基于3D打印的Ⅰ型胶原涂覆β-TCP骨组织工程支架研究
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摘要
根据骨缺损形态构建个性化的组织工程支架在骨组织工程应用中有巨大需求。基于3D打印技术制备个性化的I型胶原涂覆的β-磷酸三钙(β-TCP)骨支架。通过比较0/90°、0/60°、0/45°的填充角度,0.10、0.25、0.50 mg/m L涂覆胶原的浓度对β-TCP支架孔径、孔隙率、力学性能的影响,选定最优填充角度为0/90°及最佳涂覆胶原的浓度为0.50 mg/m L的β-TCP/胶原支架。所得支架能准确地再现设计的三维模型,具有多级孔结构,大孔平均直径为315μm,微孔直径为3~5μm,孔隙率为84%。β-TCP/胶原支架的抗压能力为(12.29±0.88)MPa,压缩弹性模量为(116.74±27.75)MPa,与成人松质骨相似。体外大鼠骨髓间充质干细胞(m BMSCs)支架培养实验结果显示,涂覆胶原的支架具有更好的生物相容性,能有效促进m BMSCs的粘附增殖,β-TCP/胶原支架上细胞具有更高的碱性磷酸酶(ALP)活性和Collagen-I、BSP相关成骨基因的表达。研究结果显示,3D打印制备的I型胶原涂覆的β-TCP支架具有匹配的外形,良好可控的孔隙率,对m BMSCs有良好成骨活性,为骨组织支架在临床上应用提供新的技术。
Fabricating individualized tissue engineering scaffolds based on the three-dimensional shape of patient bone defects is required for the successful clinical application of bone tissue engineering. In this work,type I collagen gel was coated on individuated β-TCP scaffolds through 3 D printing technique for bone repair. By comparing the influence of filling angle of 0/90°,0/60°,0/45° and concentration of coated collagen of0. 10 mg/m L,0. 25 mg/m L,0. 5 mg/m L on the pore diameter,porosity and mechanical properties of β-TCP scaffold,the β-TCP/collagen scaffold with an optimal filling angle of 0/90° and an optimal concentration of0. 5 mg/m L for coated collagen was chosen,which was able to accurately reproduce the 3 D model of design by equipping itself with multilevel pore structure whose mean diameter of megalopore and micropore were 315 μm and 3 ~ 5 μm respectively with a porosity of 84%. Meanwhile,due to the compression strength of 12. 29 ±0. 88 MPa and elasticity modulus of 116. 74 ± 27. 75 MPa,it has quite a similarity with adult cancellous bone.In vitro culturing experiments of mouse bone marrow mesenchymal stem cells( m BMSCs) demonstrated that the coated collagen promoted the bioactivity and osteogenic properties,including better cytocompatibility,cell adhesion,proliferation,alkaline phosphatase( ALP) activity,and bone-related gene expressions( Collagen-I,BSP). The results showed that the collagen gel coated β-TCP scaffoldshad the matching shape,good controllableporosity and good osteogenic activity for m BMSCs through 3 D printing technique.
Fabricating individualized tissue engineering scaffolds based on the three-dimensional shape of patient bone defects is required for the successful clinical application of bone tissue engineering. In this work,type I collagen gel was coated on individuated β-TCP scaffolds through 3 D printing technique for bone repair. By comparing the influence of filling angle of 0/90°,0/60°,0/45° and concentration of coated collagen of0. 10 mg/m L,0. 25 mg/m L,0. 5 mg/m L on the pore diameter,porosity and mechanical properties of β-TCP scaffold,the β-TCP/collagen scaffold with an optimal filling angle of 0/90° and an optimal concentration of0. 5 mg/m L for coated collagen was chosen,which was able to accurately reproduce the 3 D model of design by equipping itself with multilevel pore structure whose mean diameter of megalopore and micropore were 315 μm and 3 ~ 5 μm respectively with a porosity of 84%. Meanwhile,due to the compression strength of 12. 29 ±0. 88 MPa and elasticity modulus of 116. 74 ± 27. 75 MPa,it has quite a similarity with adult cancellous bone.In vitro culturing experiments of mouse bone marrow mesenchymal stem cells( m BMSCs) demonstrated that the coated collagen promoted the bioactivity and osteogenic properties,including better cytocompatibility,cell adhesion,proliferation,alkaline phosphatase( ALP) activity,and bone-related gene expressions( Collagen-I,BSP). The results showed that the collagen gel coated β-TCP scaffoldshad the matching shape,good controllableporosity and good osteogenic activity for m BMSCs through 3 D printing technique.
引文
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[2]辛晨,齐鑫,朱敏,等.三维打印羟基磷灰石晶须增强复合骨修复支架[J].无机材料学报,2017,32(8):837-844.
[3]Dorozhkin SV.Bioceramics of calcium orthophosphates[J].Biomaterials,2010,31(7):1465-1485.
[4]Samavedi S,Whittington AR,Goldstein AS.Calcium phosphate ceramics in bone tissue engineering:a review of properties and their influence on cell behavior[J].Acta Biomaterialia,2013,9(9):8037-8045.
[5]Mastrogiacomo M,Scaglione S,Martinetti R,et al.Role of scaffold internal structure on in vivo bone formation in macroporous calcium phosphate bioceramics[J].Biomaterials,2006,27(17):3230-3237.
[6]Kang Y,Kim S,Bishop J,et al.The osteogenic differentiation of human bone marrow MSCs on HUVEC-derived ECM andβ-TCP scaffold[J].Biomaterials,2012,33(29):6998-7007.
[7]Bose S,Vahabzadeh S,Bandyopadhyay A.Bone tissue engineering using 3D printing[J].Materials Today,2013,16(12):496-504.
[8]Leong KF,Cheah CM,Chua CK.Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs[J].Biomaterials,2003,24(13):2363-2378.
[9]赵东亮,赵移畛,金琰.骨科三维可视化医患沟通平台构建与应用[J].国际生物医学工程杂志,2015,38(2):111-113.
[10]Uchida T,Ikeda S,Oura H,et al.Development of biodegradable scaffolds based on patient-specific arterial configuration[J].Journal of Biotechnology,2008,133(2):213-218.
[11]Carrel JP,Wiskott A,Moussa M,et al.A 3D printed TCP/HA structure as a new osteoconductive scaffold for vertical bone augmentation[J].Dental Materials,2016,27(1):55-62.
[12]袁梅娟,徐铭恩,胡金夫,等.基于股骨三维重建及计算机辅助低温沉积制造研究[J].生物医学工程学杂志,2012,29(3):156-159.
[13]许国军,朱咸兵,李勃,等.3D打印β-TCP/HA/PLA骨移植支架材料的研究[J].国际生物医学工程杂志,2016,39(4):212-217.
[14]王庆利,何丹,郑晓冬,等.有机溶剂对细胞的毒害及细胞的耐受性机制[J].微生物学通报,2002,29(4):81-85.
[15]Rasperini G,Pilipchuk SP,Flanagan CL,et al.3D-printed bioresorbable scaffold for periodontal Repair[J].Journal of Dental Research,2015,94(9):153S.
[16]Oprita EI,Moldovan L,Craciunescu O,et al.A bioactive collagen-βtricalcium phosphate scaffold for tissue engineering[J].Open Life Sciences,2006,1(1):61-72.
[17]Matsuno T,Nakamura T,Kuremoto K,et al.Development of beta-tricalcium phosphate/collagen sponge composite for bone regeneration[J].Dental Materials Journal,2006,25(1):138-144.
[18]Xu Zhijun,Yang Yang,Zhao Weilong,et al.Molecular mechanisms for intrafibrillar collagen mineralization in skeletal tissues[J].Biomaterials,2015,39(1):59-66.
[19]Ju YP,Shim JH,Choi SA,et al.3D printing technology to control BMP-2 and VEGF delivery spatially and temporally to promote large-volume bone regeneration[J].Journal of Materials Chemistry B,2015,3(27):5415-5425.
[20]Wang Ling,Xu Ming-en,Luo Li,et al.Iterative feedback bioprinting-derived cell-laden hydrogel scaffolds with optimal geometrical fidelity and cellular controllability[J].Scientific Reports,2018,8:2802-2814.
[21]李艳蕾,徐铭恩,王秋君,等.血管组织工程支架的计算机辅助低温沉积制造研究[J].生物医学工程学杂志,2011,28(4):804-809.
[22]杨丽,张荣华,谢厚杰,等.建立大鼠骨髓间充质干细胞稳定分离培养体系与鉴定[J].中国组织工程研究,2009,13(6):1064-1068.
[23]Schmittgen TD,Livak KJ.Analyzing real-time PCR data by the comparative C(T)method[J].Nature Protocols,2008,3(6):1101-1108.
[24]马艺娟.三维可控微结构多孔生物活性陶瓷的制备及其性能研究[D].华南理工大学,2015.
[25]Zhao Yingdi,Tian Ke,Zhou Yan,et al.A combinatorial variation in surface chemistry and pore size of three-dimensional porous poly(ε-caprolactone)scaffolds modulates the behaviors of mesenchymal stem cells[J].Materials Science&Engineering C,2016,59(1):193-202.
[26]王迎军,杜昶,赵娜如,等.仿生人工骨修复材料研究[J].华南理工大学学报(自然科学版),2012,40(10):51-58.
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[28]Pilliar RM,Filiaggi MJ,Wells JD,et al.Porous calcium polyphosphate scaffolds for bone substitute applications-In vitro characterization.[J].Biomaterials,2001,22(9):963-972.
[29]陈大福,田伟,行勇刚,等.胶原支架增强自固化磷酸钙骨水泥的力学及成骨性能研究[J].中国矫形外科杂志,2006,14(18):1410-1412.
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[31]Zou Fen,Zhao Naru,Fu Xiaoling,et al.Enhanced osteogenic differentiation and biomineralization in mouse mesenchymal stromal cells on aβ-TCP robocast scaffold modified with collagen nanofibers[J].Rsc Advances,2016,6(28):23588-23598.