羧甲基壳聚糖锌多肽复合材料对人牙周膜成纤维细胞的毒性
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摘要
目的 :研究羧甲基壳聚糖锌多肽复合材料(carboxymethyl chitosan zinc and peptide,CMC-Zn+-P)对人牙周膜成纤维细胞(human periodontal ligament cells,HPDLCs)的细胞毒性,为材料的安全使用提供依据。方法:体外培养人牙周膜成纤维细胞,并进行形态学及免疫组织化学鉴定。使用含不同浓度材料浸提液的细胞培养液培养细胞,并进行分组,1~4组为实验组,材料浸提液浓度分别为100%、75%、50%和25%,第5组为对照组,不含材料浸提液。采用CCK-8法测定复合材料不同浓度浸提液对细胞增殖活性的影响,通过细胞相对增殖率进行细胞毒性评级。采用SPSS 17.0软件包对数据进行统计学分析。结果 :原代培养的细胞呈成纤维细胞形态,免疫组织化学染色结果显示细胞波形丝蛋白染色阳性,角蛋白染色阴性,属中胚层组织来源。不同浓度材料浸提液对细胞均有促进增殖的作用,相对增殖率均大于100%,细胞毒性分级为0级。结论:CMC-Zn+-P复合材料对体外培养的HPDLCs无细胞毒性,符合国家标准的要求。
PURPOSE: The purpose of this investigation was to study the cytotoxicity of carboxymethyl chitosan zinc and peptide to human periodontal ligament cells in vitro, in order to provide a reference for understanding materials' safety.METHODS: The primary cells were obtained from human periodontal tissues and cultured. The cultured cells were identified by observing the shape under microscope and by immunocytochemical method. HPDLCs were cultured in different concentrations of the tested materials extract and the activity of cells was evaluated using CCK-8 assay. The cytotoxicity grade was determined in term of the cell relative growth rates. The concentration of the tested materials extract was 100%, 75%, 50% and 25% in group 1-4, respectively. The fifth group had no materials. All statistical analysis was performed using SPSS 17.0 software package. RESULTS: The primary cells owned fibroblasts' shape. Immunocytochemical analysis showed the cells were stained positively to antibodies against vimentin, and negatively to antibodies against cytokeratin, which indicated that they were external embryo mesenchymal cell. The cell relative growth rates were more than100%, no matter the concentrations of materials extract were. The cytotoxicity grade of CMC-Zn+-P was 0. CONCLUSIONS:CMC-Zn+-P exhibits no cytotoxicity to HPDLCs in vitro, which meets the requirements of the national standard.
PURPOSE: The purpose of this investigation was to study the cytotoxicity of carboxymethyl chitosan zinc and peptide to human periodontal ligament cells in vitro, in order to provide a reference for understanding materials' safety.METHODS: The primary cells were obtained from human periodontal tissues and cultured. The cultured cells were identified by observing the shape under microscope and by immunocytochemical method. HPDLCs were cultured in different concentrations of the tested materials extract and the activity of cells was evaluated using CCK-8 assay. The cytotoxicity grade was determined in term of the cell relative growth rates. The concentration of the tested materials extract was 100%, 75%, 50% and 25% in group 1-4, respectively. The fifth group had no materials. All statistical analysis was performed using SPSS 17.0 software package. RESULTS: The primary cells owned fibroblasts' shape. Immunocytochemical analysis showed the cells were stained positively to antibodies against vimentin, and negatively to antibodies against cytokeratin, which indicated that they were external embryo mesenchymal cell. The cell relative growth rates were more than100%, no matter the concentrations of materials extract were. The cytotoxicity grade of CMC-Zn+-P was 0. CONCLUSIONS:CMC-Zn+-P exhibits no cytotoxicity to HPDLCs in vitro, which meets the requirements of the national standard.
引文
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[21]Zhang X,Fan C,Xiao Y,et al.Anti-inflammatory and antiosteoclastogenic activities of parthenolide on human periodontal ligament cells in vitro[J].Evid Based Complement Alternat Med,2014,2014:546097.
[22]李冉,张旗.丝氨酸蛋白酶Htr A1在人牙周膜组织中的表达及对人牙周膜细胞增殖的影响[J].中华口腔医学杂志,2016,51(2):87-92.
[23]Wang Y,Zhao Q,Hu Y,et al.Ordered nanoporous silica as carriers for improved delivery of water insoluble drugs:a comparative study between three dimensional and two dimensional macroporous silica[J].Int J Nanomedicine,2013,8:4015-4031.
[24]侯春梅,李新颖,叶伟亮,等.MTT法和CCK-8法检测悬浮细胞增殖的比较[J].军事医学科学院院刊,2009,33(4):400-401.
[25]熊建文,肖化,张镇西.MTT法和CCK-8法检测细胞活性之测试条件比较[J].激光生物学报,2007,10(5):559-562.
[2]Kim IY,Seo SJ,Moon HS,et al.Chitosan and its derivatives for tissue engineering applications[J].Biotechnol Adv,2008,26(1):1-21.
[3]Szymańska E,Winnicka K.Stability of chitosan-a challenge for pharmaceutical and biomedical applications[J].Mar Drugs,2015,13(4):1819-1846.
[4]Upadhyaya L,Singh J,Agarwal V,et al.Biomedical applications of carboxymethyl chitosans[J].Carbohydr Polym,2013,91(1):452-466.
[5]Song Y,Li Y,Liu Z,et al.A novel ultrasensitive carboxymethyl chitosan-quantum dot-based fluorescence"turn on-off"nanosensor for lysozyme detection[J].Biosens Bioelectron,2014,61:9-13.
[6]Li X,Kong X,Zhang Z,et al.Cytotoxicity and biocompatibility evaluation of N,O-carboxymethyl chitosan/oxidized alginate hydrogel for drug delivery application[J].Int J Biol Macromol,2012,50(5):1299-1305.
[7]Lynch RJ.Zinc in the mouth,its interactions with dental enamel and possible effects on caries[J].Int Dent J,2011,61(Suppl 3):46-54.
[8]张岩,吴燕燕,李来好,等.酶法制备海洋活性肽及其功能活性研究进展[J].生物技术通报,2012,28(3):42-48.
[9]巢警殳,张岚,杜娟,等.生物活性肽的研究进展[J].吉林医药学院学报,2010,31(6):359-362.
[10]中华人民共和国国家质量监督检验检疫总局.GB/T14233.2-2005医用输液、输血、注射器具检验方法.第2部分:生物学试验方法[S].北京:中国标准出版社,2005.
[11]孟焕新.牙周病学[M].第4版.北京:人民卫生出版社,2013.
[12]吉秋霞,徐全臣,于新波,等.壳聚糖及其季铵盐对牙周常见致病菌的体外抑菌活性[J].青岛大学医学院学报,2011,47(1):23-24.
[13]Duan W,Shen C,Fang H,et al.Synthesis of dehydroabietic acid-modified chitosan and its drug release behavior[J].Carbohydr Res,2009,344(1):9-13.
[14]Adhiyaman R,Basu SK.Crystal modification of dipyridamole using different solvents and crystallization conditions[J].Int J Pharm,2006,321(1-2):27-34.
[15]Sitohy MZ,Mahgoub SA,Osman AO.In vitro and in situ antimicrobial action and mechanism of glycinin and its basic subunit[J].Int J Food Microbiol,2012,154(1-2):19-29.
[16]宋佳,邓婧,吴园园,等.羧甲基壳聚糖锌多肽复合材料对四种牙周致病菌体外抑菌作用的研究[J].中华口腔医学杂志,2015,50(7):413-417.
[17]刘学玉,潘克清,张丽,等.羧甲基壳聚糖锌多肽复合材料局部应用对巴马小型猪龈沟液中IL-1、TNF-α和PGE-2含量的影响[J].上海口腔医学,2016,25(2):172-176.
[18]International Standard Orgnization.ISO10993.5-2002 Biological evaluation of medical devices.Part 5:Test for in vitro cytotoxicity[S].
[19]袁艳波.牙科生物材料细胞毒性试验方法的研究进展[J].生物医学工程学杂志,2009,26(3):688-691.
[20]Seo BM,Miura M,Gronthos S,et al.Investigation of multipotent postnatal stem cells from human periodontal ligament[J].Lancet,2004,364(9429):149-155.
[21]Zhang X,Fan C,Xiao Y,et al.Anti-inflammatory and antiosteoclastogenic activities of parthenolide on human periodontal ligament cells in vitro[J].Evid Based Complement Alternat Med,2014,2014:546097.
[22]李冉,张旗.丝氨酸蛋白酶Htr A1在人牙周膜组织中的表达及对人牙周膜细胞增殖的影响[J].中华口腔医学杂志,2016,51(2):87-92.
[23]Wang Y,Zhao Q,Hu Y,et al.Ordered nanoporous silica as carriers for improved delivery of water insoluble drugs:a comparative study between three dimensional and two dimensional macroporous silica[J].Int J Nanomedicine,2013,8:4015-4031.
[24]侯春梅,李新颖,叶伟亮,等.MTT法和CCK-8法检测悬浮细胞增殖的比较[J].军事医学科学院院刊,2009,33(4):400-401.
[25]熊建文,肖化,张镇西.MTT法和CCK-8法检测细胞活性之测试条件比较[J].激光生物学报,2007,10(5):559-562.