3D打印含铜钴基合金与PDLF细胞生物相容性初探
|
摘要
目的在现有医用钴基合金材料中加入铜元素,使用选择性激光熔化技术(selectedlasermelting,SLM)制得一种新型含铜钴基合金,并检测其生物相容性。方法 以SLM技术制得的新型含铜钴基合金作为实验组,以SLM技术制得的不含铜元素的传统医用钴基合金作为对照组,将实验材料与人牙周膜成纤维细胞(periodontalliga-mentfibroblast,PDLF)进行共培养后,使用细胞增殖活力测定(CCK-8法)及实时无标记细胞分析技术(Real Time Cellular Analysis,RTCA)-细胞毒性测定、鬼笔环肽细胞染色、细胞流式实验,来检测材料对细胞增殖、凋亡、粘附的影响,从而评价新型含铜钴基合金的生物相容性。结果与新型含铜钴基合金直接接触共培养后的PDLF细胞,其细胞相对增长率大于99%,属于零级细胞毒性。与新型含铜钴基合金间接接触共培养后的PDLF细胞,其整个生长增殖过程并未受到新型合金的影响。与新型含铜钴基合金共培养1天后,所有分组PDLF细胞凋亡率均小于1%,共培养3天后,所有分组细胞凋亡率均小于5%,实验组与对照组无显著性差异,且整体略低于对照组。鬼笔环肽染色后观察到细胞粘附正常,实验组与对照组之间无明显差异。结论经SLM技术制作的含铜钴基合金在保证拥有良好抗菌性能的同时,不会对细胞产生细胞毒性,即具有良好的细胞相容性。
The work aims to add Cu in existing medical CoCrW alloy to prepare a new Cu-bearing CoCrW alloy by selected laser melting(SLM) and test the biocompatibility. The new Cu-bearing CoCrW alloy prepared by SLM was used as the experimental group and the Cu-free CoCrW alloy prepared by SLM was regarded as the control group. After the experimental materials and periodontalliga-ment fibroblast were co-cultured, CCK-8 and Real Time Cellular Analysis(RTCA, cytotoxicity assay,Phalloidin cell staining and flow cytometry) were adopted to test the effects of materials on proliferation, apoptosis and adhesion of cells so as to evaluate the biocompatibility of new Cu-bearing CoCrW alloy. The RGR of PDLF cells directly contacting the new Cu-bearing CoCrW alloys after co-cultured was more than 99% and the cells were classified as zero-grade cytotoxicity. The growth of PDLF cells indirectly contacting the new Cu-bearing CoCrW alloys after co-cultured was not affected by the new alloy. The apoptosis rate of PDLF was less than 1% after co-cultured with CoCrW-Cu alloy for 1 day and less than 5% after 3 days of co-culture. There was no significant difference between the experimental group and the control group and the apoptosis rate of experimental group was lower than that of control group. Through the Phalloidin cell staining, the cell adhesion was normal and there was no significant difference between the experimental group and the control group. The Cu-bearing CoCrW-Cu alloy fabricated by SLM technology has good antibacterial performance and no cytotoxicity to cells, that is, good cell compatibility.
The work aims to add Cu in existing medical CoCrW alloy to prepare a new Cu-bearing CoCrW alloy by selected laser melting(SLM) and test the biocompatibility. The new Cu-bearing CoCrW alloy prepared by SLM was used as the experimental group and the Cu-free CoCrW alloy prepared by SLM was regarded as the control group. After the experimental materials and periodontalliga-ment fibroblast were co-cultured, CCK-8 and Real Time Cellular Analysis(RTCA, cytotoxicity assay,Phalloidin cell staining and flow cytometry) were adopted to test the effects of materials on proliferation, apoptosis and adhesion of cells so as to evaluate the biocompatibility of new Cu-bearing CoCrW alloy. The RGR of PDLF cells directly contacting the new Cu-bearing CoCrW alloys after co-cultured was more than 99% and the cells were classified as zero-grade cytotoxicity. The growth of PDLF cells indirectly contacting the new Cu-bearing CoCrW alloys after co-cultured was not affected by the new alloy. The apoptosis rate of PDLF was less than 1% after co-cultured with CoCrW-Cu alloy for 1 day and less than 5% after 3 days of co-culture. There was no significant difference between the experimental group and the control group and the apoptosis rate of experimental group was lower than that of control group. Through the Phalloidin cell staining, the cell adhesion was normal and there was no significant difference between the experimental group and the control group. The Cu-bearing CoCrW-Cu alloy fabricated by SLM technology has good antibacterial performance and no cytotoxicity to cells, that is, good cell compatibility.
引文
[1]SIMEL A S,SOYLU E H,?DE S,et al.Annealing of Co-Cr dental alloy:Effects on nanostructure and rockwell hardness[J].The journal of advanced prosthodontics,2013,5(4):471.
[2]TAKEDA O,TOYAMA T,WATANABE K,et al.Ameliorating effects of Juzentaihoto on restraint stress and P.gingivalis-induced alveolar bone loss[J].Archives of oral biology,2014,59(11):1130-1138.
[3]KONG L,QI X,HUANG S,et al.Theaflavins inhibit pathogenic properties of P.gingivalis and MMPs production in P.gingivalis-stimulated human gingival fibroblasts[J].Archives of oral biology,2015,60(1):12-22.
[4]HARDES J,AHRENS H,GEBERT C,et al.Lack of toxicological side-effects in silver-coated megaprostheses in humans[J].Biomaterials,2007,28(18):2869-2875.
[5]REITEMEIER B,HANSEL K,WALTER M H.Effect of posterior crown margin placement on gingival health[J].Journal of prosthetic dentistry,2002,87(2):167-172.
[6]TAYLOR R,MARYAN C,VERRAN J.Retention of oral microorganisms on cobalt-chromium alloy and dental acrylic resin with different surface finishes[J].Journal of prosthetic dentistry,1998,80(5):592-597.
[7]DYLINA T J.Contour determination for ovate pontics[J].Prosthet dent,1999,82(2):136-142.
[8]DRAKE C W,BECK J D.The oral status of elderly removable par-tial denture wearers[J].Oral rehabil,1993,20(1):53-60.
[9]YADROITSEV I,BERTRAND P,SMUROV I.Parametric analysis of the selective laser melting process[J].Applied surface science,2007,253(19):8064-8069.
[10]ANTANASOVA M,KOCJAN A,KOVA?J,et al.Influence of thermo-mechanical cycling on porcelain bonding to cobalt-chromium and titanium dental alloys fabricated by casting,milling,and selective laser melting[J].Journal of prosthodontic research,2018,62(2):184-194.
[11]STRUB J R,REKOW E D,WITKOWSKI S.Computer-aided design and fabrication of dental restorations:Current systems and future possibilities[J].Journal of the American dental association,2006,137(9):1289-1296.
[12]GALHANO G,PELLIZZER E,MAZARO J.Optical impression systems for CAD-CAM restorations[J].Journal of craniofacial surgery,2012,23(6):575-579.
[13]SCHEIBER I F,MERCER J F B,DRINGEN R.Metabolism and functions of copper in brain[J].Progress in neurobiology,2014,116(2):33-57.
[14]IBRAHIM M,WANG F,LOU M M,et al.Copper as an antibacterial agent for human pathogenic multidrug resistant burkholderiacepacia complex bacteria[J].Journal of bioscience and bioengineering,2011,112(6):570-576.
[15]RENSING C,GRASS G.Escherichia coli mechanisms of copper homeostasis in a changing environment[J].FEMSmicrobiology reviews,2003,27(2-3):197-213.
[16]GUO S,LU Y,WU S.Preliminary study on the corrosion resistance,antibacterial activity and cytotoxicity of selective-laser-melted Ti6Al4V-x Cu alloys[J].Materials science and engineering:C,2017,72(3):631-640.
[17]REN L,NAN L,YANG K.Study of copper precipitation behavior in a Cu-bearing austenitic antibacterial stainless steel[J].Mater design,2011,32(4):2374-2379.
[18]REN L,YANG K.Bio-functional design for metal implants,a new concept for development of metallic biomaterials[J].Journal of materials science&technology,2013,29(11):1005-1110.
[19]REN L,YANG K,GUO L,et al.Preliminary study of anti-infective function of a copper-bearing stainless steel[J].Materials science and engineering:C,2012,32(5):1204-1209.
[20]CHAI H W,GUO L,WANG X T,et al.Antibacterial effect of 317L stainless steel contained copper in prevention of implant-related infection in vitro and in vivo[J].Journal of materials science materials in medicine,2011,22(11):2525-2535.
[21]ZHANG D,REN L,ZHANG Y,et al.Antibacterial activity against porphyromonas gingivalis and biological characteristics of antibacterial stainless steel[J].Colloids and surfaces B:Biointerfaces,105(3):51-57.
[22]REN L,XU L,FENG J W,et al.In vitro study of role of trace amount of Cu release from Cu-bearing stainless steel targeting for reduction of in-stent restenosis[J].Journal of materials science materials in medicine,2012,23(5):1235-1245.
[23]LIU R,MEMARZADEH K,CHANG B,et al.Antibacterial effect of copper-bearing titanium alloy(Ti-Cu)against streptococcus mutans and porphyromonas gingivalis[J].Scientific reports,2016,6(1):29985.
[24]LIU R,TANG Y,ZENG L,et al.In vitro and in vivo studies of anti-bacterial copper-bearing titanium alloy for dental application[J].Dental materials,2018,34(8):1112-1126.
[25]REN L,MEMARZADEH K,ZHANG S,et al.A novel coping metal material CoCrCu alloy fabricated by selective laser melting with antimicrobial and antibiofilm properties[J].Materials science and engineering:C,2016,67(10):461-467.
[26]FLüGGE T V,SCHLAGER S,NELSON K,et al.Precision of intraoral digital dental impressions with iTero and extraoral digitization with the i Tero and a model scanner[J].American journal of orthodontics&dentofacial,2013,144(3):471-478.
[27]DA C J,PELOGIA F,HAGEDORN B,et al.Evaluation of different methods of optical impression making on the marginal gap of onlays created with CEREC 3D[J].Operative dentistry,2010,35(3):324-329.
[28]WILLIAMS R J,BIBB R,EGGBEER D,et al.Use of CAD/CAM technology to fabricate a removable partial denture framework[J].Journal of prosthetic dentistry,2006,96(2):96-99.
[29]BAE E J,KIM J H,KIM W C,et al.Bond and fracture strength of metal-ceramic restorations formed by selective laser sintering[J].The journal of advanced prosthodontics,2014,6(4):266-271.
[30]LIU Y,WANG Z,GAO B,et al.Evaluation of mechanical properties and porcelain bonded strength of nickel-chromium dental alloy fabricated by laser rapid forming[J].Lasers in medical science,2010,25(6):799-804.
[31]CRAIG R,HANKS C.Reaction of fibroblasts to various dental casting alloys[J].Journal of oral pathology and medicine,1988,17(7):341-347.
[32]BERARDEN L J,COOKE F W.Growth inhibition of cultured fibroblasts by cobalt and nickel[J].Journal of biomedical materials research,1980,14(3):289-309.
[33]KAWAHARA H,YAMAGAMI A,NAKAMURA M.Biological testing of dental materials by means of tissue culture[J].Int Dent J,1968,18:443-467.
[34]BAGHERI B,AKBARI N,TABIBAN S,et al.Serum level of copper in patients with coronary artery disease[J].American journal of the medical sciences,2015,56(1):39-42.
[35]SHEKARKHAR S,FOROUGHI M,MOATAMEDI M,et al.The association of serum parathyroid hormone and severity of coronary artery diseases[J].Coronary artery disease.2014,25(4):339-342.
[36]HASHEMIPOUR M,KELISHADI R,SHAPOURI J,et al.Effect of zinc supplementation on insulin resistance and components of the metabolic syndrome in prepubertal obese children[J].Hormones,2009,8(4):279-285.
[37]KARPENKO M,ILYICHEVA E,MURUZHEVA Z,et al.Role of copper dyshomeostasis in the pathogenesis of parkinson's disease[J].Bulletin of experimental biology and medicine,2018,164(5):596-600.
[38]ESKICI G,AXELSEN P H.Copper and oxidative stress in the pathogenesis of Alzheimer's disease[J].Biochemistry,2012,51(32):6289-6311.
[39]SHIRAI T,TSUCHIYA H,SHIMIZU T,et al.Prevention of pin tract infection with titanium-copper alloys[J].Journal of biomedical materials research part B:Applied biomaterials,2009,91(1):373-380.
[40]孙皎,薛淼,今井弘一,等.牙科金属材料的组成对细胞毒性影响的研究[J].中国生物医学工程学报,1997(2):59-64,99.SUN Jao,XUE Miao,JINJING Hong-yi,et al.Cytotoxicity of dental metal material composition[J].Chinese journal of biomedical engineering,1997(2):59-64,99.
[41]LU Y,REN L,XU X,et al.Effect of Cu on microstructure,mechanical properties,corrosion resistance and cytotoxicity of CoCrW alloy fabricated by selective laser melting[J].Journal of the mechanical behavior of biomedical materials,2018,81(3):130-141.
[42]LUO J,WU S,LU Y,et al.The effect of 3%Cu addition on the microstructure,tribological property and corrosion resistance of CoCrW alloys fabricated by selective laser melting[J].Journal of materials science:Materials in medicine,2018,29(4):37.
[43]DOGUER C,HA J H,COLLINS J F.Intersection of iron and copper metabolism in the mammalian intestine and liver[J].Comprehensive physiology,2018,8(4):1433-1461.
[2]TAKEDA O,TOYAMA T,WATANABE K,et al.Ameliorating effects of Juzentaihoto on restraint stress and P.gingivalis-induced alveolar bone loss[J].Archives of oral biology,2014,59(11):1130-1138.
[3]KONG L,QI X,HUANG S,et al.Theaflavins inhibit pathogenic properties of P.gingivalis and MMPs production in P.gingivalis-stimulated human gingival fibroblasts[J].Archives of oral biology,2015,60(1):12-22.
[4]HARDES J,AHRENS H,GEBERT C,et al.Lack of toxicological side-effects in silver-coated megaprostheses in humans[J].Biomaterials,2007,28(18):2869-2875.
[5]REITEMEIER B,HANSEL K,WALTER M H.Effect of posterior crown margin placement on gingival health[J].Journal of prosthetic dentistry,2002,87(2):167-172.
[6]TAYLOR R,MARYAN C,VERRAN J.Retention of oral microorganisms on cobalt-chromium alloy and dental acrylic resin with different surface finishes[J].Journal of prosthetic dentistry,1998,80(5):592-597.
[7]DYLINA T J.Contour determination for ovate pontics[J].Prosthet dent,1999,82(2):136-142.
[8]DRAKE C W,BECK J D.The oral status of elderly removable par-tial denture wearers[J].Oral rehabil,1993,20(1):53-60.
[9]YADROITSEV I,BERTRAND P,SMUROV I.Parametric analysis of the selective laser melting process[J].Applied surface science,2007,253(19):8064-8069.
[10]ANTANASOVA M,KOCJAN A,KOVA?J,et al.Influence of thermo-mechanical cycling on porcelain bonding to cobalt-chromium and titanium dental alloys fabricated by casting,milling,and selective laser melting[J].Journal of prosthodontic research,2018,62(2):184-194.
[11]STRUB J R,REKOW E D,WITKOWSKI S.Computer-aided design and fabrication of dental restorations:Current systems and future possibilities[J].Journal of the American dental association,2006,137(9):1289-1296.
[12]GALHANO G,PELLIZZER E,MAZARO J.Optical impression systems for CAD-CAM restorations[J].Journal of craniofacial surgery,2012,23(6):575-579.
[13]SCHEIBER I F,MERCER J F B,DRINGEN R.Metabolism and functions of copper in brain[J].Progress in neurobiology,2014,116(2):33-57.
[14]IBRAHIM M,WANG F,LOU M M,et al.Copper as an antibacterial agent for human pathogenic multidrug resistant burkholderiacepacia complex bacteria[J].Journal of bioscience and bioengineering,2011,112(6):570-576.
[15]RENSING C,GRASS G.Escherichia coli mechanisms of copper homeostasis in a changing environment[J].FEMSmicrobiology reviews,2003,27(2-3):197-213.
[16]GUO S,LU Y,WU S.Preliminary study on the corrosion resistance,antibacterial activity and cytotoxicity of selective-laser-melted Ti6Al4V-x Cu alloys[J].Materials science and engineering:C,2017,72(3):631-640.
[17]REN L,NAN L,YANG K.Study of copper precipitation behavior in a Cu-bearing austenitic antibacterial stainless steel[J].Mater design,2011,32(4):2374-2379.
[18]REN L,YANG K.Bio-functional design for metal implants,a new concept for development of metallic biomaterials[J].Journal of materials science&technology,2013,29(11):1005-1110.
[19]REN L,YANG K,GUO L,et al.Preliminary study of anti-infective function of a copper-bearing stainless steel[J].Materials science and engineering:C,2012,32(5):1204-1209.
[20]CHAI H W,GUO L,WANG X T,et al.Antibacterial effect of 317L stainless steel contained copper in prevention of implant-related infection in vitro and in vivo[J].Journal of materials science materials in medicine,2011,22(11):2525-2535.
[21]ZHANG D,REN L,ZHANG Y,et al.Antibacterial activity against porphyromonas gingivalis and biological characteristics of antibacterial stainless steel[J].Colloids and surfaces B:Biointerfaces,105(3):51-57.
[22]REN L,XU L,FENG J W,et al.In vitro study of role of trace amount of Cu release from Cu-bearing stainless steel targeting for reduction of in-stent restenosis[J].Journal of materials science materials in medicine,2012,23(5):1235-1245.
[23]LIU R,MEMARZADEH K,CHANG B,et al.Antibacterial effect of copper-bearing titanium alloy(Ti-Cu)against streptococcus mutans and porphyromonas gingivalis[J].Scientific reports,2016,6(1):29985.
[24]LIU R,TANG Y,ZENG L,et al.In vitro and in vivo studies of anti-bacterial copper-bearing titanium alloy for dental application[J].Dental materials,2018,34(8):1112-1126.
[25]REN L,MEMARZADEH K,ZHANG S,et al.A novel coping metal material CoCrCu alloy fabricated by selective laser melting with antimicrobial and antibiofilm properties[J].Materials science and engineering:C,2016,67(10):461-467.
[26]FLüGGE T V,SCHLAGER S,NELSON K,et al.Precision of intraoral digital dental impressions with iTero and extraoral digitization with the i Tero and a model scanner[J].American journal of orthodontics&dentofacial,2013,144(3):471-478.
[27]DA C J,PELOGIA F,HAGEDORN B,et al.Evaluation of different methods of optical impression making on the marginal gap of onlays created with CEREC 3D[J].Operative dentistry,2010,35(3):324-329.
[28]WILLIAMS R J,BIBB R,EGGBEER D,et al.Use of CAD/CAM technology to fabricate a removable partial denture framework[J].Journal of prosthetic dentistry,2006,96(2):96-99.
[29]BAE E J,KIM J H,KIM W C,et al.Bond and fracture strength of metal-ceramic restorations formed by selective laser sintering[J].The journal of advanced prosthodontics,2014,6(4):266-271.
[30]LIU Y,WANG Z,GAO B,et al.Evaluation of mechanical properties and porcelain bonded strength of nickel-chromium dental alloy fabricated by laser rapid forming[J].Lasers in medical science,2010,25(6):799-804.
[31]CRAIG R,HANKS C.Reaction of fibroblasts to various dental casting alloys[J].Journal of oral pathology and medicine,1988,17(7):341-347.
[32]BERARDEN L J,COOKE F W.Growth inhibition of cultured fibroblasts by cobalt and nickel[J].Journal of biomedical materials research,1980,14(3):289-309.
[33]KAWAHARA H,YAMAGAMI A,NAKAMURA M.Biological testing of dental materials by means of tissue culture[J].Int Dent J,1968,18:443-467.
[34]BAGHERI B,AKBARI N,TABIBAN S,et al.Serum level of copper in patients with coronary artery disease[J].American journal of the medical sciences,2015,56(1):39-42.
[35]SHEKARKHAR S,FOROUGHI M,MOATAMEDI M,et al.The association of serum parathyroid hormone and severity of coronary artery diseases[J].Coronary artery disease.2014,25(4):339-342.
[36]HASHEMIPOUR M,KELISHADI R,SHAPOURI J,et al.Effect of zinc supplementation on insulin resistance and components of the metabolic syndrome in prepubertal obese children[J].Hormones,2009,8(4):279-285.
[37]KARPENKO M,ILYICHEVA E,MURUZHEVA Z,et al.Role of copper dyshomeostasis in the pathogenesis of parkinson's disease[J].Bulletin of experimental biology and medicine,2018,164(5):596-600.
[38]ESKICI G,AXELSEN P H.Copper and oxidative stress in the pathogenesis of Alzheimer's disease[J].Biochemistry,2012,51(32):6289-6311.
[39]SHIRAI T,TSUCHIYA H,SHIMIZU T,et al.Prevention of pin tract infection with titanium-copper alloys[J].Journal of biomedical materials research part B:Applied biomaterials,2009,91(1):373-380.
[40]孙皎,薛淼,今井弘一,等.牙科金属材料的组成对细胞毒性影响的研究[J].中国生物医学工程学报,1997(2):59-64,99.SUN Jao,XUE Miao,JINJING Hong-yi,et al.Cytotoxicity of dental metal material composition[J].Chinese journal of biomedical engineering,1997(2):59-64,99.
[41]LU Y,REN L,XU X,et al.Effect of Cu on microstructure,mechanical properties,corrosion resistance and cytotoxicity of CoCrW alloy fabricated by selective laser melting[J].Journal of the mechanical behavior of biomedical materials,2018,81(3):130-141.
[42]LUO J,WU S,LU Y,et al.The effect of 3%Cu addition on the microstructure,tribological property and corrosion resistance of CoCrW alloys fabricated by selective laser melting[J].Journal of materials science:Materials in medicine,2018,29(4):37.
[43]DOGUER C,HA J H,COLLINS J F.Intersection of iron and copper metabolism in the mammalian intestine and liver[J].Comprehensive physiology,2018,8(4):1433-1461.
© 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 |