results show that the fluid velocity,shear stress,and pressure are uniformly distributed in the perfusion chamber and vessel,and the physiological environment of bone tissue and endothelial cells is satisfactorily simulated;therefore,the rationality of the device is verified. The device can be used for dynamic cell culture of bone tissue in vitro. It lays a foundation for the construction and vascularization of bone tissue-engineered scaffolds.
引文
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[22]Jeon J H,Jeong O C.Effect of shear stress magnitude on intracellular calcium expression in bone cells[J].Microelectronic Engineering,2012,97:329-332.
[23]Sakai K,Mohtai M,Iwamoto Y.Fluid shear stress increases transforming growth factor beta 1 expression in human osteoblast-like cells:Modulation by cation channel blockades[J].Calcif Tissue Int,1998,63(6):515-520.
[24]Xu H,Duan J,Ren L,et al.Impact of flow shear stress on morphology of osteoblast-like IDG-SW3cells[J].Journal of Bone and Mineral Metabolism,2018,36:529-536.
[25]Ho H,Mithraratne K,Hunter P.Numerical simulation of blood flow in an anatomically-accurate cerebral venous tree[J].IEEE Trans on Med Imaging,2013,32(1):85-91.
[26]Mc Cormick S M,Whitson P A,Wu K K,et al.Shear stress differentially regulates PGHS-1 and PGHS-2 protein levels in human endothelial cells[J].Ann Biomed Eng,2000,28(7):824-833.
[27]毕振庆.颅骨多孔支架的设计及其增材制造工艺研究[D].天津:天津大学机械工程学院,2018.Bi Zhenqing.Design and Additive Manufacturing for Skull Porous Scaffold[D].Tianjin:School of Mechanical Engineering,Tianjin University,2018(in Chinese).
[28]Hu K,Wang C,Zhang X.High pressure may inhibit periprosthetic osteogenesis[J].Journal of Bone and Mineral Metabolism,2010,28(3):289-298.
[2]Ben-David D,Kizhner T A,Kohler T,et al.Cellscaffold transplant of hydrogel seeded with rat bone marrow progenitors for bone regeneration[J].Journal of Cranio-Maxillofacial Surgery,2011,39(5):364-371.
[3]Ding M,Henriksen S S,Theilgaard N,et al.Assessment of activated porous granules on implant fixation and early bone formation in sheep[J].Journal of Orthopaedic Translation,2016,5:38-47.
[4]Goldstein A S,Juarez T M,Helmke C D,et al.Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds[J].Biomaterials,2001,22(11):1279-1288.
[5]Yeatts A B,Fisher J P.Bone tissue engineering bioreactors:Dynamic culture and the influence of shear stress[J].Bone,2011,48(2):171-181.
[6]Liu L,Yuan W,Wang J.Mechanisms for osteogenic differentiation of human mesenchymal stem cells induced by fluid shear stress[J].Biomechanics and Modeling in Mechanobiology,2010,9(6):659-670.
[7]Volkmer E,Drosse I,Otto S,et al.Hypoxia in static and dynamic 3D culture systems for tissue engineering of bone[J].Tissue Engineering Part A,2008,14(8):1331-1340.
[8]Du D,Furukawa K S,Ushida T.3D culture of osteoblast-like cells by unidirectional or oscillatory flow for bone tissue engineering[J].Biotechnol Bioeng,2009,102(6):1670-1678.
[9]Frohlich M,Grayson W L,Marolt D,et al.Bone grafts engineered from human adipose-derived stem cells in perfusion bioreactor culture[J].Tissue Eng Part A,2010,16(1):179-189.
[10]Kim D H,Heo S J,Kang Y G,et al.Shear stress and circumferential stretch by pulsatile flow direct vascular endothelial lineage commitment of mesenchymal stem cells in engineered blood vessels[J].Journal of Materials Science:Materials in Medicine,2016,27:DOI10.1007/s10856-016-5670-0.
[11]Santoro R,Olivares A L,Brans G,et al.Bioreactor based engineering of large-scale human cartilage grafts for joint resurfacing[J].Biomaterials,2010,31(34):8946-8952.
[12]Bhaskar B,Owen R,Bahmaee H,et al.Design and assessment of a dynamic perfusion bioreactor for large bone tissue engineering scaffolds[J].Applied Biochemistry and Biotechnology,2018,185(2):555-563.
[13]Jagodzinski M,Breitbart A,Wehmeier M,et al.Influence of perfusion and cyclic compression on proliferation and differentiation of bone marrow stromal cells in 3-dimensional culture[J].Journal of Biomechanics,2008,41(9):1885-1891.
[14]Berner A,Woodruff M A,Lam C X F,et al.Effects of scaffold architecture on cranial bone healing[J].International Journal of Oral and Maxillofacial Surgery,2014,43(4):506-513.
[15]Sinikovic B,Schumann P,Winkler M,et al.Calvaria bone chamber-A new model for intravital assessment of osseous angiogenesis[J].Journal of Biomedical Materials Research Part A,2011,99A(2):151-157.
[16]刘洁,郑淑贤,李佳.颅骨组织工程血管支架的参数化设计[J].机械工程学报,2018,54(1):178-187.Liu Jie,Zheng Shuxian,Li Jia.Parametric design for skull tissue engineering vascular scaffold[J].Chinese Journal of Mechanical Engineering,2018,54(1):178-187(in Chinese).
[17]Whittaker R J,Booth R,Dyson R,et al.Mathematical modelling of fibre-enhanced perfusion inside a tissueengineering bioreactor[J].Journal of Theoretical Biology,2009,256(4):533-546.
[18]Guyot Y,Papantoniou I,Luyten F P,et al.Coupling curvature-dependent and shear stress-stimulated neotissue growth in dynamic bioreactor cultures:A 3D computational model of a complete scaffold[J].Biomechanics and Modeling in Mechanobiology,2016,15(1):169-180.
[19]邹盛铨.血流动力学与心血管人工器官[M].成都:成都科技大学出版社,1991.Zou Shengquan.Hemodynamics and Cardiovascular Artificial Organ[M].Chengdu:Chengdu University of Science and Technology Press,1991(in Chinese).
[20]Freitas D,Almeida H A,Bártolo P J.Perfusion bioreactor fluid flow optimization[J].Procedia Technology,2014,16:1238-1247.
[21]喀蔚波.医用物理学[M].北京:高等教育出版社,2008.Ka Weibo.Medical Physics[M].Beijing:Higher Education Press,2008(in Chinese).
[22]Jeon J H,Jeong O C.Effect of shear stress magnitude on intracellular calcium expression in bone cells[J].Microelectronic Engineering,2012,97:329-332.
[23]Sakai K,Mohtai M,Iwamoto Y.Fluid shear stress increases transforming growth factor beta 1 expression in human osteoblast-like cells:Modulation by cation channel blockades[J].Calcif Tissue Int,1998,63(6):515-520.
[24]Xu H,Duan J,Ren L,et al.Impact of flow shear stress on morphology of osteoblast-like IDG-SW3cells[J].Journal of Bone and Mineral Metabolism,2018,36:529-536.
[25]Ho H,Mithraratne K,Hunter P.Numerical simulation of blood flow in an anatomically-accurate cerebral venous tree[J].IEEE Trans on Med Imaging,2013,32(1):85-91.
[26]Mc Cormick S M,Whitson P A,Wu K K,et al.Shear stress differentially regulates PGHS-1 and PGHS-2 protein levels in human endothelial cells[J].Ann Biomed Eng,2000,28(7):824-833.
[27]毕振庆.颅骨多孔支架的设计及其增材制造工艺研究[D].天津:天津大学机械工程学院,2018.Bi Zhenqing.Design and Additive Manufacturing for Skull Porous Scaffold[D].Tianjin:School of Mechanical Engineering,Tianjin University,2018(in Chinese).
[28]Hu K,Wang C,Zhang X.High pressure may inhibit periprosthetic osteogenesis[J].Journal of Bone and Mineral Metabolism,2010,28(3):289-298.