| [Background and objective]Since the discovery of osseointegration of titanium,titanium and its alloys have been increasing used in clinic for biomedical application.However,their bio-inert surface challenges application in patients with compromised surgical conditions(weakened immunity,open injury,systemic diseases),and their use still faces two risk factors:bacterial infections and lack of local tissue integration.If bacteria existed in the microenvironment,bacteria will compete with cell to occupy the surface once the material is implanted,thus function of osteoblast will be influenced and finally disturb osseointegration.So,strict control of bacterial infection is vital for a successful implant.In addition,sufficient vascular network is also prerequisite for osseointegration,which can provide oxygen and nutrient to guarantee the proliferation,differentation of osteoblast.In order to get the ideal titanium biomedical material,numerous studies have been attempted to modify its surface topography and chemical composition for the purpose of obtaining multifunctional properties of antibacterial ability,angiogenic and osteogenic activity.In our previous work,we have successfully prepared the chitosan/gelatin(CSG)composite coatings on titanium via single-step electrophoretic deposition(EPD),which has three-dimensional porous scaffold like structure,and showed favorable mechanical properties,biodegradability,biocompatibility and osteogenic properties.What's more,this CSG coating was a promising candidate for further loading of functional agents like bioactive molecules and metal ions.Among the variety of functional agents,we have found that metal copper(Cu)ion may have antibacterial,angiogenic,and osteogenic activities simultaneously at certain condition.Taking the above background into consideration,the present study aim to introduce copper element into the CSG coating via EPD,thus fabricating the Cu/CSG composite coatings.The surface morphology,physicochemical properties,copper entrapment and release profile,swelling and degradation property,and mechanical property were investigated.The antibacterial ability of the coatings against E.coli and S.aureus was examined.Furthermore,rat bone marrow stromal cells were used to investigate the cytocompatibility,angiogenic,and osteogenic potential of the coatings.This study may provide theoretical and experimental basis for the potential use of the Cu-doped CSG coatings in clinical application.In addition,in our previous study,we have already found that strontium and silver can be loaded into the CSG coatings,respectively,and the Sr-containing coatings have osteogenic property,while the Ag-doped coatings have antibacterial activity.Under this background,in the second part,strontium and silver had been co-deposited into the CSG coatings,thus preparing Sr/Ag/CSG coatings.The physicochemical properties,ion entrapment and release profile,degradation ratio were analyzed.In addition,bacteria and BMSCs were used to evaluate its biological activity.This investigation may provide certain basis for its future use.Part I Fabrication and characterization of Cu-doped CSG coatings Exp.l Fabrication of Cu-doped CSG coatings[Materials and Methods]The chitosan/gelatin solution was prepared as our previous study.Copper chloride was added into the CSG solution to obtain the final electrophoretic solution with Cu concentrations of 0,0.01,0.1,1,and 10 mM.The obtained Cu/CSG coatings were labeled as CSG,Cu ?,Cu ?,Cu ?,and Cu ?.[Results]With increasing amount of Cu in the EPD suspension,the color of the solution changed gradually from transparent to light blue,while color of the coating changed from transparent to purple.Porous hydrogel coatings were uniformly deposited on the substrates after EPD.After being air-dried,the coatings showed a thin film-like structure on the Ti disks.Exp.2 The physicochemical properties of Cu-doped CSG coatings[Materials and Methods]Microstructure of the freshly deposited coatings was observed by TEM and fluorescence microscopy.After being air-dried,the coatings were examined by FESEM.For qualitative elemental analysis of the coatings,EDS was used.Surface chemistry was investigated with ATR-FTIR;In addition,the chemical state of Cu in the coatings was studied by XPS.The copper entrapment and release profile were analyzed by AAS,and the morphology of the release compounds was observed by TEM.The swelling ratio and weight loss of the coating in the PBS solution were measured.Mechanical tests were conducted to evaluate the mechanical property of the coatings(tensile bond strength and shear bond strength).[Results]TEM results showed that nanoscale particles were deposited onto the titanium surface during EPD.Fluorescence images of the wet coatings displayed macroporous morphology.Under FESEM,microscale particles were distributed in the Cu-doped coatings,which were composed of smaller nanoparticles.EDS analysis confirmed the presence of Cu in the coatings.The ATR-FT1R spectra of all coatings exhibited characteristic peaks of chitosan and gelatin.XPS result showed bivalent copper ions were reduced to metallic Cu in the coating.AAS results showed that Cu entrapment was positively correlated to the Cu concentration in the EPD solutions.All Cu-containing nanocomposite coatings presented a biphasic release of Cu with an initial burst release followed by sustained slow release.TEM images display the morphology of the nanocomposites released from the coatings.The study on the swelling ratio of the coatings indicated that all the coatings could recover to three-dimensional macroporous structure.Degradation results showed that the weight of all the samples decreased sharply in the first few days,followed by relatively little change in the following days.Mechanical tests showed that the tensile bond strength of the coatings increased when Cu was introduced,while there was no significant difference for the shear bond strength.Exp.3 In vitro antibacterial study of the Cu-doped CSG coatings[Materials and Methods]FESEM was used to observe the bacteria adhered on the specimens,in addition,Live/Dead staining assay was applied to analyze the bacterial solution,and the number of live and dead bacterial cells was quantitatively measured by software.The bacteria adhered on the sample surface and suspended in the surrounding medium were counted by spread plate method,thus quantitatively calculated the antibacterial rate.The protein content in the medium after co-culture was measured by bicinchoninic acid(BCA)protein assay kit.[Results]FESEM showed that the amounts of bacteria on the surfaces decreased with increasing Cu concentration in the coatings,in addition,the bacteria were shriveled and deformed at high Cu concentration groups.Live/Dead staining assay displayed that as Cu concentration increased,the green fluorescent intensity(live bacterial colonies)decreased,while the red fluorescent intensity(dead bacterial colonies)increased.The bacteria spread counting method also verified the antibacterial activity of the coatings against the bacteria adhered on the sample surface and suspended in the surrounding medium,and the antibacterial rate was positively correlated with the copper concentration.BCA kit assay showed that Cu-doped coatings undermined the permeability of bacterial membranes,and led to the leakage of bacterial proteins.Exp.4 In vitro cellular study of the Cu-doped CSG coatings[Materials and Methods]FESEM and fluorescence microscopy were used to observe the cell morphology on the coated Ti samples.Cell counting kit-8(CCK-8)was used to analyze the cell proliferation.In addition,cytotoxicity of copper-organic compounds and pure copper ion were compared by the CCK-8 kit.Real-time quantitative polymerase chain reaction(RT-qPCR)was executed to assess angiogenesis-related gene(including HIF-la and VEGF)expressions of various samples in BMSCs.Moreover,ALP(alkaline phosphatase)activity of the cells were measured by pNPP method,and the osteogenesis-related gene(including ALP,OCN,Runx2,and Col-1)expressions were examined by RT-qPCR.[Results]FESEM results indicated that Cu-incorporated coatings could support BMSCs attachment and spreading.In addition,immunofluorescence staining of all groups exhibited a polygonal shape with extended filopodia,through which the cells were connected.Cell density of the Cu ? group was less than all the other groups.CCK-8 assay showed that Cu ?,?,and ? groups could support cell proliferation,while Cu IV group displayed certain cytotoxicity.CCK-8 tests showed that cell activity of the Cu organic compounds group was significantly higher than the corresponding Cu ion group.In addition,the cell activity decreased as the concentration of copper increased.In the RT-qPCR assay,Cu ? and Cu ? groups demonstrated significantly up-regulated gene expressions of angiogenic factors compared with the CSG group,while the Cu IV group showed remarkably lower expressions of these genes.ALP activity of BMSCs in Cu ?,Cu ?,and Cu ? groups increased as Cu loading ascended,and it was significantly higher in the Cu ? group than CSG group.Meanwhile,RT-qPCR analysis showed that BMSCs expressions of osteogenesis-related genes were dramatically up regulated in Cu ? samples compared with other groups,especially on day 14.In addition,Col-1 expression was apparently lower in the Cu ?group than in the CSG group.[Conclusions]1.For the first time,copper-doped inorganic-organic(Cu/CSG)composite coatings were successfully deposited on titanium substrates via EPD,and the coatings had a three-dimensional porous structure;2.Through physical-chemical analysis of the coatings,it was verified that the bivalent copper ions were reduced to metallic Cu,the combined Cu-CSG nanocomposites were deposited on the Ti substrate,and Cu compounds released from the coatings displayed nano-scale structure.Incorporation of Cu into CSGcoatings did not influence the swelling property significantly,but decrease the degradation ratio,and improve the mechanical property;3.In vitro antibacterial study showed that the antibacterial property of copper was preserved.The coatings have antibacterial ability against the bacteria adhered on the sample surface and suspended in the surrounding medium.In addition,the antibacterial rate was positively correlated with the copper concentration;4.As for the in vitro cellular study,Cu ? group displayed improved angiogenic and osteogenic activities,while the Cu IV group showed certain cytotoxicity;5.Considering the performance of coatings systemically,multifunctional Cu ?group coatings could guarantee considerable antibacterial activity and benefit angiogenic and osteogenic differentiation,this study may provide theoretical and experimental basis for the potential use of the Cu-doped CSG coatings in clinical application.Part ? Fabrication and characterization of Sr/Ag-doped CSG coatings Exp.5 Fabrication and material property of Sr/Ag-doped CSG coatings[Materials and Methods]Sr/Ag solution was added into the CSG solution to get the final electrophoretic solution with Sr/Ag concentrations of 0,10/1 mM,50/1 mM,and 50/5 mM,the obtained coatings were labeled as CSG,Sr10/Agl,Sr50/Agl,and Sr50/Ag5.Fluorescence microscopy FESEM,EDS,XRD,AAS,and TEM were used to evaluate their property.[Results]After EPD,hydrogel coatings were uniformly deposited on the Ti surface.Homogeneously distributed white particles appeared in the Sr/Ag/CSG coatings under fluorescence microscopy.Under FESEM,microscale particles were distributed in the Sr/Ag/CSG coatings,and some brighter nanoparticles appeared in the microparticles.EDS verified the presence of Sr/Ag in the coatings,while the XRD showed that these two elements mainly existed in the form of SrCO3 and AgC1.AAS results showed that Sr/Ag entrapment was positively correlated to the Sr/Ag concentration in the EPD solutions.All coatings displayed.a biphasic release of Sr/Ag with an initial burst release followed by sustained slow release.TEM images presented that nanocomposites released from the coatings.Degradation results showed that the weight of all the samples decreased greatly in the first few days,followed by relatively little change in the following days.Exp.6 In vitro antibacterial study of the Sr/Ag-doped CSG coatings[Materials and Methods]Live/Dead staining assay was used to obeserve the bacterial solution.Spread plate method was applied to count the bacteria on the sample surface and suspended in the surrounding medium,thus quantitatively calculated the antibacterial rate.[Results]Live/Dead staining assay showed that as Ag concentration increased,the green fluorescent intensity(live bacterial colonies)decreased.The bacteria spread counting method also verified the antibacterial activity of the coatings against the bacteria adhered on the sample surface and suspended in the surrounding medium.In addition,the antibacterial rate was positively correlated with the silver concentration.Exp.7 In vitro cellular study of the Sr/Ag-doped CSG coatings[Materials and Methods]FESEM and fluorescence microscopy were used to observe the cell morphology.Cell counting kit-8(CCK-8)was used to analyze the cell proliferation after seeding on the coatings.Moreover,ALP activity of the cells was measured,and the osteogenesis-related gene expressions were examained by RT-qPCR.[Results]FESEM results indicated that Sr/Ag-incorporated coatings could support BMSCs attachment and spreading except for the Sr50/Ag5 group.Under fluorescence microscopy,cells exhibited a polygonal shape with extended filopodia,while only some debris could be observed in the Sr50/Ag5 group.CCK-8 assay showed that Sr10/Agl and Sr50/Agl groups could promote proliferation of the cells,while the Sr50/Ag5 group displayed significantly cytotoxicity.ALP activity assay and RT-qPCR analysis showed that expressions of osteogenesis-related genes in BMSCs were enhanced in Sr10/Agl and Sr50/Agl groups,and the effect of Sr50/Agl group is greater than Sr10/Agl group.[Conclusions]1.Sr/Ag-doped inorganic-organic(Sr/Ag/CSG)composite coatings were successfully fabricated on titanium substrates via EPD;2.Sr/Ag mainly existed in the coatings as the form of SrCO3 and AgCl,and the compounds released from the coatings displayed nano-scale structure.Incorporation of Sr/Ag into CSG coatings decrease the degradation ratio;3.Sr/Ag/CSG coatings could inhibite the bacteria adhered on the sample surface and suspended in the surrounding medium,and the antibacterial rate was positively correlated with the silver concentration;4.Sr10/Agl and Sr50/Agl group could promote proliferation and osteogenic differentiation of the cells,and the effect of Sr50/Agl group is greater than Sr10/Agl group.In addition,Sr50/Ag5 group displayed significantly cytotoxicity;5.In total,Sr50/Agl group coating is multifunctional,which could have antibacterial activity and osteogenic property simultaneously,this investigation may provide certain basis for the future use of Sr/Ag/CSG coatings. |
PDF Full Text Request