纯钛表面TiO_2纳米管结合Ⅰ型胶原促进成骨细胞黏附和骨结合
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摘要
背景:有研究报道,单纯纳米管改性的钛种植体表面可促进体外成骨细胞的黏附、增殖、分化,胶原涂层也具有增强成骨细胞黏附和体内骨结合的作用。目的:在纯钛表面制备TiO_2纳米管结构表层并结合Ⅰ型胶原,观察改性后的纯钛表面对体外成骨细胞黏附和体内骨结合的影响。方法:采用阳极氧化处理在纯钛表面制备TiO_2纳米管涂层,并结合Ⅰ型胶原,采用扫描电镜观察、接触角测定对纯钛(纯钛组)、TiO_2纳米管(纳米管组)和TiO_2纳米管结合Ⅰ型胶原(胶原/纳米管组)3种表面进行表征。将MC3T3-E1小鼠前成骨细胞株分别接种于3组材料上,培养4h后,利用扫描电镜观察细胞黏附形态,倒置荧光显微镜观察细胞黏附计数,激光共聚焦显微镜观察细胞骨架与黏附斑蛋白表达,实时荧光定量PCR分析黏附斑蛋白与护骨素基因表达。将3组试样分别植入SD大鼠(中国医学科学院放射医学研究所实验动物中心提供)胫骨内,4周后取胫骨标本,分别进行生物力学推出实验与组织学观察。结果与结论:①扫描电镜显示,纯钛表面仅可见机械打磨的划痕;纳米管组表面形成了可控、均一的垂直排列的纳米管状结构,管径约70 nm;胶原/纳米管组表面纳米管结构周围可见胶原附着,封闭了部分管口;②胶原/纳米管组的亲水性大于纳米管组、纯钛组(P<0.05);③与纯钛组、纳米管组比较,胶原/纳米管组成骨细胞黏附数目增加(P <0.05),细胞伸展完全,骨架结构明显,黏附斑蛋白表达强度高(P <0.05),黏附斑蛋白和护骨素基因表达升高(P <0.05);④动物体内植入实验显示,胶原/纳米管组最大推出力显著高于纯钛组、纳米管组(P<0.05);苏木精-伊红染色显示,纯钛组种植体周围骨质较少,可见较多的纤维结缔组织;纳米管组种植体周围新生骨较多,纤维结缔组织较少;胶原/纳米管组种植体周围形成结构致密的新生骨,仅残留菲薄的纤维结缔组织;⑤结果表明,纯钛表面TiO_2纳米管结合Ⅰ型胶原的新型改性方法,可有效增强体外成骨细胞的黏附,促进体内骨结合。
BACKGROUND: Simple nanotube surface modification of titanium implant has been shown to promote adhesion, proliferation and differentiation of osteoblasts. Collagen coating can promote osteoblast adhesion and osseointegration in vivo. OBJECTⅠVE: To observe the effects of type collagen combined titanium dioxide nanotube composite coating modified titanium surface on osteoblast adhesion in vitro and osseointegration in vivo. METHODS: The titanium dioxide nanotube was fabricated on the pure titanium surface, then type Ⅰ collagen was combined with the nanotube structure to form composite coating. Scanning electron microscope observation was used to characterize the surface topography of the pure titanium, titanium dioxide nanotube and type Ⅰ collagen combined titanium dioxide nanotube surfaces. Contact angle test was employed to evaluate the hydrophilicity of different samples. MC3 T3-E1 murine preosteoblasts were seeded on the three kinds of materials for 4 hours. Cell adhesion morphology was examined by scanning electron microscope. Adherent cell counting was detected under inverted fluorescence microscope. Expression of actin cytoskeleton and vinculin was observed under laser scanning confocal microscope. The gene expression of vinculin and osteoprotegerin mRNA was detected by real-time quantitative PCR. The three kinds of samples were implanted into the tibia of Sprague-Dawley rats(provided by Laboratory Animal Center, Ⅰnstitute of Radiation Medicine, Chinese Academy of Medical Sciences), and tibia samples were removed after 4 weeks of implantation for biological push-out test and histological observation. RESULTS AND CONCLUSⅠON:(1) Scanning electron microscope: There was mechanical scratch on the pure titanium surface. There was controllable, and uniform vertical arrangement of nanotubular structures with a diameter of approximately 70 nm on the titanium dioxide nanotube surface, and collagen adhered surrounding the nanotubular structures on the type Ⅰ collagen combined titanium dioxide nanotube substrate, and partial tubule orifices were closed.(2) The hydrophicility of type Ⅰ collagen combined titanium dioxide nanotube was significantly larger than those of the other two materials(P < 0.05).(3) Compared with the pure titanium and titanium dioxide nanotube surfaces, the type Ⅰ collagen combined titanium dioxide nanotube substrate displayed increased adherent cell number, much well-organized cytoskeleton, enhanced immunofluorescence intensity of vinculin protein staining, and increased expression levels of vinculin and osteoprotegerin mRNA levels(all P < 0.05).(4) Ⅰn vivo test revealed that the maximum push-out force in the type Ⅰ collagen combined titanium dioxide nanotube group was significantly higher than that in the pure titanium and titanium dioxide nanotube groups(P < 0.05). Hematoxylin-eosin staining results showed that there were few bones, but many fibrous connective tissue surrounding the implant in the pure titanium group; there were more newly-born bones, and less fibrous connective tissue surrounding the implant in the titanium dioxide nanotube group; there were dense newly-born bones, and few thin fibrous connective tissue surrounding the implant in the type Ⅰ collagen combined titanium dioxide nanotube group.(5) These results indicate that type Ⅰ collagen combined titanium dioxide nanotube surface can facilitate osteoblast cell adhesion and promote osseointegration in vivo.
BACKGROUND: Simple nanotube surface modification of titanium implant has been shown to promote adhesion, proliferation and differentiation of osteoblasts. Collagen coating can promote osteoblast adhesion and osseointegration in vivo. OBJECTⅠVE: To observe the effects of type collagen combined titanium dioxide nanotube composite coating modified titanium surface on osteoblast adhesion in vitro and osseointegration in vivo. METHODS: The titanium dioxide nanotube was fabricated on the pure titanium surface, then type Ⅰ collagen was combined with the nanotube structure to form composite coating. Scanning electron microscope observation was used to characterize the surface topography of the pure titanium, titanium dioxide nanotube and type Ⅰ collagen combined titanium dioxide nanotube surfaces. Contact angle test was employed to evaluate the hydrophilicity of different samples. MC3 T3-E1 murine preosteoblasts were seeded on the three kinds of materials for 4 hours. Cell adhesion morphology was examined by scanning electron microscope. Adherent cell counting was detected under inverted fluorescence microscope. Expression of actin cytoskeleton and vinculin was observed under laser scanning confocal microscope. The gene expression of vinculin and osteoprotegerin mRNA was detected by real-time quantitative PCR. The three kinds of samples were implanted into the tibia of Sprague-Dawley rats(provided by Laboratory Animal Center, Ⅰnstitute of Radiation Medicine, Chinese Academy of Medical Sciences), and tibia samples were removed after 4 weeks of implantation for biological push-out test and histological observation. RESULTS AND CONCLUSⅠON:(1) Scanning electron microscope: There was mechanical scratch on the pure titanium surface. There was controllable, and uniform vertical arrangement of nanotubular structures with a diameter of approximately 70 nm on the titanium dioxide nanotube surface, and collagen adhered surrounding the nanotubular structures on the type Ⅰ collagen combined titanium dioxide nanotube substrate, and partial tubule orifices were closed.(2) The hydrophicility of type Ⅰ collagen combined titanium dioxide nanotube was significantly larger than those of the other two materials(P < 0.05).(3) Compared with the pure titanium and titanium dioxide nanotube surfaces, the type Ⅰ collagen combined titanium dioxide nanotube substrate displayed increased adherent cell number, much well-organized cytoskeleton, enhanced immunofluorescence intensity of vinculin protein staining, and increased expression levels of vinculin and osteoprotegerin mRNA levels(all P < 0.05).(4) Ⅰn vivo test revealed that the maximum push-out force in the type Ⅰ collagen combined titanium dioxide nanotube group was significantly higher than that in the pure titanium and titanium dioxide nanotube groups(P < 0.05). Hematoxylin-eosin staining results showed that there were few bones, but many fibrous connective tissue surrounding the implant in the pure titanium group; there were more newly-born bones, and less fibrous connective tissue surrounding the implant in the titanium dioxide nanotube group; there were dense newly-born bones, and few thin fibrous connective tissue surrounding the implant in the type Ⅰ collagen combined titanium dioxide nanotube group.(5) These results indicate that type Ⅰ collagen combined titanium dioxide nanotube surface can facilitate osteoblast cell adhesion and promote osseointegration in vivo.
引文
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[14]Morra M,Cassinelli C,Meda L,et al.Surface analysis and effects on interfacial bone microhardness of collagen-coated titanium implants:A rabbit model.Int J Oral Maxillofac Implants.2005;20(1):23-30.
[15]Sartori M,Giavaresi G,Parrilli A,et al.Collagen type i coating stimulates bone regeneration and osteointegration of titanium implants in the osteopenic rat.Int Orthop.2015;39(10):2041-2052.
[16]Liu P,Hao Y,Zhao Y,et al.Surface modification of titanium substrates for enhanced osteogenetic and antibacterial properties.Colloids Surf B Biointerfaces.2017;160:110-116.
[17]Tang H,Li Y,Ma J,et al.Improvement of biological and mechanical properties of titanium surface by anodic oxidation.Biomed Mater Eng.2016;27(5):485-494.
[18]Song Y,Ma A,Ning J,et al.Loading icariin on titanium surfaces by phase-transited lysozyme priming and layer-by-layer self-assembly of hyaluronic acid/chitosan to improve surface osteogenesis ability.Int J Nanomedicine.2018;13 6751-6767.
[19]Geissler U,Hempel U,Wolf C,et al.Collagen type i-coating of ti6al4v promotes adhesion of osteoblasts.J Biomed Mater Res.2000;51(4):752-760.
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[22]Itoh S,Kikuchi M,Takakuda K,et al.The biocompatibility and osteoconductive activity of a novel hydroxyapatite/collagen composite biomaterial,and its function as a carrier of rhbmp-2.J Biomed Mater Res.2001;54(3):445-453.
[23]Andres JL,DeFalcis D,Noda M,et al.Binding of two growth factor families to separate domains of the proteoglycan betaglycan.J Biol Chem.1992;267(9):5927-5930.
[24]Brighton CT,Albelda SM.Identification of integrin cell-substratum adhesion receptors on cultured rat bone cells.J Orthop Res.1992;10(6):766-773.
[25]Ruoslahti E,Pierschbacher MD.New perspectives in cell adhesion:Rgd and integrins.Science.1987;238(4826):491-497.
[26]Salasznyk RM,Williams WA,Boskey A,et al.Adhesion to vitronectin and collagen i promotes osteogenic differentiation of human mesenchymal stem cells.J Biomed Biotechnol.2004;2004(1):24-34.
[27]Rammelt S,Schulze E,Bernhardt R,et al.Coating of titanium implants with type-i collagen.J Orthop Res.2004;22(5):1025-1034.
[28]Rammelt S,Schulze E,Witt M,et al.Collagen type i increases bone remodelling around hydroxyapatite implants in the rat tibia.Cells Tissues Organs.2004;178(3):146-157.
[29]Mathews S,Bhonde R,Gupta PK,et al.A novel tripolymer coating demonstrating the synergistic effect of chitosan,collagen type 1 and hyaluronic acid on osteogenic differentiation of human bone marrow derived mesenchymal stem cells.Biochem Biophys Res Commun.2011;414(1):270-276.
[30]Chen S,Guo Y,Liu R,et al.Tuning surface properties of bone biomaterials to manipulate osteoblastic cell adhesion and the signaling pathways for the enhancement of early osseointegration.Colloids Surf B Biointerfaces.2018;164:58-69.
[31]Bays JL,DeMali KA.Vinculin in cell-cell and cell-matrix adhesions.Cell Mol Life Sci.2017;74(16):2999-3009.
[32]Zhao J,Watanabe T,Bhawal UK,et al.Transcriptome analysis of beta-tcp implanted in dog mandible.Bone.2011;48(4):864-877.
[33]Zhan X,Zhang C,Dissanayaka WL,et al.Storage media enhance osteoclastogenic potential of human periodontal ligament cells via rankl-independent signaling.Dent Traumatol.2013;29(1):59-65.
[34]Sul YT.Electrochemical growth behavior,surface properties,and enhanced in vivo bone response of tio2 nanotubes on microstructured surfaces of blasted,screw-shaped titanium implants.Int J Nanomedicine.2010;5:87-100.
[35]von Wilmowsky C,Bauer S,Roedl S,et al.The diameter of anodic tio2 nanotubes affects bone formation and correlates with the bone morphogenetic protein-2 expression in vivo.Clin Oral Implants Res.2012;23(3):359-366.
[36]Popat KC,Leoni L,Grimes CA,et al.Influence of engineered titania nanotubular surfaces on bone cells.Biomaterials.2007;28(21):3188-3197.
[2]Shi Q,Qian Z,Liu D,et al.Surface modification of dental titanium implant by layer-by-layer electrostatic self-assembly.Front Physiol.2017;8:574.
[3]Decuzzi P,Ferrari M.Modulating cellular adhesion through nanotopography.Biomaterials.2010;31(1):173-179.
[4]Meswania IM,Bousdras VA,Ahir SP,et al.A novel closed-loop electromechanical stimulator to enhance osseointegration with immediate loading of dental implant restorations.Proc Inst Mech Eng H.2010;224(10):1221-1232.
[5]李莺,李长义.钛种植体表面改性策略及对骨整合的影响[J].中国组织工程研究,2013,17(29):5395-5402.
[6]Mendonca G,Mendonca DB,Aragao FJ,et al.Advancing dental implant surface technology--from micron-to nanotopography.Biomaterials.2008;29(28):3822-3835.
[7]Li B,Li Y,Li J,et al.Influence of nanostructures on the biological properties of ti implants after anodic oxidation.J Mater Sci Mater Med.2014;25(1):199-205.
[8]Minagar S,Wang J,Berndt CC,et al.Cell response of anodized nanotubes on titanium and titanium alloys.J Biomed Mater Res A.2013;101(9):2726-2739.
[9]Li Y,Li B,Fu X,et al.Anodic oxidation modification improve bioactivity and biocompatibility of titanium implant surface.JHard Tissue Biol.2013;22(3):351-358.
[10]Avila G,Misch K,Galindo-Moreno P,et al.Implant surface treatment using biomimetic agents.Implant Dent.2009;18(1):17-26.
[11]川叶,马敏先,张弢,等.Ⅰ型胶原修饰纯钛片促进人脂肪间充质干细胞增殖[J].中国组织工程研究,2014,18(25):4032-4037.
[12]Ricard-Blum S,Ruggiero F.The collagen superfamily:From the extracellular matrix to the cell membrane.Pathol Biol(Paris).2005;53(7):430-442.
[13]李赛娜,康跻耀,高建萍,等.胶原涂层对3d打印种植体表面生物相容性的影响[J].中国组织工程研究,2017,21(10):1558-1564.
[14]Morra M,Cassinelli C,Meda L,et al.Surface analysis and effects on interfacial bone microhardness of collagen-coated titanium implants:A rabbit model.Int J Oral Maxillofac Implants.2005;20(1):23-30.
[15]Sartori M,Giavaresi G,Parrilli A,et al.Collagen type i coating stimulates bone regeneration and osteointegration of titanium implants in the osteopenic rat.Int Orthop.2015;39(10):2041-2052.
[16]Liu P,Hao Y,Zhao Y,et al.Surface modification of titanium substrates for enhanced osteogenetic and antibacterial properties.Colloids Surf B Biointerfaces.2017;160:110-116.
[17]Tang H,Li Y,Ma J,et al.Improvement of biological and mechanical properties of titanium surface by anodic oxidation.Biomed Mater Eng.2016;27(5):485-494.
[18]Song Y,Ma A,Ning J,et al.Loading icariin on titanium surfaces by phase-transited lysozyme priming and layer-by-layer self-assembly of hyaluronic acid/chitosan to improve surface osteogenesis ability.Int J Nanomedicine.2018;13 6751-6767.
[19]Geissler U,Hempel U,Wolf C,et al.Collagen type i-coating of ti6al4v promotes adhesion of osteoblasts.J Biomed Mater Res.2000;51(4):752-760.
[20]Roehlecke C,Witt M,Kasper M,et al.Synergistic effect of titanium alloy and collagen type i on cell adhesion,proliferation and differentiation of osteoblast-like cells.Cells Tissues Organs.2001;168(3):178-187.
[21]Caiazza S,Colangelo P,Bedini R,et al.Evaluation of guided bone regeneration in rabbit femur using collagen membranes.Implant Dent.2000;9(3):219-225.
[22]Itoh S,Kikuchi M,Takakuda K,et al.The biocompatibility and osteoconductive activity of a novel hydroxyapatite/collagen composite biomaterial,and its function as a carrier of rhbmp-2.J Biomed Mater Res.2001;54(3):445-453.
[23]Andres JL,DeFalcis D,Noda M,et al.Binding of two growth factor families to separate domains of the proteoglycan betaglycan.J Biol Chem.1992;267(9):5927-5930.
[24]Brighton CT,Albelda SM.Identification of integrin cell-substratum adhesion receptors on cultured rat bone cells.J Orthop Res.1992;10(6):766-773.
[25]Ruoslahti E,Pierschbacher MD.New perspectives in cell adhesion:Rgd and integrins.Science.1987;238(4826):491-497.
[26]Salasznyk RM,Williams WA,Boskey A,et al.Adhesion to vitronectin and collagen i promotes osteogenic differentiation of human mesenchymal stem cells.J Biomed Biotechnol.2004;2004(1):24-34.
[27]Rammelt S,Schulze E,Bernhardt R,et al.Coating of titanium implants with type-i collagen.J Orthop Res.2004;22(5):1025-1034.
[28]Rammelt S,Schulze E,Witt M,et al.Collagen type i increases bone remodelling around hydroxyapatite implants in the rat tibia.Cells Tissues Organs.2004;178(3):146-157.
[29]Mathews S,Bhonde R,Gupta PK,et al.A novel tripolymer coating demonstrating the synergistic effect of chitosan,collagen type 1 and hyaluronic acid on osteogenic differentiation of human bone marrow derived mesenchymal stem cells.Biochem Biophys Res Commun.2011;414(1):270-276.
[30]Chen S,Guo Y,Liu R,et al.Tuning surface properties of bone biomaterials to manipulate osteoblastic cell adhesion and the signaling pathways for the enhancement of early osseointegration.Colloids Surf B Biointerfaces.2018;164:58-69.
[31]Bays JL,DeMali KA.Vinculin in cell-cell and cell-matrix adhesions.Cell Mol Life Sci.2017;74(16):2999-3009.
[32]Zhao J,Watanabe T,Bhawal UK,et al.Transcriptome analysis of beta-tcp implanted in dog mandible.Bone.2011;48(4):864-877.
[33]Zhan X,Zhang C,Dissanayaka WL,et al.Storage media enhance osteoclastogenic potential of human periodontal ligament cells via rankl-independent signaling.Dent Traumatol.2013;29(1):59-65.
[34]Sul YT.Electrochemical growth behavior,surface properties,and enhanced in vivo bone response of tio2 nanotubes on microstructured surfaces of blasted,screw-shaped titanium implants.Int J Nanomedicine.2010;5:87-100.
[35]von Wilmowsky C,Bauer S,Roedl S,et al.The diameter of anodic tio2 nanotubes affects bone formation and correlates with the bone morphogenetic protein-2 expression in vivo.Clin Oral Implants Res.2012;23(3):359-366.
[36]Popat KC,Leoni L,Grimes CA,et al.Influence of engineered titania nanotubular surfaces on bone cells.Biomaterials.2007;28(21):3188-3197.