SLM含铜钴基合金的抗菌性能及细胞相容性的初步探索
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摘要
目的以SLM-CoCrW为对照,评价SLM-CoCrCu的抗菌性及生物相容性,为SLM-CoCrCu的潜在临床应用提供理论依据。方法 选择金黄色葡萄球菌为研究对象,采用平板法验证合金的抗菌性能。选择MC3T3-E1细胞为研究对象,采用CCK-8检测法评价合金对于细胞增殖的影响和细胞毒性等级。采用细胞骨架荧光染色标记法评价细胞在合金表面的粘附及生长,结合增殖率,反映合金的细胞毒性。采用流式细胞术评价材料对细胞早期凋亡的影响。结果 SLM-CoCrCu对金黄色葡球菌的生长有明显抑制作用,对金黄色葡萄球菌抗菌率可达98%,与SLM-CoCrW比较有显著性差异(P<0.01)。随培养时间的延长,SLM-CoCrCu细胞数量逐步增长,表现出对细胞增殖的促进作用。SLM-CoCrCu和SLM-CoCrW在与细胞共培养1、3、7 d后,细胞增殖率有显著差异性(P=0.01)。共培养1 d后,不含铜组细胞增殖率优于含铜组;共培养3 d及7d后,含铜组对细胞增殖影响明显优于不含铜组。两者对于细胞粘附及形态上的影响无明显差异。SLM-CoCrCu和SLM-CoCrW对于细胞凋亡的影响无明显差异。结论 SLM-CoCrCu相较于SLM-CoCrW有良好的抗菌性能。同时,SLM-CoCrCu具有良好的生物相容性,对细胞增殖、粘附及形态都无明显影响。
The work aims to use SLM-CoCrW as a control to evaluate the antimicrobial and biocompatibility of SLM-CoCrCu, and then provide a theoretical basis for the potential clinical application of SLM-CoCrCu. Staphylococcus aureus was selected as the research object, and the antimicrobial properties of the alloy were tested by plate method. MC3T3-E1 cells were selected as the research object. CCK-8 assay was used to evaluate the effect of alloy on cell proliferation and cytotoxicity.The adhesion and growth of cells on the alloy surface were evaluated by cytoskeleton fluorescence staining, and the cytotoxicity of the alloy was reflected by the proliferation rate. Flow cytometry was used to evaluate the effect of materials on early cell apoptosis. SLM-CoCrCu obviously inhibited the growth of staphylococcus aureus, and the antimicrobial rate of SLM-CoCrCu was 98%. There was a significant difference between SLM-CoCrCu and SLM-CoCrW(P<0.01). With the prolongation of culture time, the number of SLM-Co Cr Cu cells increased gradually, and promoted cell proliferation. SLM-CoCrCu and SLM-CoCrW co-cultured with cells for 1, 3 and 7 days showed significant difference in cell proliferation rate(P=0.01). After co-culture for 1 day, the cell proliferation rate of copper-free group was better than that of copper-free group. After co-culture for 3 days and 7 days, the effect of copper-containing group on cell proliferation was significantly better than that of copper-free group. There was no significant difference in cell adhesion and morphology between the two groups. There was no significant difference between SLM-CoCrCu and SLM-CoCrW on apoptosis. Compared with SLM-CoCrW, SLM-CoCrCu has better antimicrobial properties. At the same time, SLM-CoCrCu has good biocompatibility and has no significant effect on cell proliferation, adhesion and morphology.
The work aims to use SLM-CoCrW as a control to evaluate the antimicrobial and biocompatibility of SLM-CoCrCu, and then provide a theoretical basis for the potential clinical application of SLM-CoCrCu. Staphylococcus aureus was selected as the research object, and the antimicrobial properties of the alloy were tested by plate method. MC3T3-E1 cells were selected as the research object. CCK-8 assay was used to evaluate the effect of alloy on cell proliferation and cytotoxicity.The adhesion and growth of cells on the alloy surface were evaluated by cytoskeleton fluorescence staining, and the cytotoxicity of the alloy was reflected by the proliferation rate. Flow cytometry was used to evaluate the effect of materials on early cell apoptosis. SLM-CoCrCu obviously inhibited the growth of staphylococcus aureus, and the antimicrobial rate of SLM-CoCrCu was 98%. There was a significant difference between SLM-CoCrCu and SLM-CoCrW(P<0.01). With the prolongation of culture time, the number of SLM-Co Cr Cu cells increased gradually, and promoted cell proliferation. SLM-CoCrCu and SLM-CoCrW co-cultured with cells for 1, 3 and 7 days showed significant difference in cell proliferation rate(P=0.01). After co-culture for 1 day, the cell proliferation rate of copper-free group was better than that of copper-free group. After co-culture for 3 days and 7 days, the effect of copper-containing group on cell proliferation was significantly better than that of copper-free group. There was no significant difference in cell adhesion and morphology between the two groups. There was no significant difference between SLM-CoCrCu and SLM-CoCrW on apoptosis. Compared with SLM-CoCrW, SLM-CoCrCu has better antimicrobial properties. At the same time, SLM-CoCrCu has good biocompatibility and has no significant effect on cell proliferation, adhesion and morphology.
引文
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[23]JEVREMOVIC D,PUSKAR T,KOSEC B,et al.The analysis of the mechanical properties of F75 Co-Cr alloy for use in selective laser melting(SLM)manufacturing of removable partial dentures(RPD)[J].Metalurgija,2012,51(2):171-174.
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[2]XIANG N,XIN X Z,CHEN J,et al.Metal-ceramic bond strength of Co-Cr alloy fabricated by selective laser melting[J].Journal of dentistry,2012,40(6):453-457.
[3]TAKAICHI A,SUYALATU,NAKAMOTO T,et al.Microstructures and mechanical properties of Co-29Cr-6Mo alloy fabricated by selective laser melting process for dental applications[J].Journal of the mechanical behavior of biomedical materials,2013,21(3):67-76.
[4]白晓峰.口腔颌面外科学(9)口腔颌面部畸形和缺损[J].中国实用口腔科杂志,2010,3(8):511-512.BAI Xiao-feng.Oral and maxillofacial surgery(9)deformity and defect in oral and maxillofacial region[J].Chinese journal of practical dentistry,2010,3(8):511-512.
[5]JABBARI Y S A.Physico-mechanical properties and prosthodontic applications of Co-Cr dental alloys:A review of the literature[J].Journal of advanced prosthodontics,2014,6(2):138-145.
[6]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.
[7]GODOYGALLARDO M,MANZANARESCéSPEDESM C,SEVILLA P,et al.Evaluation of bone loss in antibacterial coated dental implants:An experimental study in dogs[J].Materials science&engineering C,2016,69:538-545.
[8]SUZUKI S,NAKAMURA S,MIYAKUSU K.Antimicrobial activity of Cu contained austenitic stainless steels[J].Current advances in materials and processes,1999(12):518-518.
[9]REN L,YANG K.Bio-functional design for metal implants,a new concept for development of metallic biomaterials[J].Journal of materials science&technology,2013,20:1005-1110.
[10]REN L,MA Z,LI M,et al.Antibacterial properties of Ti-6Al-4V-x Cu alloys[J].Journal of materials science&technology,2014,30(7):699-705.
[11]ZHANG D,REN L,ZHANG Y,et al.Antibacterial activity against Porphyromonas gingivalis and biological characteristics of antibacterial stainless steel[J].Colloids surfaces B:Biointerfaces,2013,105(4):51-57.
[12]REN L,NAN L,YANG K.Study of copper precipitation behavior in a Cu-bearing austenitic antibacterial stainless steel[J].Materials&design,2011,32:2374-2379.
[13]REN L,ZHU J,NAN L,et al.Differential scanning calorimetry analysis on Cu precipitation in a high Cu austenitic stainless steel[J].Materials&design,2011,32(7):3980-3985.
[14]REN L,YANG K,GUO L,et al.Preliminary study of anti-infective function of a copper-bearing stainless steel[J].Materials science&engineering C,2012,32(5):1204-1209.
[15]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.
[16]浦素云.金属植入材料及其腐蚀[M].北京:北京航空航天大学出版社,1990.PU Su-yun.Metal implant materials and their corrosion[M].Beijing:Beijing University of Aeronautics and Astronautics Press,1990.
[17]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&engineering C,2016,67:461-467.
[18]马政.新型含铜抗菌钛合金的制备与性能研究[D].大连:大连理工大学,2015.MA Zheng.Study on preparation and properties of new-type copper-containing antibacterial titanium alloy[D].Dalian:Dalian University of Technology,2015.
[19]WANG S,YANG C,REN L,et al.Study on antibacterial performance of Cu-bearing cobalt-based alloy[J].Materials letters,2014,129(32):88-90.
[20]李小宇,郑美华,王洁琪,等.3D打印和铸造钴铬合金耐蚀性及力学稳定性比较[J].中华口腔医学研究杂志,2016,10(5):327-332.LI Xiao-yu,ZHENG Mei-hua,WANG Jie-qi,et al.3DPrinting and casting the corrosion resistance and mechanical stability of cobalt-chromium alloy[J].Chinese journal of stomatology research,2016,10(5):327-332.
[21]邹洁,胡滨.选择性激光熔融技术所制牙科合金的研究进展[J].北京口腔医学,2014(5):292-294.ZOU Jie,HU Bin.Research progress of dental alloys made by selective laser melting technology[J].Beijing oral medicine,2014(5):292-294.
[22]VANDENBROUCKE B,KRUTH J P.Selective laser melting of biocompatible metals for rapid manufacturing of medical parts[J].Rapid prototyping journal,2007,13(4):196-203.
[23]JEVREMOVIC D,PUSKAR T,KOSEC B,et al.The analysis of the mechanical properties of F75 Co-Cr alloy for use in selective laser melting(SLM)manufacturing of removable partial dentures(RPD)[J].Metalurgija,2012,51(2):171-174.
[24]段小冬.银?铜离子对人角质形成细胞增殖的影响及机制探索[D].重庆:第三军医大学,2015.DUAN Xiao-dong.Effects of silver and copper ions on the proliferation of human keratinocyte cells and its mechanism[D].Chongqing:Third Military Medical University,2015.