Exploration of the enhanced performances for silk fibroin/sodiu alginate composite coatings on biodegradable Mg?Zn?Ca alloy

Hui Fng,Chenxi Wng,*,Shicheng Zhou,Ge Li,Ynhong Tin,Tdtomo Sug

a State Key Laboratory of Advanced Welding and Joining,Harbin Institute of Technology,Harbin 150001,China

b Collaborative Research Center,Meisei University,Hino,Tokyo 191-8506,Japan

Abstract To expand the future clinic applications of biodegradable magnesium alloy,polymer coatings with excellent biocompatibility are the keys to solve the local alkalinity and rapid hydrogen release.Natural?organic silk fibroi provides an approach to fabricate a protective coating on biomedical Mg?Zn?Ca alloy,however,the adhesion force and mechanical properties of the coating on substrates are ought to be further improved without any chemical conversion/intermediate layer.Hereby,based on VUV/O3 surface activation,a hybrid of silk fibroi and sodium alginate is proposed to enhance the adhesion force and mechanical properties of the composite coatings on hydrophilic Mg?Zn?Ca alloy surfaces.Various mass ratios of sodium alginate addition were investigated to achieve the optimum coating strategy.The nanoscratch test and nanoindentation test confirme that the adhesion force was tripled and mechanical properties index was significantl improved when the mass ratio of silk fibroin/sodiu alginate was 70/30 compared to pure silk fibroi or sodium alginate coatings.Meanwhile,the corrosion rate of the coated Mg?Zn?Ca alloy was significantl delayed with the addition of sodium alginate,resulting in a reaction layer during corrosion process.Furthermore,the mechanisms for both adhesion and corrosion processes were discussed in detail.Our finding offer more possibilities for the controllable surface performance of degradable metals.? 2020 Chongqing University.Publishing services provided by Elsevier B.V.on behalf of KeAi Communications Co.Ltd.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/)Peer review under responsibility of Chongqing University

Keywords:Mg?Zn?Ca alloy;Silk fibroin Sodium alginate;Adhesion force;Mechanical property;Corrosion resistance.

1.Introduction

Motivated by the outstanding mechanical properties and biocompatibility,biodegradable magnesium and its alloys have been explored for biomedical use especially in the fabrication of orthopedic fixatio devices[1-3].In order to maintain the support of surrounding bone tissue,the mechanical integrity of magnesium and its alloy had better stabilize within the matching bone healing period of 2?4 months,which is also expected to promote the growth of osteoblasts to avoid the innate immune response[4,5].However,the badly local alkalosis and constantly released hydrogen caused by the rapid degradation of magnesium alloy probably lead to many problems such as emphysema,osteonecrosis and separation of implant from tissues[6,7].Many researchers have added various metal element(Al,Mn,Ce,Zr and Cd)into magnesium to enhance the corrosion resistance of the substrates,nevertheless,the biosafety of these elements in human body still called in question[8-10].As essential elements of human body,Zn and Ca elements have been reported to possess excellent biocompatibility.In addition,the alloying of the two elements in magnesium substrates has the advantages of refinin grains,enhancing strength and reducing corrosion rate[11,12].Thus,Mg?Zn?Ca alloy(MgZnCa)has been considered as one of the most promising biomaterials in the current research upsurge.Unfortunately,the corrosion resistance of Mg?Zn?Ca alloy is hard to meet the clinical requirements for bone implants as well,and resulted in the development of extensive surface modificatio methods,among which preparing polymer coatings is most common.

Silk fibroi(SF)is a natural biodegradable polymer,which have recently drawn the most attention due to its excellent biocompatibility and tunable biodegradability[13].With unique properties,natural?organic silk fibroi(particularly regenerated silk that derived from the silkwormBombyx mori)[14]has been widely applied in drug delivery,tissue engineering and biodegradable electronic devices[15].Although coating silk fibroi on the surface of magnesium alloy is a feasible strategy to improve the corrosion resistance,there are still some issues to be solved,which impedes its clinical application.First issue is related to the insufficien adhesion between organic silk and inorganic magnesium,always resulting in delamination of the coating under mechanical shearing or long?term service[16].The chemical conversion treatment[17,18]such as fluorin treatment[19]and alkaline treatment[20]have been reported to modify the magnesium alloy surface to generate magnesium fluorid and magnesium hydroxide intermediate layer,thus improving the adhesion force of coating.However,their use is limited by inherent weak mechanical properties,immunogenicity,lot?to?lot variability and structural complexity.Moreover,pure SF coating is subject to the poor mechanical strength and fl xibility because of its internal structures(water?soluble random coils)[20-22].Thus,another issue brings out the problems in mechanical properties of coating,which are easily worn out when bearing mechanical movement.In view of these,there is an urge demand for the development of non?hazardous and environmentally?friendly coating strategy without any chemical conversion layer to improve the adhesion force and mechanical properties of coating,meanwhile realizing the functionalization in corrosion resistance.

Sodium alginate(SA)extracted from the brown algae with extensive sources,excellent biocompatibility,biodegradability,antibacterial property,and high ionic absorbability is commonly used as a food thickener especially due to its viscosity[23-27].Based on the performance complementary principle of composite materials,blending SA with SF can cause the change of internal structures and viscosity of SF/SA composite coating to improve the adhesion force and mechanical properties[28,29].Adequate reactive groups in both SF and SA also can easily form hydrogen bonding and chemical bonding between the two molecules,resulting in the different structure and properties compared to pure SF and pure SA.It has been reported that physical properties of SF/SA composite fil such as tensile strength,elongation,water absorption and moisture permeability were improved by blending SA with SF[30].Up to now,the novelty of applying SF/SA composite coating on biodegradable magnesium alloy has been rarely reported regardless of the numerous studies on the blends of silk fibroi and sodium alginate.

In this work,vacuum ultraviolet?ozone(VUV/O3)activation is introduced to pretreat the surface of magnesium alloy,which has been confirme as an effective method to increase surface hydrophilicity and available functional groups in our previous study[31-33].There are multiple advantages that can be fully utilized such as removing surface contaminants,surface disinfection and sterilization[34,35].We aim at coating a composite SF/SA fil on Mg?Zn?Ca alloy combined with vacuum ultraviolet?ozone(VUV/O3)activation to enhance the adhesion force and mechanical properties of protective film At the same time,the corrosion resistance of coated structure is expected to be improved.These finding we explored presents a new simple and feasible technique for the surface functionalization of biodegradable magnesium alloy,providing insight into the factors underlying the successful clinical outcome of biodegradable engineering based on the composite structure.

2.Materials and methods

2.1.Mg alloy and specimens

Hot extruded Mg?Zn?Ca alloy(Mg?2.0 wt%Zn?1.0 wt%Ca)with a diameter of 12mm was cut into 2mm thick specimens by wire?electrode cutting and used as substrates in this work.Prior to the VUV/O3activation,the specimens were mechanically polished with 1000#-5000# grit SiC papers;subsequently ultrasonically cleaned in acetone,ethanol and deionized water for 5min,respectively;and then dried in fl wing nitrogen,which were available for the experiments.

2.2.VUV/O3 surface activation

To improve adhesion force between the coating and the substrate,a simple one?step VUV/O3irradiation was used to activate the magnesium alloy surfaces as our previous work[31].The polished Mg substrates were loaded in a chamber with a Xe excimer lamp(SUS713,Ushio,Japan),providing a wavelength of 172nm and a full width at half?maximum(FWHM)of 14nm.The VUV/O3chamber was maintained at a vacuum pressure of<1kPa and the irradiance was 10mW/cm2.Then,the substrates were directly placed under the ultraviolet lamp source and irradiated for 15min in an atmospheric environment.After VUV/O3activation,the substrates were removed from the radiation chamber and placed in ambient air for use.

2.3.SF/SA composite coating

The silk fibroi solution was prepared by mixing lyophilized silk fibroi sponges and deionized water.The SA power was used to dissolved in the deionized water to obtain a uniformly SA solution.Both concentration of SF solution and SA solution were 2.0 wt%,which were mixed with different mass ratios by stirring.The various mass ratios of SF/SA solution were 100/0,80/20,70/30,60/40,50/50 and 0/100,respectively.Coating process was carried out using a micro?drop deposition method with a pipetting gun to quantitatively and directly drop the solution onto the activated substrates.This process avoids the disadvantages of fin operation in the smearing method and the waste of raw materials in the spin coating method.Usually,the prepared coatings are very thin,which are obtained by smearing method and spin coating method,resulting in a low efficien y in production.In this work,a uniform coating with the same thickness can be obtained by directly dropping a quantitative coating solution on the substrate surface.After the surfaces of substrates were covered evenly by SF/SA solution,all the samples were dried at 60°C and immersed in 80 wt% ethanol solution to change silk fibroi into insoluble structure[36,37].The procedure of fabricating SF/SA composite coating on Mg substrates is briefl illustrated in Fig.1.

Fig.1.Schematic drawings of the silk fibroin/sodiu alginate composite coatings fabricated on MgZnCa alloy.

2.4.Characterization methods

The morphologies of surfaces and cross?section were characterized by scanning electron microscopy(SEM,MERLIN Compact,ZEISS,Germany).The functional groups of the activated surface and coating were analyzed by flourie transform infrared(FT?IR,Nicolet is50,Thermo Fisher)with the resolution of 1 cm?1and the wavenumber from 500 cm?1to 4000 cm?1.X-ray photoelectron spectroscopy(XPS,ESCALAB 250Xi,Thermo Fisher)was used to analyze the chemical bonding of the composite coatings.The surface roughness and 3D morphologies with size of 5×5μm2was investigated by atomic force microscopy(AFM,Dimension Fastscan,Bruker).Five measurements were performed on each sample.The water contact angles of the coated surfaces were measured at room temperature(25°C)by a contact angle tester(DataPhysics,OCA 25?HTV).Adhesion force between coatings and substrates was evaluated by nanoscratch tests(Agilent Nano Indenter G200)and the loaded force of diamond indenter was increased from 0 to 100 mN with the scratch velocity of 30μm/s,which resulted in a scratching length of 300μm on each sample.Meanwhile,the mechanical properties of coatings were investigated by nanoindentation test using Agilent Nano Indenter G200 to obtain the hardness and modulus.The surface approach velocity was 10nm/s,the surface approach distance was 3000nm and the depth limit was 1000nm.Six measurements were carried out on each sample surface during the tests.

2.5.Corrosion behavior

Immersion experiments were conducted in Hank’s solution(KCl 0.4g/L,NaCl 8.0g/L,CaCl20.14g/L,KH2PO40.06g/L,Na2HPO4?12H2O 0.12g/L,MgSO4?7H2O 0.2g/L,NaHCO30.35g/L,pH=7.4)at 37°C.The samples were divided into seven groups(bare MgZnCa and coated MgZnCa fabricated by pure SF coating,pure SA coating and SF/SA composite coatings with mass ratios of 80/20,70/30,60/40 and 50/50,respectively)and each group was performed with 5 duplicates,respectively.After immersing for 10 days,the samples were rinsed with deionized water and dried in air.The morphologies of surface and cross-section after immersion were observed by SEM combined with EDS analysis.X-ray diffraction(XRD,X’PERT,Panalytical)scans were collected on an X-ray diffractometer in the 2θrange of 5-90° to ascertain the structure of corrosion products.During immersion,the Hank’s solution was refreshed every 2 days.The pH values of the solution were recorded by a pH meter(PHS?3C,Leici,Shanghai)and hydrogen released volume of all groups of samples were collected using drainage method,both of which were measured every 12 h during the tests.

Electrochemical measurements were conducted in an electrochemistry workstation(CHENHUA CHI660E,Shanghai)with a standard three?electrode system.The samples,the saturated calomel electrode(SCE)and the platinum electrode were used as working electrode,reference electrode,and counter electrode in Hank’s solution at 37°C,respectively.The electrochemical impedance spectroscopy(EIS)measurement was performed at a scanning frequency range of 105～10?2Hz with a single amplitude of 10mV.The EIS spectra were presented by Nyquist plots and Bode plots,which were quantitatively analyzed using corresponding equivalent circuit models.Fitting curves were performed with the ZSimpwin 3.30 software.For the experimental data statistics,fi e duplicates were made for each group.

3.Results

3.1.Surface and cross-sectional morphology

Fig.2 shows the surface morphologies of the coating fabricated on MgZnCa alloy,including the pure SF coating,pure SA coating and various SF/SA composite coatings.All coated samples exhibited the homogeneous surface morphologies without any cracks,which indicated that these film had covered the MgZnCa substrates uniformly and closely.In addition,the pure SF coated sample showed no obvious defects,indicating a good adhesion behavior as shown in Fig.2a.It has been previously reported that pure silk fibroi coating was brittle due to the poor mechanical properties and exhibited insufficien adhesion,always resulting in delamination and easily peeled off from the substrates[38].But after VUV/O3activation,the pure SF coating in our work showed a uniform and intact surface morphology,which suggested an improved adhesion force.Moreover,the SF/SA composite coating with the addition of different SA mass ratios had relatively rough surface morphologies compared to pure SF coating,which may be beneficia to cells adhesion in biocompatibility.With the increase of SA content in composite coating,the surface morphologies displayed certain differences but no defects,which may be attributed to the existence of intermolecular and intramolecular hydrogen bonds and interactions between SF and SA molecules[39,40].The apparent viscosity of obtained coating and the relative strength of intermolecular bond were different as the SA content changed in blend solution.However,phase separation characteristics could appear in the morphologies when the content of SA was too high.

Fig.2.SEM images for surface morphologies of(a)pure SF coating,(b)pure SA coating and SF/SA composite coatings with mass ratios of(c)80/20,(d)70/30,(e)60/40 and(f)50/50 on MgZnCa substrates.

Fig.3 shows the cross?sectional SEM images of coated samples,which were encapsulated by resin to observe the interface between coatings and substrates.The average thickness of the coatings was 5.0±0.5μm,and all the coatings completely covered onto the MgZnCa substrates.As shown in Fig.3a and b,obvious cracks occurred at the interface between coatings and substrates when directly coating pure SF or pure SA on magnesium alloy surface.However,there were no obvious defects appearing at the interface of VUV/O3-activated samples,indicating an excellent adhesion between coatings and substrates.Compact interface was profite by strong adhesion force,which could lead to an excellent corrosion resistance and stability during long?term service according to our previous studies[41].

Fig.3.SEM images for cross-sectional morphologies of(a)pure SF coating(non-activation),(b)pure SA coating(nonactivation),(c)pure SF coating,(d)pure SA coating and SF/SA composite coatings(VUV/O3 activation)with mass ratios of(e)80/20,(f)70/30,(g)60/40 and(h)50/50 on MgZnCa substrates.

Fig.4.AFM images(5×5μm2)for surface morphologies of(a)pure SF coating,(b)pure SA coating and SF/SA composite coatings with mass ratios of(c)80/20,(d)70/30,(e)60/40 and(f)50/50 on MgZnCa substrates.

3.2.The surface roughness and contact angle

Fig.4 clearly shows the 3D AFM images of the coated sample surfaces with the size of 5×5μm2,which reflecte the distinctive structures of various coatings.The surfaces of these coatings all presented uniform structure,and the pure SF coating surface was relatively smooth.However,the pure SA coating and SF/SA composite coating exhibited rougher structures,which corresponded to the SEM morphologies in Fig.2.Thus,surface roughness presented in Fig.5a directly determines the coating morphologies and the real exposed area of the coating.The measured surface roughness was expressed by root mean?square(RMS)values[42,43].The initial roughness of the MgZnCa substrate after VUV/O3activation was relatively high.After coating process,the pure SF coating had lowest RMS value,and the RMS value of SF/SA composite coating initially increased then decreased with the addictive mass ratios of SA.When the mass ratio of SF/SA reached 70/30,the surface roughness was the highest,which could be beneficia to the adhesion force at interface.The adhesion force between coating and substrate is mainly related to mechanical interlock at interface.Generally speaking,when the surface roughness is high,the mechanical interlock is strong,thus the adhesion force at the interface will be high[44].Under the same MgZnCa substrates,the RMS value of coating surface was directly coherent with the adhesion force.Therefore,compared to pure SF or pure SA coating,SF/SA composite coatings possessed higher RMS values,resulting in bigger exposed area and rougher morphologies.These coated samples with composite SF/SA coatings not only achieved the improvement of adhesion force at interface,but also profite the adhesion of more issues and cells.

Fig.5.(a)Surface roughness and(b)water contact angle for the surfaces of VUV activated substrate and pure SF coating,pure SA coating and SF/SA composite coatings with mass ratios of 80/20,70/30,60/40 and 50/50 on MgZnCa substrates.

Water contact angle(CA)is a very important evaluation for surface wettability(hydrophilicity or hydrophobicity),which was measured by sessile drop method[16].Generally,lower CA reflect better hydrophilicity.Fig.5b shows the CA of the substrate surface after VUV/O3activation and various coating surfaces on MgZnCa substrates,in which captured images of water drop are presented.All the measured CA were lower than 90.0 °,exhibiting the hydrophilic coatings.Especially,a super-hydrophilic surface on MgZnCa substrate was achieved with a CA value less than 2.0 ° after VUV/O3activation.This could be attributed to the cleaning effect of VUV radiation[34,35],based on which the droplets of the coating solution were completely spreading across the substrate surface,thus fabricating a uniform coating surface and controlling the thickness of the coatings.Moreover,poor hydrophilicity appeared with the increase of SA addition.When the mass ratios of SF/SA reached 50/50,the CA was almost 90 °,which indicated that the composite coating tended to show“hydrophobicity”so as the pure SA coating.The surface hydrophilicity is always related to the factors like surface roughness and functional groups.When the coating carries more functional groups,it would show better hydrophilicity,which is conducive to the formation of growth factor bond and adhesion,thus promoting the growth of cells and tissues.Silk fibroi is a kind of natural organic polymer protein,which has many kinds of amino acids.There are a lot of hydrophilic functional groups such as amino group,hydroxyl group,carboxyl group on the coating surface[45,46].Therefore,the SF/SA composite coating with high SF ration also shows good hydrophilicity in favor of the adhesion of growth factors.

Fig.6.FTIR patterns of(a)bare MgZnCa,VUV/O3?activated MgZnCa,pure SF coating,pure SA coating and(b)SF/SA composite coatings with mass ratios of 80/20,70/30,60/40 and 50/50;(c)and(d)are partially enlarged zoom from(b).

3.3.Chemical composition of the coating

The chemical states of the pure SF coating,pure SA coating and SF/SA composite coating were investigated by FTIR in the wavenumber ranges from 500 cm?1to 4000 cm?1.For bare MgZnCa substrate as shown in Fig.6a,three main peaks at 1102 cm?1,1388 cm?1and 3331 cm?1appeared and markedly enhanced after VUV/O3activation(red line).The absorption peaks at 1102 cm?1and 1388 cm?1were related to?OH in metal hydroxide stretching,and the absorption peak at 3331 cm?1was corresponding to O-H stretching of the H2O binding to the surface[47,48].This indicated the increased hydroxyl groups on the surface of magnesium alloy,resulting in better hydrophilicity of the substrates.It has been confirme that the increased functional groups and hydrophilicity of MgZnCa substrate could contribute the adhesion of coating on substrate surfaces.It has been reported that insufficien adhesion between organic silk and inorganic magnesium without any surface treatment always resulted in delamination of the coating under mechanical shearing or long?term service[16].Compared to the un-activated MgZnCa substrate,the VUV/O3activation process achieved super-hydrophilic surface,resulting in the improvement of functional groups on substrate.Thus,the adhesion force at the interface was strengthened.Moreover,the characteristic peak at 1051 cm?1was attributed to the C-N stretching vibration absorption[30].The characteristic peaks at 1633 cm?1,1529 cm?1and 1233 cm?1were assigned to the amide I,amide II of theβ-sheet conformation(Silk II)and amide III of the random coil structure(Silk I),respectively[49].The sharp peak at 3287 cm?1appeared in pure SF coating was related to the stack stretching vibration of N-H and O-H.For pure SA coating,the strong absorption peaks for six-member ring stretching vibration absorption,C-H bending vibration absorption and carbonyl group absorption appeared at approximately 1032 cm?1,1412 cm?1and 1601 cm?1,respectively.Similarly,the hydroxyl group of SA was detected by the strong absorption peak at 3308 cm?1.

Fig.7.(a)XPS spectra of(b)Mg 1s,(c)O 1s,(d)N 1s and(e)C 1s for bare,VUV/O3 activated MgZnCa and coated samples with pure SF coating,pure SA coating and SF/SA composite coating.

Fig.6b shows the FTIR spectra of the SF/SA composite coatings,which exhibits different absorption signals compared to the pure SF coating and pure SA coating.There were two main peaks appearing at around 1100 cm?1and the range of 3000-3800 cm?1.The two new peaks might be generated from the 1051 cm?1in pure SF coating combined with 1030 cm?1in pure SA coating and 3287 cm?1in pure SF coating combined with 3308 cm?1in pure SA coating,respectively.Four groups of the SF/SA composite coatings with different mass ratios had almost the same range of characteristic peak positions,but a clear distinction in the absorption peak width and intensity especially in partially enlarged zoom in Fig.6c and d.Fig.6c shows that the peak width at around 1100 cm?1became wider when the additional mass ration increased to 30% and 40%,and peak intensity was much sharper when the additional mass ration increased to 50% compared to that of SF/SA=80/20 composite coating.Moreover,the absorption intensity was significantl enhanced at the range of 3000-3800 cm?1with the addition of SA content as shown in Fig.6d.It is evident that the absorption intensity enhanced when the addition of SA content increased.This could be explained by the chemical bonding reaction and hydrogen bonding interaction between the SF molecules and SA molecules,which both contains abundant functional groups[29,50].Different kinds of chemical bonds and hydrogen bonds were formed in SF/SA composite coatings,resulting in different chemical states.As the addition ration of SA increased,the reaction of chemical bonds and proportion of hydrogen bonds were enhanced in SF and SA blended system.This suggested that the intermolecular interactions between the SF molecules and SA molecules were stronger,resulting in the stable composite coating system and in favor of in vitro and in vivo performance.

XPS analysis were employed to investigate the chemical bonding of samples,as shown in Fig.7.The survey spectra in Fig.7a shows that the major elements of these sample were Mg,O,N and C,which appeared on bare MgZnCa,VUV activated MgZnCa and coated samples with pure SF coating,pure SA coating and SF/SA composite coating,respectively.The content of various chemical bonds was concluded in Table 1.For uncoated MgZnCa,Mg 1s spectra(Fig.7b)showed characteristic peaks at 1303.0eV(Mg metal),1304.0eV(Mg(OH)2),1305.2eV(MgO)and 1306.3eV(MgCO3)[51].The O-H bond increased and the content of MgO decreased after VUV/O3activation,indicating the improved hydrophilicity of substrate surfaces.Moreover,the content of Mg(OH)2reduced and the chemical bond of Mg?O improved after coating,which suggested that the Mg?O?C bonds were generated after coating.For coated samples,the decomposed spectrum of O 1s(Fig.7c)showed characteristic peaks at 531.2eV(C-O-C),531.8eV(C-O)and 532.8eV(O-H)corresponding to the typical peaks of silk fibroi and sodium alginate[52].In addition to the N?related peaks at 399.5eV(N-C)and 400.0eV(N-H)[53]in Fig.7d,there was a significan Mg?Nchemical bond was detected at 397.8eV[54]for the samples coated by SF/SA composite coating.Combined the divided peaks in XPS spectra,at least two chemical bonds of Mg?O-C and Mg?N were newly formed at the bonding interface between MgZnCa substrates and SF/SA composite coatings.Furthermore,the C 1s spectrum was decomposed as shown in Fig.7e,and the fitte peaks for C 1s at 284.8eV,286.0eV,288.1eV and 289.8eV,respectively corresponded to C-C/C-H,C-O,COO-and CO32?binding in organic contaminants[55,56,57].After VUV/O3activation,the width of C 1s(organic related groups on substrate)significantl decreased,indicating the cleaning effect of vacuum ultraviolet?ozone on surface.During the mechanical polishing of magnesium alloy,the substrate surface absorbed organic contaminants in ambient air,which impeded the adhesion of sub-sequent coatings.Advantageously,VUV/O3activation removed the contaminants from substrates,and robust chemical bonds were formed at the interface,contributing to an excellent adhesion performance.

Table 1The content of various chemical bonds divided from XPS spectra.(total 100%).

3.4.Scratch tests

Adhesion force between coating and substrates was commonly estimated by nanoscratch testing,which was reflecte from the critical lateral force when appearing the initial coating failure[58].Fig.8 shows the critical lateral force corresponding to the scratch resistance of the coated MgZnCa samples,in which the morphologies of scratches resulted from different coatings were detected by SEM.As shown in Fig.8a and b,the adhesion force of direct-coated samples was very low due to the poor physical adhesion.However,after VUV/O3activation,the adhesion force of pure SF coating and pure SA coating on MgZnCa substrates increased to approximately 4.9 mN and 5.6 mN at around 10s,respectively.This could be attributed to the generated chemical bonds at the interface according to our previous work[31,44].Moreover,the adhesion force of SF/SA composite coatings was enhanced with the addition of SA,but fell back to a lower value when the SA content was high,which could be attributed to the phase separation.The adhesion force of composite coating with the mass ratio of 70/30 was higher than 15 mN,which was almost three times of the pure SF coating and pure SA coating.The smooth and undamaged morphology in nanoscratch SEM image also reflecte a superior adhesion on the substrate,as shown in Fig.8d.In contrast,other scratches exhibited obvious damages,such as cracks,tears and other defects,which indicated failure of coating and poor adhesion force between coating and substrate.According to the chemical state of the coatings,the intermolecular interactions were enhanced with the increased SA content,resulting in more viscous composite coatings.The viscosity of the blended solution would affect the adhesion performance of the coating[59,60].Appropriate viscosity of coating was conducive to the improvement of adhesion force under the same conditions.

3.5.Mechanical properties

For the coatings,the ability of anti-wear and deformation usually depends on the improvement of hardness and modulus.The higher the hardness and elastic modulus are,the stronger the anti-wear performance and the smaller the deformation of the sample under high stress or high contact pressure are.Generally,the method of improving the strength and modulus of the material is to increase the ratio of the reinforcing phase in the coating.However,this strategy will lead to the decrease of the toughness of the coating.When the coating material is used in the environment of high impact load,it will crack because of its brittleness.In addition to high hardness,modulus is also an important factor to evaluate the mechanical properties of the coating.The ability of coating to resist external force aging and failure can be evaluated byH3/E2(H and E represent the hardness and modulus of the coating)[61].A highH3/E2ratio(the ability to resist failure due to strain)means that the coating material resists failure due to strain for a longer period of time to allow redistribution of applied loads over a large area,thereby delaying failure of the coating.Therefore,H3/E2marks the ability to resist deformation from external force.Fig.9 shows the hardness and modulus of different coatings measured by nanoindentation method.Compared to pure SF coating,both the hardness and modulus were improved after blending SA into SF,which indicated that the composite SF/SA coating had enhanced ability to resist external force and wear in the long?term service in vivo and in vitro.The calculatedH3/E2value were concluded in Table 2.The composite SF/SA coating with the mass ratio of 70/30 had the highestH3/E2value,which was significantl improved than the other coatings.Therefore,the addition of SA effectively improved the mechanical properties of the coating to ensure the stability of the coated structure in long?term service.

Table 2The hardness and modulus of various coatings on MgZnCa alloy.

Fig.8.Adhesion force and nanoscratch SEM images of(a)pure SF coating(non-activation),(b)pure SA coating(non-activation),(c)pure SF coating,(d)pure SA coating and SF/SA composite coatings(VUV/O3 activation)with mass ratios of(e)80/20,(f)70/30,(g)60/40 and(h)50/50 on MgZnCa substrates.

Fig.9.Hardness and modulus of(a)pure SF coating,(b)pure SA coating and SF/SA composite coatings with mass ratios of(c)80/20,(d)70/30,(e)60/40 and(f)50/50 on MgZnCa substrates.

3.6.Immersion test

The overfast corrosion of biodegradable magnesium alloys is a serious concern especially in the environment containing chlorine,which impedes its clinical application.The most serious problem mainly includes the local alkaline environment and rapid release of hydrogen,resulting in subcutaneous emphysema and even tissue necrosis.Therefore,the reduction of pH value and hydrogen releasing during immersion are two important factors for evaluating the corrosion rate of magnesium alloys.The pH values of bare MgZnCa samples exhibited a sharply increased trend,which reached to a highest point of 10.0 at the end of 10th day,as shown in Fig.10.The pH values of bare MgZnCa samples exhibited a sharply increased trend,which reached to a highest point of 10.0 at the end of 10th day.However,coated MgZnCa samples showed much lower pH values than that of bare samples.Moreover,the composite SF/SA coatings effectively protected the substrates from the corrosion of ions,especially when SA content increased,the pH values presented fewer changes.Among these coatings,the fina pH of pure SA coating was the lowest at the 10th day,which could be attributed to many positive factors such as the existence of sodium ion and chelating reaction of SA[62].The volume of released hydrogen had a similar trend,which agreed with the pH value results.When the immersion time came to 10 days,the coated samples generated much lower hydrogen compared to bare MgZnCa.The measured volume of released hydrogen was all less than 2.0ml/cm2for coated samples,which indicated superior corrosion resistance in immersion test.According to the reported literature[19,63],the pH changes and released hydrogen were dramatically delayed,and the corrosion resistance of composite SF/SA coatings were also confirme to be advantageous compared to pure SF coating during the long-term immersion.

Fig.10.Variation of(a)pH values and(b)volume of evolved hydrogen for bare and coated samples fabricated by pure SF coating,pure SA coating and various SF/SA composite coatings.

Fig.11.Surface and cross-sectional morphologies of(a)bare and(b-g)coated samples fabricated by pure SF coating,pure SA coating and various SF/SA composite coatings after immersion in Hank’s solution for 10 days,respectively.The surface residual compositions in white line frame were characterized with EDS.

The surface and cross-sectional morphologies of the bare and coated samples after immersing for 10 days were investigated by SEM observation,as shown in Fig.11.After 10 days’immersion,the bare samples and coated samples exhibited obvious distinction in surface and cross-sectional morphologies.According to our previous work[41,44],due to the poor adhesion,part of the coating would fall off from the unactivated substrate,resulting in the failure of the protective effect.However,in this work,the coatings were intact and completely covered the substrate,which could be attributed to the excellent adhesion after VUV/O3activation.This indicated that the tightness of the interface directly affected the adhesion,which in turn determined the corrosion resistance of the sample.On the basis of the immersion test,big cracks were formed in the surface of bare MgZnCa samples,and thick corrosion product layer could be clearly noticed at the cross-section.According to previous studies[19,31,41,63],the corrosion products were mainly phosphate.There were thinner corrosion product layers appearing underneath the pure SF coating,pure SA coating and composite SF/SA coatings.However,the surface morphologies of various coatings were significantl different especially when the SA content was high(the mass ratio of 60/40 and 50/50),a thin reaction layer newly formed on the surface.This reaction layer combined with corrosion products,which gave rise to a more compact layer with the“coal cinder”morphology to delay the corrosion of magnesium alloy[62].Similar reaction layer was also observed on the cross-section of the samples coated with pure SA coating.There is no obvious reaction layer appearing at the interface of the composite coatings with the mass ratio of 80/20 and 70/30,but bulk reaction products could be observed in the surface morphologies.This reaction originated from the property of SA in the aggressive Hank’s solution,which contained sodium and chloride ions in electrolyte,resulting in cross-linking with divalent and polyvalent metallic ions in aqueous solution to form complex compounds[64-66].The interactions between Mg2+ions and cross-linking agent of SA occurred by the carboxylate and hydroxyl terminate groups,where chelating reaction took place,thus resulting SA-Mg complexes combined with the corrosion products formed on sample surfaces[67].This reaction layer exhibited a more compact structure in favor of the protection of Mg substrates.The EDS results of the seven groups of samples after immersion for 10 days were concluded in Table 3,including the elements of C,O,Mg,Na and P collected from the corroded surfaces.The highest P element on bare sample surface indicated the most serious corrosion of Mg alloy,whereas,the lower P content detected from the composite coating surfaces reflecte less phosphate products.The results of P combined with Mg and C suggested that the reaction layer was likely a mixture of[SA-Mg]+Mg3(PO4)2/Mg(OH)2.In the mixed corrosion products,Mg(OH)2was a porous structure,which could not protect the substrate in the subsequent corrosion process.Lower P and O indicated less phosphate and magnesium hydroxide products,indicating that the corrosion rate was delayed.Therefore,thicker reaction layer of[Mg-SA]with compact structure was generated to protect the substrate from corrosion.Such a reaction layer could effectively inhibit corrosion of MgZnCa alloy because of the smaller pore size compared to the loose and porous corrosion product layer of bare samples,but cannot terminate it completely.

Table 3The EDS results of the surfaces corresponding to Fig.10 in Hank’s solution for 10 days.

To further confir the structure of the corrosion products,the XRD pattern of the uncoated and the coated samples after immersion was shown in Fig.12.For bare MgZnCa alloy,the main products were the reaction products of magnesium and Hank’s solution including Mg(OH)2and Mg3(PO4)2[68,69].Moreover,due to the largest exposure area of the uncoated magnesium alloy,the samples underwent the most serious corrosion process,which resulted in the typical Mg(OH)2and Mg3(PO4)2with the stronger intense diffraction peaks of 2θmainly located around 15-40°[70,71].XRD pattern of coated samples with pure SF coatings and pure SA coatings not only showed the corrosion products of Mg(OH)2and Mg3(PO4)2but also indicated the diffraction peaks representing the their own characteristic structure.The diffraction peak at approximately 12.1° and 19.7° suggested the silk I structure of silk fibroi[50,72],SA typical structure were detected at 13.3° and 21.9°[73,74].The XRD diffraction peaks for SF and SA were very close,but the coated samples with composite SF/SA coatings exhibited distinct diffraction peaks around 14.4° and 17.5°,which demonstrated the[SA-Mg]reaction products existed in corrosion products after immersion.Therefore,the corrosion products of the samples with SF/SA composite coatings were likely a mixture of[SAMg]+Mg3(PO4)2/Mg(OH)2,which agreed with the element analysis in EDS results.

Fig.12.XRD patterns of bare samples and coated samples fabricated by pure SF coating,pure SA coating and SF/SA composite coatings after immersion in Hank’s solution for 10 days.

3.7.Electrochemical measurements

EIS is considered as a non-destructive method to assess the corrosion resistance and investigate the corrosion mechanism[75,76].Fig.13 shows the Nyquist plots and Bode plots of the seven groups of samples in Hank’s solution,which was fit ted by corresponding equivalent circuits(EC).In Nyquist EIS spectra,there was an obviously resistive-capacitive(RC)loop and resistive-inductive(RL)loop.Generally,the larger the diameter of the RC semicircle is,the better the corrosion resistance of the samples have.Bare MgZnCa samples exhibited smallest RC semicircle,indicating poor corrosion resistance,whereas,the coated samples with much larger RC loop were not easily corroded by Hank’s solution.Moreover,the corrosion resistance of the samples coated with pure SA coating was the best among these groups,exhibiting the impedance with an increased order of magnitude,which suggested that SA was a reinforcing phase with good performance to retard corrosion.With the increased SA content adding into SF,the composite coatings presented improved anticorrosion ability.Therefore,according to the different shapes of the EIS spectra,the equivalent circuit Rs(Q1(Q2R2)R1)and Rs(Q1(R1(Q2R2(RL(L))))were used to fi the data to compare the corrosion behavior of various samples,as embedded in Fig.13e and f respectively[77].In these ECs,Rswas the solution resistance.R1andQ1were related to the double layer resistance and capacitance corresponding to interfacial charge transfer reaction respectively.R2andQ2were indicative of the surface coatings on MgZnCa alloy.RLandLrepresented the inductance resistance and inductance corresponding to the inductive loop appearing at low frequency,respectively[78,79].All the parameters were fitte and concluded in Table 4.The reaction resistance value(R1)was the most important assessment indicator to determine the relative protection effect of the samples[80].TheR1value of the seven groups of samples from EIS fitte data followed the order:bare MgZnCa

Table 4Fitting results of EIS spectra for all groups of samples.

Fig.13.(a,b)Nyquist plots and(c,d)Bode plots of bare and coated samples fabricated by pure SF coating,pure SA coating and various SF/SA composite coatings;Equivalent circuit model for fittin the EIS data of(e)bare and(f)coated samples fabricated by pure SF coating,pure SA coating and various SF/SA composite coatings.

Fig.14 shows the calculatedRpvalues for all groups of samples,which exhibited distinct differences between bare MgZnCa alloy and coated MgZnCa alloys with various coatings.HigherRpvalue implies lower corrosion rate of the materials[81].Therefore,the corrosion resistance was enhanced when the SA was high in composite coating,which could be attributed to the superior anti-corrosion ability of SA layer with a much higher polarization resistance.The data of electrochemical measurement corroborated the results of immersion test well.

Fig.14.Polarization resistance of bare and coated samples fabricated by pure SF coating,pure SA coating and various SF/SA composite coatings.

As concluded,though the coated samples with the mass ratio of SF/SA=70/30 possessed the optimum mechanical properties and adhesion force,the corrosion resistance was not the best.This did not affect the application of the samples with composite SF/SA coatings in the medical treatment of bone implantation.The purpose of adding sodium alginate into silk fibroi is to use its superiority to improve the performance,thus we could control it.In other words,for medical implant degradable materials,especially bone implant materials,with the healing of bone,the implant is required to have an appropriate and matching degradation rate.Therefore,there is a long way to go and more research is needed to investigate the matching degradation rate of corrosion resistance in human body in the fiel of degradable materials.

4.Discussion

4.1.Adhesion mechanism

According to the results of chemical analysis,the schematic diagram of adhesion mechanism is proposed for the coated MgZnCa alloy fabricated by SF/SA composite coatings,as shown in Fig.15.Because the surface of primary MgZnCa substrate contained organic contaminants and lacked functional groups,the poor physical adhesion of coatings always resulted in peeling or delamination.However,the substrate surfaces were cleaned and modifie by hydroxyl groups after VUV/O3activation(compare Fig.6a black line with red line).This could be explained by the two aspects of VUV/O3effect,that was,surface cleaning and surface activation.The firs process was related to the Reactions(1)and(2)during ultraviolet irradiation as follows[35].Oxygen were converted into oxygen radicals under the ultraviolet light,and then reacted with oxygen to form ozone,as presented in Fig.15a.Oxygen converted into oxygen radicals under the ultraviolet light,and then reacted with oxygen to form ozone.In this way,the organic contaminants on the substrate surface would be decomposed into water and carbon dioxide by them,which were pumped out of the system by vacuum pump,thus completing the removal of contaminants.

After VUV/O3activation,the samples were directly placed in atmospheric environment and the substrate surface was exposed to air.Because the MgZnCa surface was super-clean and super-hydrophilic,the surface energy was very high.In this case,the substrate surface was easy to adsorb water molecules in air.Moreover,Mg metal is very active and can easily react with water molecules to generate Mg(OH)2.After removing the contaminants on the surfaces,cleaned substrates were exposed to ambient air,which contributed to absorb the water molecules and resulted in an improved hydrophilicity.Meanwhile,oxygen radicals generated by the 200% photo efficien y of VUV irradiation underwent interaction with water molecules(Reaction(3))[82,83],which contributed to an increased hydroxyl groups on MgZnCa surfaces,as depicted in Fig.15b.Therefore,there were more opportunities for MgZnCa alloy to bond with various coatings without any conversion layer.

Fig.15.Schematic diagram of adhesion mechanism for SF/SA composite coating on MgZnCa via(a)VUV/O3 surface activation,(b)exposing to air and(c)coating process.

When SF/SA composite coatings spread on the surface of magnesium alloy,the increased hydroxyl groups caused by VUV/O3activation promoted the chemical reactions with function groups(hydroxyl,amino and carboxyl groups)in SF and SA,significantl improving the adhesion force between the coating and substrate[84].Thus,robust chemical bonds were formed at the interface after dehydration.Meanwhile,hydrogen bonds and van der Waals were also components of adhesion force.Moreover,according to the FTIR spectra in Fig.6b,the abundant functional groups in SF molecules and SA molecules promoted that the chemical reaction and intermolecular interactions occurred when the composite coatings adhered on MgZnCa surface,as shown in Fig.15c.This indicated that the SF/SA coatings had more compact structure compared to pure coatings,and this was the reason for the improved corrosion resistance with the increased SA content.However,when the content of SA was too high,the phase separation led to the decreased adhesion force.Therefore,the appropriate proportion of SA was conducive to the increase of adhesion,and the SF/SA mass ratio of about 70/30 was optimal in this work.

4.2.Corrosion mechanism

Fig.16 illustrates the corrosion mechanism of the coated MgZnCa samples fabricated by SF/SA composite coatings in Hank’s solution.The degradation process mainly included four stages when the coated samples were immersed in the electrolyte containing corrosive ions,such as Cl-,,HCO?3 and H2PO4?.Mg alloy is a very active metal,which could be easily react with water molecules in solution,resulting in the beginning of magnesium alloy as shown in Fig.16a.The generated OH?and H2brought out the change of pH value and release of hydrogen in immersion tests[85],corresponding to the investigation in Fig.10.Reaction(4)shows the initial Mg alloy corrosion process.After that,the initial corrosion product,magnesium hydroxide(Mg(OH)2),was formed on the surface of magnesium alloy by the combination of Mg2+and OH?,and gradually deposited on the coating surfaces(Reaction(5)),as shown in Fig.16b.However,the corrosion product layer was loose and porous,which could not resist the aggressive corrosion process.Fortunately,the SA added in composite coating had the property of crosslinking with divalent metal ions(Mg2+)to form complex compounds[64-66],as expressed in Reaction(6).Thus,newly generated[SA-Mg]with more compact structure was formed by chelating reaction to improve the corrosion resistance,making up for the shortage of magnesium hydroxide.And this process was mainly attributed to the binding of Mg2+with the G-G blocks and some particular parts of the G-M blocks in the molecular chain of SA[62].Meanwhile,the H+was released through this reaction,contributing to the fewer changes in pH of alkaline environment.This could explain the pH results in Fig.9a,corresponding to the lower pH values with the increased SA content.The cross-sectional morphologies in Fig.11 indeed presented obvious[SA-Mg]product layer,which confirme that the EIS results of the samples with composite coatings exhibited enhanced corrosion resistance especially when the mass ratio was 60/50 and 50/50.According to the EDS analysis of the sample surfaces,P element was detected and had a decreased trend with the addition of SA content.This indicated that the magnesium phosphate was also formed(Reaction(7))on the surface of the samples and gradually deposited in the fina reaction layer after the third stage(Fig.16c).Thus,Fig.16d illustrates that the mixture of[SA-Mg]+Mg3(PO4)2/Mg(OH)2]constituted the fina reaction layer to protect the substrates from the corrosion,which gave rise to the significantl improved anti-corrosion ability during service.

Fig.16.Schematic illustration of the corrosion mechanism:(a)initial Mg corrosion,(b)corrosion products deposition,(c)SA chelating reaction and(d)fina reaction layer for coated MgZnCa alloy fabricated by various SF/SA composite coatings.

5.Conclusions

In this work,we successfully fabricated SF/SA composite coatings on biodegradable MgZnCa alloys via simple VUV/O3surface activation method without any chemical conversion layer.The surface characterization suggested that the surface morphologies,surface roughness,surface wettability and surface functional groups were different with various mass ratios of SA addition,resulting in discrepant performance in adhesion force,mechanism property and corrosion resistance of composite coatings.The models for adhesion and corrosion processes were respectively established to illustrate the mechanisms of the improved performances.The main conclusions are as following.

?The adhesion force of composite coating was significantl enhanced when with the mass ratio of SF/SA was 70/30,which showed three times of the pure SF coating or pure SA coating.This superiority mainly came from the improved surface hydrophilicity of magnesium alloy,robust chemical bonds at the interface and intermolecular interactions between SF and SA molecules.

?The composite SF/SA coating with the mass ratio of 70/30 had the highestH3/E2value,which was significantl improved than the other coatings.The addition of SA could effectively improve the mechanical properties of the coating to ensure the stability of the coated structure in favor of resisting deformation from external force.

?The improved corrosion resistance of coated magnesium alloys was confirme by immersion tests and electrochemical measurement,indicating that the enhanced anticorrosion with increased SA content.Corrosion mechanism suggested that the superior corrosion resistance not only attributed to the addition of SA in composite coating,but also related to the reaction layer formed during corrosion process.

Declaration of Competing Interests

The authors declare that they do not have any conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China(Grant No.51975151),the China Postdoctoral Science Foundation(Grant No.2017M610207),the Heilongjiang Provincial Natural Science Foundation of China(Grant No.LH2019E041)and the Fundamental Research Funds for Central Universities(Grant No.HIT.NSRIF.2019005).