灭活细菌生物膜杂化Ti金属材料的生物活性研究
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摘要
为调控生物活性Ti金属材料的生物活性,使用灭活的细菌生物膜对其进行杂化。金黄色葡萄球菌(S.aureus)在纯Ti(P-Ti)和酸碱处理Ti(AA-Ti)表面培养5 d后在121℃、0.13 MPa条件下灭活30 min,获得细菌生物膜杂化的纯Ti(BP-Ti)和酸碱处理Ti(BAA-Ti)。BCA蛋白分析和苯酚-硫酸分析表明,在BAA-Ti表面的细菌生物膜的细胞外基质较BP-Ti表面细菌生物膜的细胞外基质含较多蛋白,而后者含有较多的多糖。酶免疫分析表明,在BP-Ti和BAA-Ti表面的肠毒素都低于阈值。模拟体液(SBF)浸泡实验表明,BAA-Ti和BP-Ti在5 d内均可诱导磷灰石形成,但BAA-Ti诱导磷灰石能力低于BP-Ti。AA-Ti在1 d内可诱导磷灰石生成,但在5 d内P-Ti表面无磷灰石生成。表明生物膜提高了BP-Ti的生物活性,但降低了BAA-Ti的生物活性。这可能源于生物膜中多糖有利于促进磷灰石的形成,而其中的蛋白抑制磷灰石的形成。MG-63细胞培养实验表明,细胞增殖能力顺序为:BAA-Ti Bacterial biofilm contained with proteins and polysaccharides, which made it have the potential to mimic extracellular matrix for tissue repair. It also has the properties to attach the substrate firmly.In order to regulate the bioactivity of titanium metal, inactivated bacterial biofilm was hybridized on the metal surfaces. Staphylococcus aureus(S.aureus) was cultured for 5 d on pure titanium metal(P-Ti) and thatsubjected to acid-alkali treatment(AA-Ti), and then the bacteria was inactivated at 121 ℃ and 0.13 MPa for 30 min to get hybridized titanium metals BP-Ti and BAA-Ti respectively. BCA(bicinchoninic acid) assay and phenol-sulfuric acid analysis showed the extracellular matrix(ECM) in the biofilm on BAA-Ti contented less polysaccharide and more protein than that on BP-Ti. Enzyme immunoassay showed the staphylococcal enterotoxins in both biofilms were lower than the threshold value. SBF(simulated body fluid) soaking experiments showed both BAA-Ti and BP-Ti could induce apatite formation after 5 d, and BAA-Ti induced less apatite than the BP-Ti did. The AA-Ti could induce apatite formation at 1 d, but there is no apatite formation on P-Ti even after 5 d. It indicated that the biofilm decreased the bioactivity of BAA-Ti, but increased the bioactivity of BP-Ti. This effect might depend on the roles of proteins and polysaccharide in the biofilms which could restrain the apatite formation for the former, and accelerate the apatite formation for the later. When osteosarcoma cell MG-63 was cultured on the metals for 3 d, the cell proliferation ability on the metals followed by the order of BAA-Ti
引文
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[4]Wen H B,Liu Q,De Wijn J R,et al.Preparation of bioactive microporous titanium surface by a new two-step chemical treatment[J].J.Mater.Sci.:Mater.Med.,1998,9:121
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[6]Xiao M,Chen Y M,Biao M N,et al.Bio-functionalization of biomedical metals[J].Mater.Sci.Eng.,2017,C70:1057
[7]Trindade M C D,Schurman D J,Maloney W J,et al.G-protein activity requirement for polymethylmethacrylate and titanium particleinduced fibroblast interleukin-6 and monocyte chemoattractant protein-1 release in vitro[J].J.Biomed.Mater.Res.,2000,51:360
[8]Cai K Y,Rechtenbach A,Hao J Y,et al.Polysaccharide-protein surface modification of titanium via a layer-by-layer technique:Characterization and cell behaviour aspects[J].Biomaterials,2005,26:5960
[9]Wei A L,Liu S Q,Peng H,et al.An experimental study on repairing bone defect with composite of b-tricalcium phosphate-hyaluronic acid-type I collagen-marrow stromal cells[J].Chin.J.Reparat.Reconstruct.Surg.,2005,19:468(卫爱林,刘世清,彭昊等,磷酸三钙-透明质酸-I型胶原-骨髓基质细胞复合修复骨缺损的实验研究[J].中国修复重建外科杂志,2005,19:468)
[10]Costerton J W,Stewart P S,Greenberg E P.Bacterial biofilms:A common cause of persistent infections[J].Science,1999,284:1318
[11]Donlan R M,Costerton J W.Biofilms:Survival mechanisms of clinically relevant microorganisms[J].Clin.Microbiol.Rev.,2002,15:167
[12]Mah T F C,O'Toole G A.Mechanisms of biofilm resistance to antimicrobial agents[J].Trends Microbiol.,2001,9:34
[13]Sutherland I W.Biofilm exopolysaccharides:A strong and sticky framework[J].Microbiology,2001,147:3
[14]Ye X,Li J,Lu M X,et al.Identification and molecular typing of Streptococcus agalactiae isolated from pond-cultured tilapia in China[J].Fish.Sci.,2011,77:623
[15]Masuko T,Minami A,Iwasaki N,et al.Carbohydrate analysis by a phenol-sulfuric acid method in microplate format[J].Anal.Biochem.,2005,339:69
[16]Cuesta G,Suarez N,Bessio M I,et al.Quantitative determination of pneumococcal capsular polysaccharide serotype 14 using a modification of phenol-sulfuric acid method[J].J.Microbiol.Methods,2003,52:69
[17]Du Bois M,Gilles K A,Hamilton J K,et al.Colorimetric method for determination of sugars and related substances[J].Anal.Chem.,1956,28:350
[18]Yang X N,Cui F Y,Guo X C,et al.Effects of nanosized titanium dioxide on the physicochemical stability of activated sludge flocs using the thermodynamic approach and Kelvin probe force microscopy[J].Water Res.,2013,47:3947
[19]Kokubo T,Takadama H.How useful is SBF in predicting in vivo bone bioactivity?[J].Biomaterials,2006,27:2907
[20]Mosmann T.Rapid colorimetric assay for cellular growth and survival:Application to proliferation and cytotoxicity assays[J].J.Immunol.Methods,1983,65:55
[21]Hegyi F,Zalán Z,Halász A.Improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)colorimetric assay for measuring the viability of lactic acid bacteria[J].Acta Aliment.,2012,41:506
[22]Tadic D,Epple M.A thorough physicochemical characterisation of14 calcium phosphate-based bone substitution materials in comparison to natural bone[J].Biomaterials,2004,25:987
[23]Din?er M,Teker D,Sa?C P,et al.Enhanced bonding of biomimetic apatite coatings on surface-modified titanium substrates by hydrothermal pretreatment[J].Surf.Coat.Technol.,2013,226:27
[24]Wi S,Pancoska P,Keiderling T A.Predictions of protein secondary structures using factor analysis on Fourier transform infrared spectra:effect of Fourier self-deconvolution of the amide I and amide II bands[J].Biospectroscopy,1998,4:93
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[26]Miyahara T,Nyan M,Shimoda A,et al.Exploitation of a novel polysaccharide nanogel cross-linking membrane for guided bone regeneration(GBR)[J].J.Tissue Eng.Regen.Med.,2012,6:666
[27]Travan A,Marsich E,Donati I,et al.Polysaccharide-coated thermosets for orthopedic applications:From material characterization to in vivo tests[J].Biomacromolecules,2012,13:1564
[28]Tagaya M,Ikoma T,Takeguchi M,et al.Interfacial serum protein effect on biological apatite growth[J].J.Phys.Chem.,2011,115C:22523
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