阴极弧沉积掺硅二氧化锆膜对MG63细胞相关因子的影响
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摘要
背景:采用各种表面改性方法提高医用钛及钛合金植入物的骨整合,是目前人工关节假体领域研究的重点之一。目的:在纯钛表面制备掺硅二氧化锆膜,研究其对成骨样MG63细胞相关因子的影响。方法:采用磁过滤真空阴极弧沉积技术在纯钛表面分别制备掺硅二氧化锆膜与二氧化锆膜。将成骨样MG63细胞分别接种于掺硅二氧化锆膜、二氧化锆膜及纯钛片表面,接种后第1,4,7,10天,以定量RT-PCR法检测细胞骨保护素和核因子κB受体活化因子配体基因表达情况,ELISA检测细胞分泌骨保护素和核因子κB受体活化因子配体蛋白量。结果与结论:(1)接种第1天,3组骨保护素基因及蛋白表达无差异;接种第4天,掺硅二氧化锆膜组、二氧化锆膜组骨保护素基因及蛋白表达高于纯钛组(P<0.05),掺硅二氧化锆膜组、二氧化锆膜组骨保护素基因及蛋白表达无差异;接种第7,10天,掺硅二氧化锆膜组骨保护素基因及蛋白表达高于二氧化锆膜组、纯钛组(P<0.05);(2)接种第1天,3组核因子κB受体活化因子配体基因及蛋白表达无差异;接种第4,7天,掺硅二氧化锆膜组核因子κB受体活化因子配体基因及蛋白表达低于二氧化锆膜组、纯钛组(P<0.05);接种第10天,掺硅二氧化锆膜组、二氧化锆膜组基因及蛋白表达低于纯钛组(P<0.05),掺硅二氧化锆膜组、二氧化锆膜组骨保护素基因及蛋白表达无差异;(3)结果表明,在纯钛表面阴极弧沉积掺硅二氧化锆膜,能够上调成骨样MG63细胞内骨保护素表达水平,同时下调核因子κB受体活化因子配体表达水平。
BACKGROUND: Various surface modification techniques have been utilized as an attempt to improve the osteointegration of Ti-based implants, which is a hotspot research concerning the artificial joint prosthesis. OBJECTIVE: To evaluate the effect of silicon doped zirconia film on the titanium surface on relevant factors of osteoblast-like MG63 cells. METHODS: Silicon doped zirconia and zirconia films were respectively prepared on the titanium surface by cathodic arc deposition. Osteoblast-like MG63 cells were cultured on silicon doped zirconia film, zirconia film and pure titanium, respectively. After 1, 4, 7, 10 days of incubation, samples were collected for assay. Osteoprotegerin(OPG) and receptor activator of nuclear factor κB ligand(RANKL) at m RNA and protein levels were detected using real-time PCR and ELISA, respectively. RESULTS AND CONCLUSION:(1) At 1 day of inoculation, there was no significant difference in the OPG m RNA and protein expression among the three groups. At 4 days of inoculation, the expression of OPG m RNA and protein in the silicon doped zirconia film and zirconia film groups was significantly higher than that in the pure titanium group(P < 0.05), but there was no significant difference between the former two groups. At 7 and 10 days of inoculation, the expression of OPG m RNA and protein in the silicon doped zirconia film growp was highest among the three groups(P < 0.05).(2) At 1 day of inoculation, there was no significant difference in the RANKL m RNA and protein expression among the three groups. At 4 and 7 days of inoculation, the expression of RANKL m RNA and protein in the silicon doped zirconia film group was significantly lower than that in the other two groups(P < 0.05). At 10 days of inoculation, the expression of OPG m RNA and protein in the silicon doped zirconia film and zirconia film groups was significantly higher than that in the pure titanium group(P < 0.05), but there was no significant difference between the former two groups. To conclude, silicon doped zirconia film fabricated by cathodic arc deposition can enhance the expression of OPG and reduce the expression of RANKL at the same time.
BACKGROUND: Various surface modification techniques have been utilized as an attempt to improve the osteointegration of Ti-based implants, which is a hotspot research concerning the artificial joint prosthesis. OBJECTIVE: To evaluate the effect of silicon doped zirconia film on the titanium surface on relevant factors of osteoblast-like MG63 cells. METHODS: Silicon doped zirconia and zirconia films were respectively prepared on the titanium surface by cathodic arc deposition. Osteoblast-like MG63 cells were cultured on silicon doped zirconia film, zirconia film and pure titanium, respectively. After 1, 4, 7, 10 days of incubation, samples were collected for assay. Osteoprotegerin(OPG) and receptor activator of nuclear factor κB ligand(RANKL) at m RNA and protein levels were detected using real-time PCR and ELISA, respectively. RESULTS AND CONCLUSION:(1) At 1 day of inoculation, there was no significant difference in the OPG m RNA and protein expression among the three groups. At 4 days of inoculation, the expression of OPG m RNA and protein in the silicon doped zirconia film and zirconia film groups was significantly higher than that in the pure titanium group(P < 0.05), but there was no significant difference between the former two groups. At 7 and 10 days of inoculation, the expression of OPG m RNA and protein in the silicon doped zirconia film growp was highest among the three groups(P < 0.05).(2) At 1 day of inoculation, there was no significant difference in the RANKL m RNA and protein expression among the three groups. At 4 and 7 days of inoculation, the expression of RANKL m RNA and protein in the silicon doped zirconia film group was significantly lower than that in the other two groups(P < 0.05). At 10 days of inoculation, the expression of OPG m RNA and protein in the silicon doped zirconia film and zirconia film groups was significantly higher than that in the pure titanium group(P < 0.05), but there was no significant difference between the former two groups. To conclude, silicon doped zirconia film fabricated by cathodic arc deposition can enhance the expression of OPG and reduce the expression of RANKL at the same time.
引文
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[17]Lynn AK,Duquesnay DL.Hydroxyapatite-coated Ti-6Al-4V Part 2:the effects of post-deposition heat treatment at low temperatures.Biomaterials.2002;23(9):1947-1953.
[18]Suchanek W,Yoshimura M.Processing and properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants.J Mater Res.1998;13(1):94-117.
[19]Yang YC,Chang E,Lee SY.Mechanical properties and Young's modulus of plasma-sprayed hydroxyapatite coating on Ti substrate in simulated body fluid.J Biomed Mater Res A.2003;67(3):886-899.
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[21]Carlisle EM.Silicon:a possible factor in bone calcification.Science.1970;167(916):279-280.
[22]Schwarz K,Milne DB.Growth-promoting effects of silicon in rats.Nature.1972;239(5371):333-334.
[23]Xynos ID,Edgar AJ,Buttery LD,et al.Ionic products of bioactive glass dissolution increase proliferation of human osteoblasts and induce insulin-like growth factor II m RNA expression and protein synthesis.Biochem Biophys Res Commun.2000;276(2):461-465.
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[25]Keeting PE,Oursler MJ,Wiegand KE,et al.Zeolite A increases proliferation,differentiation,and transforming growth factor beta production in normal adult human osteoblast-like cells in vitro.J Bone Miner Res.1992;7(11):1281-1289.
[26]Reffitt DM,Ogston N,Jugdaohsingh R,et al.Orthosilicic acid stimulates collagen type 1 synthesis and osteoblastic differentiation in human osteoblast-like cells in vitro.Bone.2003;32(2):127-135.
[27]Arumugam MQ,Ireland DC,Brooks RA,et al.The effect orthosilicic acid on collagen type I,alkaline phosphatase and osteocalcin m RNA expression in human bone-derived osteoblasts in vitro.Key Eng Mater.2006;309-311:121-124.
[28]Shepherd JH,Shepherd DV,Best SM.Substituted hydroxyapatites for bone repair.J Mater Sci Mater Med.2012;23(10):2335-2347.
[29]Hu HJ,Liu XY,Ding CX.Preparation and cytocompatibility of Si-incorporated nanostructured TiO 2 coating.Surf Coat Technol.2010;204(20):3265-3271.
[30]Zhang Z,Sun J,Hu H,et al.Osteoblast-like cell adhesion on porous silicon-incorporated Ti O2 coating prepared by micro-arc oxidation.J Biomed Mater Res B Appl Biomater.2011;97(2):224-234.
[31]Matsuo K,Irie N.Osteoclast-osteoblast communication.Arch Biochem Biophys.2008;473(2):201-209.
[32]Boyle WJ,Lacey DL,Timms E,et al.Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.Cell.1998;93(2):165-176.
[33]Roodman GD.Advances in bone biology:the osteoclast.Endocr Rev.1996;17(4):308-332.
[34]Suda T,Nakamura I,Jimi E,et al.Regulation of osteoclast function.J Bone Miner Res.1997;12(6):869-879.
[35]Suda T,Udagawa N,Nakamura I,et al.Modulation of osteoclast differentiation by local factors.Bone.1995;17(2 Suppl):87S-91S.
[36]Hofbauer LC,Heufelder AE.Clinical review 114:hot topic.The role of receptor activator of nuclear factor-kappa B ligand and osteoprotegerin in the pathogenesis and treatment of metabolic bone diseases.J Clin Endocrinol Metab.2000;85(7):2355-2363.
[37]Hofbauer LC,Khosla S,Dunstan CR,et al.The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption.J Bone Miner Res.2000;15(1):2-12.
[38]Hsu H,Lacey DL,Dunstan CR,et al.Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.Proc Natl Acad Sci U S A.1999;96(7):3540-3545.
[39]Kulkarni RN,Bakker AD,Everts V,et al.Inhibition of osteoclastogenesis by mechanically loaded osteocytes:involvement of MEPE.Calcif Tissue Int.2010;87(5):461-468.
[40]黎彦龙,明何,陈秉雄,等.OPG-RANKL-RANK信号系统是调节破骨细胞及骨质疏松症的重要途径[J].中国组织工程研究,2015,19(24):3894-3898.
[2]Fini M,Cigada A,Rondelli G,et al.In vitro and in vivo behaviour of Ca-and P-enriched anodized titanium.Biomaterials.1999;20(17):1587-1594.
[3]Sugino A Ohtsuki C,Tsuru K,et al.Effect of spatial design and thermal oxidation on apatite formation on Ti-15Zr-4Ta-4Nb alloy.Acta Biomater.2009;5(1):298-304.
[4]Kasuga T,Nogami M,Niinomi M,et al.Bioactive calcium phosphate invert glass-ceramic coating on beta-type Ti-29Nb-13Ta-4.6Zr alloy.Biomaterials.2003;24(2):283-290.
[5]Yan YY,Han Y.Structure and bioactivity of micro-arc oxidized zirconia films.Surf Coat Techno.2007;201(9-11):5692-5695.
[6]Wang G,Meng F,Ding C,et al.Microstructure,bioactivity and osteoblast behavior of monoclinic zirconia coating with nanostructured surface.Acta Biomater.2010;6(3):990-1000.
[7]Sollazzo V,Pezzetti F,Scarano A,et al.Zirconium oxide coating improves implant osseointegration in vivo.Dent Mater.2008;24(3):357-361.
[8]Zhang S,Sun J,Xu Y,et al.Biological behavior of osteoblast-like cells on titania and zirconia films deposited by cathodic arc deposition.Biointerphases.2012;7(1-4):60.
[9]Zhang S,Sun J,Xu Y,et al.Adhesion,proliferation and differentiation of osteoblasts on zirconia films prepared by cathodic arc deposition.Biomed Mater Eng.2013;23(5):373-385.
[10]Thian ES,Huang J,Best SM,et al.Magnetron co-sputtered silicon-containing hydroxyapatite thin films--an in vitro study.Biomaterials.2005;26(16):2947-2956.
[11]Thian ES,Huang J,Best SM,et al.Silicon-substituted hydroxyapatite thin films:effect of annealing temperature on coating stability and bioactivity.J Biomed Mater Res A.2006;78(1):121-128.
[12]Thian ES,Huang J,Best SM,et al.A new way of incorporating silicon in hydroxyapatite(Si-HA)as thin films.J Mater Sci Mater Med.2005;16(5):411-415.
[13]Liu XY,Huang AP,Ding CX,et al.Bioactivity and cytocompatibility of zirconia(Zr O2)films fabricated by cathodic arc deposition.Biomaterials.2006;27(21):3904-3911.
[14]Hench LL.Bioactive materials:the potential for tissue regeneration.J Biomed Mater Res.1998;41(4):511-518.
[15]Buser D,Nydegger T,Oxland T,et al.Interface shear strength of titanium implants with a sandblasted and acid-etched surface:a biomechanical study in the maxilla of miniature pigs.J Biomed Mater Res.1999;45(2):75-83.
[16]Jing W,Zhang M,Jin L,et al.Assessment of osteoinduction using a porous hydroxyapatite coating prepared by micro-arc oxidation on a new titanium alloy.Int J Surg.2015;24(Pt A):51-56.
[17]Lynn AK,Duquesnay DL.Hydroxyapatite-coated Ti-6Al-4V Part 2:the effects of post-deposition heat treatment at low temperatures.Biomaterials.2002;23(9):1947-1953.
[18]Suchanek W,Yoshimura M.Processing and properties of hydroxyapatite-based biomaterials for use as hard tissue replacement implants.J Mater Res.1998;13(1):94-117.
[19]Yang YC,Chang E,Lee SY.Mechanical properties and Young's modulus of plasma-sprayed hydroxyapatite coating on Ti substrate in simulated body fluid.J Biomed Mater Res A.2003;67(3):886-899.
[20]章筛林,成翔宇,纪斌.硅在生物材料领域的应用:增加材料生物活性不影响其机械性能[J].中国组织工程研究,2017,21(2):296-301.
[21]Carlisle EM.Silicon:a possible factor in bone calcification.Science.1970;167(916):279-280.
[22]Schwarz K,Milne DB.Growth-promoting effects of silicon in rats.Nature.1972;239(5371):333-334.
[23]Xynos ID,Edgar AJ,Buttery LD,et al.Ionic products of bioactive glass dissolution increase proliferation of human osteoblasts and induce insulin-like growth factor II m RNA expression and protein synthesis.Biochem Biophys Res Commun.2000;276(2):461-465.
[24]Darley WM,Volcani BE.Role of silicon in diatom metabolism.A silicon requirement for deoxyribonucleic acid synthesis in the diatom Cylindrotheca fusiformis Reimann and Lewin.Exp Cell Res.1969;58(2):334-342.
[25]Keeting PE,Oursler MJ,Wiegand KE,et al.Zeolite A increases proliferation,differentiation,and transforming growth factor beta production in normal adult human osteoblast-like cells in vitro.J Bone Miner Res.1992;7(11):1281-1289.
[26]Reffitt DM,Ogston N,Jugdaohsingh R,et al.Orthosilicic acid stimulates collagen type 1 synthesis and osteoblastic differentiation in human osteoblast-like cells in vitro.Bone.2003;32(2):127-135.
[27]Arumugam MQ,Ireland DC,Brooks RA,et al.The effect orthosilicic acid on collagen type I,alkaline phosphatase and osteocalcin m RNA expression in human bone-derived osteoblasts in vitro.Key Eng Mater.2006;309-311:121-124.
[28]Shepherd JH,Shepherd DV,Best SM.Substituted hydroxyapatites for bone repair.J Mater Sci Mater Med.2012;23(10):2335-2347.
[29]Hu HJ,Liu XY,Ding CX.Preparation and cytocompatibility of Si-incorporated nanostructured TiO 2 coating.Surf Coat Technol.2010;204(20):3265-3271.
[30]Zhang Z,Sun J,Hu H,et al.Osteoblast-like cell adhesion on porous silicon-incorporated Ti O2 coating prepared by micro-arc oxidation.J Biomed Mater Res B Appl Biomater.2011;97(2):224-234.
[31]Matsuo K,Irie N.Osteoclast-osteoblast communication.Arch Biochem Biophys.2008;473(2):201-209.
[32]Boyle WJ,Lacey DL,Timms E,et al.Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.Cell.1998;93(2):165-176.
[33]Roodman GD.Advances in bone biology:the osteoclast.Endocr Rev.1996;17(4):308-332.
[34]Suda T,Nakamura I,Jimi E,et al.Regulation of osteoclast function.J Bone Miner Res.1997;12(6):869-879.
[35]Suda T,Udagawa N,Nakamura I,et al.Modulation of osteoclast differentiation by local factors.Bone.1995;17(2 Suppl):87S-91S.
[36]Hofbauer LC,Heufelder AE.Clinical review 114:hot topic.The role of receptor activator of nuclear factor-kappa B ligand and osteoprotegerin in the pathogenesis and treatment of metabolic bone diseases.J Clin Endocrinol Metab.2000;85(7):2355-2363.
[37]Hofbauer LC,Khosla S,Dunstan CR,et al.The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption.J Bone Miner Res.2000;15(1):2-12.
[38]Hsu H,Lacey DL,Dunstan CR,et al.Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.Proc Natl Acad Sci U S A.1999;96(7):3540-3545.
[39]Kulkarni RN,Bakker AD,Everts V,et al.Inhibition of osteoclastogenesis by mechanically loaded osteocytes:involvement of MEPE.Calcif Tissue Int.2010;87(5):461-468.
[40]黎彦龙,明何,陈秉雄,等.OPG-RANKL-RANK信号系统是调节破骨细胞及骨质疏松症的重要途径[J].中国组织工程研究,2015,19(24):3894-3898.