Srny.K, Bhuvneswri.S, Suvro Chtterjee, Rjendrn.N,*

a Department of Chemistry, College of Engineering Guindy Campus, Anna University, Chennai, Tamil Nadu 600 025, India

b Vascular Biology Lab, AU-KBC Research Centre, Anna University, Chromepet, Chennai, Tamil Nadu 600 044, India

Abstract The rapid degradation of Mg alloy was reduced by incorporating titanate on a fluorine-base anodized layer.The coating shows (i)excellent biomineralization ability, (ii) improved local and periodical corrosion behavior and (iii) enhanced expression of osteogenic factors(Runx2, Col 1, OCN and OPN) along with (iv) the antibacterial property.The fluorid and magnesium ions dissolution from the anodized layer is responsible for the better expression of osteogenic factors and antibacterial behavior.The preparation of the titanate incorporated anodized Mg alloy (Ti-AMg) is a facile solution to overcome the implant-associated bacterial infections with required biological functions including progression of bone ingrowth and biocompatibility.

? 2021 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: AZ31 Magnesium alloy; Titanate; Anodization; Localized electrochemical impedance spectroscopy; Osteogenic factor; Anti-bacterial.

1.Introduction

A great deal of research focusing on biodegradable implants composed of essential elements of the body has been carried out to improve the biocompatibility and to avoid the complications associated with long-lasting permanence of the implant [1,2].Magnesium (Mg), iron (Fe) and zinc (Zn) have gained attention as biodegradable implants.Magnesium is preferred as it is naturally present in the human body.It has an inevitable role in body metabolisms such as transport of potassium and calcium ions, increases the levels of osteocalcin in osteoblast cells, stabilizes the structure of RNA and DNA by interacting with the negatively charged O and N,and also acts as a cofactor for many enzymes [3,4].In addition, Mg and its alloys have mechanical properties (Young's modulus and specifi density) closer to that of cortical bone [5,6].

Despite its adorable advantages, it possesses certain foreseeable risks too which need to be rectifie when employing them in orthopedics.The rapid and uneven corrosion of the surface along with hyper hydrogen production and localized pH rise had always been a hindrance for its usage in biomedical applications [7,8].

Surface modificatio is the preferred solution to prevent rapid corrosion of Mg and its alloys [9].Among the various surface modificatio techniques, anodization is highly desirable because of its ease of operation, less time consumption and being cost-effective.This technique is well established in valve metals viz., Ti, Nb, Zr, etc.[10].However, anodization of magnesium is a complex process as the anodized layer formed acts as a barrier and prevents further oxidation [11].

Fluorides are widely used in the formation of the protective layer on the magnesium substrate as it is effective in protecting the surface from corrosion [12].Additionally, it reduces the bacterial burden on implant along with immune enhancement and incorporation of fluorid in the bones reduces the acidic dissolution [13].Titanates, are the type of ceramic material, facilitates the apatite formation with ion-exchange reactions.It is known for its osteoconductivity and antibacterial effect with the contained application on Ti with chemicalconversion coating [14,15].In the present work, we have attempted to incorporate magnesium titanates on the fluorine based anodized layer (Ti-AMg) to enhance the corrosion resistance and osteoconductivity.The surface chemistry analysis of the material will not provide sufficien information towards the viability of the implant in the body.Hence, we have extended the studies onin-vitrocell culture, ELISA, gene expression analysis and antibacterial evaluation.The gene and protein expressions of the samples were analyzed to understand the cellular mechanism behind the bone metabolism.In the opinion, cytocompatibility and antibacterial activity introduced in the coating supports more towards implant success.

2.Materials and methods

2.1.Anodization of magnesium alloy

AZ31 magnesium alloy sheets were obtained from Seoul National University, South Korea.The sheets were cut into a rectangular dimension of 25mm×15mm×2mm.The samples were mechanically polished with silicon carbide paper up to 2000 grits.The polished substrates were ultrasonically cleaned using acetone and double-distilled water.A DC source was connected in series to the multimeter (Keithley Instrument) which was directly controlled by the computer to record the current transient curve.The specimens were anodized at selected voltages ranging from 60V to 100V in ethylene glycol electrolyte containing 2mM concentration of HF and hexafluorotitani acid (H2TiF6).

2.2.Surface characterization

Scanning Electron Microscopy with Energy-dispersive Xray spectroscopy (SEM/ EDX, Hitachi S3400), Attenuated total reflectanc infrared spectroscopy (ATR-IR, Perkin Elmer Spectrum two,USA),X-ray Diffractometer(Empyrean,Malvern Panalytical) and X-ray photoelectron spectroscopy(XPS, ESCALAB 250XI) were employed to characterize the surface morphology, elemental composition, microstructural analysis and functional groups of the AZ31 Mg alloy and anodized.

2.3.Electrochemical analysis

The electrochemical workstation (PGSTAT model 302N,AUTOLAB) with Nova 2.0 software was employed for corrosion measurements.Three electrode cell system with a saturated calomel electrode (reference electrode), a platinum foil(counter electrode) and the test material (working electrode)were used.The potentiodynamic polarization curves were recorded for ± 250mV at a scan rate of 1mVs-1.All the corrosion studies were performed in SBF solution with the exposed surface area of 1 cm2[16].The corrosion rate was calculated using the Stern and Geary equation [17].

Where, ba- Anodic slope; bc- Cathodic slope

The local corrosion behavior of the samples was analyzed using localized electrochemical impedance spectroscopy(Scanning electrochemical workstation model 470).The electrochemical impedance data were obtained with a fi e electrode system.The working electrode was either bare or Ti-AMg sample with the specifi surface area of 1.96 cm2.The reference electrode was saturated calomel electrode (SCE)and the counter electrode was a platinum ring placed with the bi-electrode.The electrolyte was SBF solution at room temperature and in contact with air.The local electrochemical impedance measurements were performed at the corrosion potential of the system.The surface heterogeneity of the working electrode was analyzed at 11.3Hz in a surface area of 600×1000μm.The tip-to-surface distance was set at about 150μm and the step size used was 25μm.

2.4.Immersion studies

The osteoconductivity of the bare and Ti-AMg samples was assessed by immersing it in SBF solution for 7 days with the surface to volume ratio as 40mL/cm2.The SBF solution was changed on daily basis.The samples were analyzed using SEM and ATR-IR spectroscopy.

2.5.Cell culture studies

2.5.1.Cytotoxicity and cell morphology

The extracts were prepared according to EN ISO standards 10,993:5[18]and 10,993:12[19].The samples were sterilized by sonication for 20min in ethanol followed by 20min UV exposure.The samples were incubated in Minimum Essential Medium (MEM) for 24, 48 and 72h.

Mouse mesenchymal stem cells, MSCs (C3H10T1/2) and human osteoblast-like cells (MG63) were procured from the National centre for Cell Sciences (NCCS), Pune, India.The cells were cultured under standard culture conditions (5%CO2, 37 °C) in MEM containing 10% fetal bovine serum(FBS).The cytotoxicity of MG63 and MSC cells was assessed using 3-(4, 5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) in phosphate-buffered saline (PBS).Various concentrations (1%, 2%, 10%, 20%, 50% and 100%) of the extract were added to 96 well plates seeded with cells and incubated for 24h.The MTT assay was performed as described earlier [20].

The samples were exposed to various surface area to volume of the media viz., 3:2, 2:1 and 5:2 for cell morphology analysis.The cells were washed with PBS after 24h of incubation and stained with propidium iodide (PI) and fluorescei diacetate (FDA) solution respectively.Cell morphology was examined by a fluorescen microscope with a 20×objective.

2.5.2.Immunochemistry

The levels of osteopontin and osteocalcin were detected using the Bio-rad assay kit.The cell suspension was prepared by lysing the cells completely.The cell particulates were removed by centrifuging the samples at 1000rpm for 5min.Thequantification were done according to the manufacturer's instructions.

2.5.3.Protein estimation and western blotting

MG 63 cells were treated with the prepared implant extract for 7 days.The cells were lysed with RIPA buffer (Sigma-Aldrich, MO, USA) and protease inhibitor mixture (Sigma-Aldrich, USA).The total protein content of the cell lysate was estimated by Bradford assay.25μg of the protein was added to each well and separated with a polyacrylamide gel which was transferred into a nitrocellulose membrane (Bio-Rad, CA) by electroblotting.Then the secondary antibody conjugated with horseradish peroxidase was probed on the membrane and finall photographed.The mouse monoclonal antibody Runx2 (1:1000) and the secondary antibodies conjugated with horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology, CA.

2.5.4.Quantitative and semi-quantitative real-time RT- PCR analysis

After 7 days of culture, Trizol reagent was employed in isolating the total RNA of the cells [21].According to the manufacturer's protocol, cDNA was synthesized with a reverse transcriptase (RT) kit (Invitrogen, CA, USA).Kapa sybr fast qPCR kits (Kapa Biosystem, MA) in the Bio-Rad system was used real-time PCR analysis.The PCR procedure used was the initial denaturation at 95°C for 2min and the primer hybridization at 58°C for 15s followed by elongation at 68°C for 20s.The cycle was repeated for 40 times.The quantitative amount of mRNAs and miRNA expression was calculated using theΔΔCtmethod as reported earlier [22].Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)was used as a reference.All experiments were performed in triplicate.The primers employed in this study for real-time PCR are synthesized by Eurofin and are given below:

collagen

2.6.Antibacterial studies

Escherichia coli(E.coli, ATCC 1555) andStaphylococcus aureus(S.aureus, ATCC 547), were used in the study.The strains were cultured for 24h at 37°C in Luria broth (LB)medium.3ml of the bacterial suspension was added to each sample and incubated at 37°C for 24h in an orbital shaker.The bacteria in the culture medium were analyzed by the spread plate method.

Fig.1.Current transient (i-t) curves at the anodization potentials of 60, 80 and 100V for AZ31 Mg alloy.

3.Results and discussions

3.1.Anodization of magnesium alloy

The substrates were electrochemically anodized at 60, 80 and 100V, and the current transient (i-t) curves were recorded and shown in Fig.1.A sudden drop in current was observed initially for the substrates anodized at various potentials, due to the formation of the oxide layer [23].At the anodization potential of 60V, the sample showed fluctuation in current density and at 80V, a steady-state current density was achieved due to the concurrent formation and passivation of the surface layer.However,the higher anodization potential of 100V showed a fluctuatin behavior to a certain extent, after the initial drop, could be due to over-etching of the surface and passivation of the etched surface.

The surface morphology of the Mg alloy anodized at 60,80 and 100V is given in Fig.2 (a-c).The sample anodized at 60V showed a cracked surface, which is in correlation with the fluctuatio in current density.As the coating cracks,Mg ions from the underneath layer moves outwards causing a slightly fluctuatin current density.The sample anodized at 80V showed a uniform surface with small patches of the nanoporous structure due to the uniform anodized layer formation on the Mg alloy.However, the anodization potential is increased to 100V, the entire surface has pits and pores.The fluorin ions under the influenc of potential cause more pitting and migrate inwards and simultaneously react with the nearby active surface leading to the formation of the porous anodized layer.From the above observations, the sample anodized at 80V is found to be optimum.

At the optimized potential of 80V, the anodized fil thickness is measured using the SEM cross-sectional analysis which is shown in Fig.3.The coating thickness was found to be ～5μm.During anodization, the formation of the anodized layer offers resistance to the movement of ions as the thickness increases, which in turn reduces the growth rate of the anodized layer.The formed anodized layer breaks af-ter reaching the critical thickness due to increased resistance[24,25].To prevent the breaking of the anodized layer, hexafluorotitani acid was added in drops.The acid dissociates into H3O+and [TiF6]2-ion which helps in counteracting the resistance of the anodized layer and promote the growth [26].

Fig.2.SEM images of Ti-AMg at (a) 60V, (b) 80V, (c) 100V.

Fig.3.SEM image of Ti-AMg at 80V and its EDX profiles

To fin out the elements present on the uniform and smooth surface,EDX experiments were carried out and shown in Fig.3 (b & c).The EDX profil of the uniform surface of the Ti-AMg sample shows the presence of magnesium, oxygen and fluorin whereas the EDX profil of the nanoporous patches in the anodized fil showed the presence of magnesium, oxygen, fluorine zinc and aluminum.The difference in the EDX profil confirm that the presence of smooth and nanoporous structures was seen on theαandβphase of the alloy, respectively.The nanostructures might be formed on the secondary phase of the Mg alloy which is rich in aluminum [27].The ratio of Mg and F for the smooth surface and the nanoporous structure was found to be 0.55 and 2.65,respectively.The fluorin content was less on the nanoporous surface of the Ti-AMg sample at 80V as compared to that of the porous morphology.It could be attributed to the formation of other fluorin compounds viz.aluminum fluorid and zinc fluorid which are soluble in the electrolyte.On the whole, the anodized layer obtained at a potential difference of 80V was used for further studies, as it was compact and nanoporous.

Fig.4.ATR-IR spectrum of Ti-AMg.

Fig.5.GI-XRD analysis of Ti-AMg.

The formation of magnesium fluorid and magnesium hydroxyfluorid on Ti-AMg sample was confirme by ATRIR analysis and the spectrum was displayed in Fig.4.The Ti-AMg sample exhibits a peak at 3270 cm-1due to the stretching vibration of the O-H group, attributed to water absorption from the atmosphere.The band appear at 3700-3600 cm-1corresponds to interaction of Mg and OH [28,29].The characteristic absorption peaks at 1352 and 1610 cm-1are attributed to the presence of MgF2[30].The peak at 1440 cm-1is due to the interaction of Mg and O [31].The peak at 760 cm-1depicts the interaction of OH-with fluo rine [32].The broad peak observed in the range of 650-400 cm-1is due to the merging of MgO, MgF2and Mg(OH)xF1-xpeaks [20].

The GI-XRD spectrum of Ti-AMg alloy is displayed in Fig.5.The crystallineαandβphases of AZ31 Mg alloy demonstrates (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0),(1 0 3), (1 1 2), (2 0 1), (0 0 4) and (1 0 4) planes.The peaks of MgF2is in concordance with the JCPDS No.38-0882 [20].The titanate peaks are not seen because of their minimal quantity in the coating.

The XPS survey scan was carried out for Ti-AMg sample and the spectrum is given in Fig.6.The presences of magnesium, aluminum, oxygen, zinc and fluorin are observed, which are in good agreement with the EDX spectrum.A minute quantity of ～3% of Ti was seen.Further,the narrow spectral analysis was done for Mg, F, O and Ti.Fig.6b shows the narrow spectra of Mg 1s which comprises of triplet peaks at 1304.3, 1305.7 and 1306.6eV corresponds to magnesium hydroxyfluoride MgF2and Mg-OH [33,34].The relative peak area of the component magnesium hydroxyfluorid occupies 37.9% and 37.4% by MgF2.This indicates that most of the magnesium ions on the surface reacts with fluorin spontaneously and forms MgF2.

XPS core scan of the O 1s spectrum is displayed as Fig.6d.It consists of three peaks at 529.5,531.5 and 532.8eV which correspond to Mg-O, -OH and magnesium hydroxyflu oride, respectively [35,36].During the anodization, the oxygen from the atmosphere diffuses into the electrolytic solution or the water present in the HF acid reacts with Mg and forms Mg-O.The presence of hydroxyl ion peak may be due to the absorption of atmospheric water or the interaction between Mg and OH that formed during the anodized layer formation.The deconvoluted F (1s) spectra (given in Fig.6c) shows a doublet peak at 685.45 and 687.85eV which are typical for magnesium hydroxyfluorid and Mg-F interactions[37].From the XPS analysis, the mechanism of anodized layer formation can be given as,

Titanium (2p) narrow spectra shows a doublet peak at 457.8 and 463.8eV which corresponds to Mg-titanate [38].Titanium ion complexes are stable at low pH values.When the pH increases at the metal/solution interface induced as a result of hydronium ions reduction lead to the precipitation of Ti on the surface [39].On the whole, the anodized coating comprises of magnesium oxide, magnesium hydroxide,magnesium hydroxyfluorid and magnesium fluorid with the minimum quantity of magnesium titanate.

Fig.6.(a) XPS Survey scan of Ti-AMg.Core spectra of (b) Magnesium, (c) Fluorine, (d) Oxygen and (e) Titanium.

3.2.Apatite formation ability

The bare and Ti-AMg sample were immersed in SBF solution for 7 days to determine the osteoconductivity and are displayed in Fig.7.The bare exhibits pits, pores and agglomerated structures of calcium phosphate compounds and the corresponding EDX spectrum shows the presence of calcium and phosphorus with the ratio of 0.76.The degradation of Mg is faster in the bare and the released Mg2+ions stimulate the deposition of calcium phosphate compound promoting further deposition [40].Howbeit, the researchers have confirme that the crystallinity of the apatite is greatly affected by the Mg.The incorporation of Mg in the apatite crystal lattice reduces the crystallinity and makes it easily soluble [41].

The Ti-AMg sample exhibits agglomerated nanospheres of apatite.The presence of calcium,phosphorus along with magnesium and fluorin are observed.The Ca/P ratio was 1.52 which is lower than hydroxyapatite (1.67) because calcium ions could have been replaced by magnesium ions.Furthermore, the peak of fluorin and magnesium assures F-ion incorporation in HAp and the prevalence of the coating even after the immersion in SBF solution for 7 days.From ATR-IR studies (Fig.8), the Ti-AMg sample exhibits peaks at 1020 and 556 cm-1due to the stretching and bending modes of PO43-groups, respectively [42].The absorption peak located at 774 cm-1is assigned to the OH…F group [32].The carbonate ion exhibits peaks at 868 and 1419 cm-1.

XPS analysis has been carried out to further confir the apatite formation on the Ti-AMg sample, which was immersed in SBF solution, is given in Fig.9.The survey scan identifie the presence of calcium, phosphorous and oxygen in addition to other elements.The narrow XPS spectra of Ca(2p) at 347.4eV owes to Ca10(PO4)F2and, Ca (2p 1/2) at 351 and 352.47eV are assigned to CaCO3and Ca-OH of hydroxyapatite (HAp), respectively [43].The core-level binding energy position of P (2p) at 133.4eV corresponds to HAp or fluorapatit (FAp).The same is confirme with the O (1s)peak at 531.4eV [44].The other two O (1s) peaks at 529and 533.9eV correspond to Mg-O and C-O interaction in carbonate ion, respectively [45].It is reported that magnesium is readily replaced by Ca at the surface by adsorption, thereby increasing the formation of HAp [46].The ratio of Ca/P is 1.49 which agrees with EDX results.The F(1s)peak at 685.1 and 687.4eV can be identifie as fluorinate HAp and F-P interaction in FAp, respectively [47,48].The presence of magnesium and fluorin after immersion in SBF solution indicates the formation of magnesium incorporated fluorapatit which enhances the stimulation of bone cell response and reduces the corrosion rate by forming large crystals of HAp.The carbonate peaks present in the narrow scan spectra of Ca and O indicate that the apatite is carbonated which mimics the bone apatite [49].

Fig.8.ATR-IR spectrum of Ti-AMg after 7 days of immersion in SBF solution.

3.3.Electrochemical studies

Understanding the application background of magnesium alloy as a temporary bone implant, it is essential to analyze the corrosion behavior.As a bone implant, the degradation rate of magnesium should be compatible with the healing rate.At the initial stage, the implant should be corrosion resistant and support the cell adhesion.Later on,the degradation rate will be controlled by the cells adhered on the implant,thereby fetching sufficien time to the complete wound healing[50].

The bare and Ti-AMg samples were immersed in SBF solution for 7 days and the potentiodynamic polarization studies were done on 0th, 3rd, 5th and 7th day and are shown in Fig.10.The anodic and cathodic polarization branch of the Ti-AMg sample has shifted towards the lower current density indicating that the formed Ti-incorporated anodized layer was uniform and protects the substrate.The corrosion rate of the Ti-AMg sample had reduced significantl to 0.001mm/yr from 0.544mm/yr (bare).The results reveal that the Ti-AMg sample exhibits better corrosion resistance than the bare.

A considerable decrease in the icorrvalue was observed for the bare on the 3rd and 5th day of immersion due to the formation of Mg incorporated apatite.The icorrvalue of the bare drastically increased on the 7th day.Magnesium incorporation in the apatite destabilized the HAp structure and allowed dissolution at a faster rate than the normal HAp.The increased icorrvalue thereupon is possibly due to the dissolution of the formed apatite and breakage of the protective oxide layer.Significan oscillation in the polarization curve was observed due to the loosely bound or cracked passive fil on the bare[51].In the Ti-AMg sample, the icorrvalue was increasing on the 3rd day of immersion due to coating dissolution.Later on, icorrsteadily decreases as a result of fluorid incorporation during apatite formation [52].From the potentiodynamic polarization studies, it was observed that the Ti-AMg sample exhibited better corrosion resistance than the bare.Moreover,the corrosion resistance of the Ti-AMg sample has increased several folds.

Though, pitting is the major problem associated with Mg and its alloys.A closer observation of the surface with advanced corrosion techniques will help in analyzing the samples effectively [53].LEIS technique helps in analyzing the effectiveness of the coating in a discrete interval of distance.The samples were scanned for an area of 600×1000μm.From the area and line scan of the bare (Fig.11a & c), it is observed that the resistance varies from 1.72 to 15.6 KΩ.The localized resistance of the sample varies at every point.The loosely bound oxide layer formation starts on the bare immediately after immersion in SBF solution.0.5% of chlorine in the electrolyte reacts with the oxide layer and forms MgCl2,which is highly soluble in water, thereby exposing more fresh active Mg surface [54].The formation and dissolution of the oxide layer were not uniform on the entire surface.This contributes to the fluctuatio in the resistance of the bare.The phase segregation of the alloy may also contribute to the resistance variation of the bare.Therefore, the anodic activity of bare is intensive due to the faster degradation and Mg ions precipitation.

Fig.11 (b and d) show area and line scan of the Ti-AMg sample exhibited the localized resistance ranges from 780 to 859 KΩ.As it is clearly seen from the XPS result, the composition of the coating consists of MgO, MgF2and magnesium hydroxyfluorid with a minute quantity of Ti.Depending on the composition of the anodized layer at each point, the dissolution range varies and hence the variation in resistance is observed.The solubility of MgF2is 0.13g/l in water whereas MgO converted into MgCl2dissolves completely at the rate of 523g/l.Hence, the variation in the chemical constitution in Ti-AMg influence the local resistance.The barrier effect of Ti-AMg has the ability to delay the corrosion rate and offers a stable protection to the substrate.However, the local corrosion resistance of the Ti-AMg sample is several folds higher than the bare.

Fig.9.(a) XPS survey scan of Ti-AMg alloy after immersion in SBF solution for 7 days.Core spectra of (b) Magnesium, (c) Oxygen, (d) Fluorine, (e)Calcium and (f) Phosphorous.

Fig.10.Potentiodynamic polarization curve of the (a) Bare and (b) Ti-AMg immersed in SBF solution at different time periods.(c) The corresponding potentiodynamic polarization data.

3.4. In-vitro studies

The bare and Ti-AMg samples were incubated in MEM medium with all the supplements except the FBS for various time periods (24, 48 and 72h).The prepared extracts were added in the ratio of 1, 5, 10, 20, 50 and 100% along with the culturing medium and the viability of MG63 cells was checked using MTT assay is given in Fig.12.The cells grown without adding the implant extract is taken as control(PC).On 24 and 48h bare extract, as the concentration increases, the cell viability was improved by 12% with respect to control and there is no prominent change in the cell viability due to the increase in the extract concentration.On 72h bare extract, at higher concentrations, the proliferation rate had significantl increased by 15%.The addition of Mg accelerated the proliferation rate of the MG63 osteoblast-like cells [55].

Interestingly,the proliferation rates of the Ti-AMg samples in lower extract concentrations (24, 48 and 72h) are similar to the positive control.The 50 and 100% extract of the Ti-AMg sample showed a 5% higher proliferation rate than the control in 24 and 48h.The dissolution behavior of the fluorin from the coating affects thein vitrocellular response behavior.Literature reveals that fluorid ions rise the proliferation rate by activating the Ras-Raf-MAPK signaling pathway [56].In 72h extract, the proliferation rates decreased by 10% in the higher concentrations, howbeit it is in the agreeable range as the proliferation rate was above 70% [57].

For further confirmation MSC cells were used to confir the material's ability to support the proliferative behavior.The proliferation rate of bare had significantl increased to around 30% for all time periods.In the Ti-AMg sample, at 24 and 48h, the cell number was elevated by 10% in comparison with the control.The proliferation rate was dropped by 15%at higher concentrations in 72h extract.These results strongly suggest that the Ti-AMg sample supports cell proliferation.

For detailed understanding about the cultured MSC cells on Ti-AMg samples, observations are made under fluorescen microscopy after 24h of cell seeding as shown in Fig.13.The morphological analysis of the Ti-AMg sample was compared with the bare shown in the previous literature [20].In the Ti-AMg sample, the MSC cells were oriented and exhibit triangular polygonal shape.The extension of lamellopodia was seen.The ratio of minor and macro- axis is greater than one which confirm that the cells were flattene and adhered to the bare with increased contact ratio.The presence of the nano-morphological structures and release of fluorid and magnesium have enhanced the attachment of MSC cells in comparison to control [44,58,59].As the surface to volume ratio increases from 3:2 to 2:1, the cells attachments were good and the cell death is minimal.When the ratio is 5:2,the cells were attached to the surface with reduced spread-ing.The number of cell to cell communications was reduced along with increased cell death.

Fig.11.Localized electrochemical impedance spectroscopic images: Line scan and area scan images of Bare sample (a & c) and Ti-AMg (b & d).

Fig.12.MTT assay graphs representing the cell percent viability of (a) MG63 and (b) MSC cells at different extract concentrations.

3.4.Osteogenic marker expression

Runx2 is a major osteogenic transcription factor that is essential for the expression of various bone-related genes which includes osteocalcin, osteopontin, type 1 collagen and mineralization of bone [60].To directly address the effect of the chemical constituents of the implant extract at the molecular level on osteoblast differentiation, the expression of Runx2 was analyzed by semi-quantitative and quantitative RT-PCR,and western blot (Fig.14a,c and e).The mRNA of the Runx2 was down-regulated by 0.35 times in the bare and upregulated in the Ti-AMg sample (0.27 times).The total protein content was estimated by Bradford assay and found to be 8, 14 and 16μg/ml.Using western blot, the expression of the Runx2 protein was analyzed.The results show that the expression of Runx2 was increased in the Ti-AMg sample as compared to the control.

Fig.13.Morphology of MSC cells cultured on Ti-AMg with various surface to volume ratios.

Collagen acts as a micro-architectural base for bone mineralization and hence used as a marker [61].Fig.14 (b & c)shows the quantitative and semiquantitative PCR estimation of Col 1 genes.The expression of the Col 1 was reduced by 0.21 times in the bare and increased by 1.4 times in the Ti-AMg sample.

The imbalance in the expression of anti-calcificatio markers like OPN and pro-osteogenic markers like OCN results in calcification The expression of OCN and OPN levels was analyzed on the 3rd and 7th day using ELISA kit (Fig.15).The OCN levels were increasing from 13 to 19ng/ml in the control, whereas it was decreasing from 12 to 9ng/ml in the bare and increasing from 13 to 21ng/ml in the Ti-AMg sample, respectively.The OPN levels were elevated from 8.7 to 10.5ng/ml, 7.9 to 10.8ng/ml and 3.2 to 6.8mg/ml in the control, bare and Ti-AMg samples.The rise in OPN levels was seen on all the samples.Howbeit, the Ti-AMg sample exhibited lower OPN level compared to bare and control.

Fluorine is an essential element in the maintenance and growth of bone.Higher dosage of the fluorin leads to bone damages and bone-related disorders.Mg and F are the major constituents of the corrosion product in the Ti-AMg sample.Hence, the corrosion rate of the Ti-AMg sample determines the mechanisms involved in bone remodeling.The expression level of the osteogenic markers (Runx2, OCN, OPN and Col 1) helps in determining the osteoblast behavior of the bare and Ti-AMg sample.Overall, the expression level of the phenotypic markers was less in the bare and considerably high in the Ti-AMg sample except for OPN.The activation of Runx2 in the Ti-AMg sample leads to the increased expression of osteogenic markers.Col 1 expression is associated with the extracellular matrix synthesis and its increased level in the Ti-AMg suggests that F-ions play a key role in mineralization.The accumulation of OCN and OPN markers is observed inthe mineralization.However,OPN expression is also observed during the proliferation [62-64].The higher proliferation rate of the bare is responsible for the up-regulation of the OPN.The higher OCN levels in the Ti-AMg sample correlate to the apatite formation and enhancement of osteogenesis.The increased Mg concentrations in the bare improve the cell viability and decrease the Ca2+mediated mineralization through matrix Gla protein [65].The expression of OCN, Col 1 and ALP expression is influence by the soluble factors, whereas Runx2 regulation was free from the biological response [66].Results suggest that the Ti-AMg sample enhances the expression of osteogenic markers.

Fig.14.The gene expression analysis (a) Runx2, (b) Type 1 Col mRNA expression at days 7 using quantitative RT-PCR, (c) PCR Amplifie DNA run on agarose gel electrophoresis.(d) Total protein content of the cells using Bradford assay, (e) Western blot showing Runx2 protein expression.[** and *** indicate that the results were analyzed with Annova one way software to evaluate the statistical significanc value p＜0.02 and p＜0.05, respectively].

Fig.15.Quantificatio of (a) osteocalcin and (b) osteopontin using ELISA kit.*, ** and *** represent the results analyzed with Annova one way software to evaluate the statistical significanc value p＜0.01, p＜0.02 and p＜0.05, respectively.

Fig.16.The antibacterial effect of Bare and Ti-AMg.

3.5.Antibacterial studies

Microbial infection is the major cause of implant failure and causes a menace to the patient's life.The eradication of the infection is very difficult once the biofil was formed on the implant surface.In addition, the bacterial endotoxins activate the macrophages, which in turn activate the toll-likereceptors and finall leading to infection mediated osteolysis[67].These problems lead to implant failure and require subsequent surgeries.Hence,the implant with inherent antimicrobial property is preferred.The bacterial prohibition ability of the bare and Ti-AMg sample was studied with a colony count method and the bacterial reduction rate (BR) was shown in Fig.16.In gram-negative bacteria(E.coli),the BR was around 30% in the bare and 95% in the Ti-AMg sample.The BR of the gram-positive bacteria was 27% and 88% in the bare and Ti-AMg sample, respectively.

The corrosion rate of the bare and Ti-AMg sample determines the degree of antibacterial effect.The exact mechanism of the antibacterial activity of bare was unknown.The increase in alkalinity destabilizes the bacterial cell wall causing cell death [68].Mg2+ion released from the bare interacts with the negatively charged microbial cell wall.This alters themembrane integrity causing the leakage of the proteinaceous and intracellular constituents [69,70].The different standard potentials of the primary and secondary phases of AZ31 Mg alloy (bare) forms micro galvanic cells.The proton depleted region of the micro galvanic cell can inactive the synthesis of ATP and ion transport, which kills the bacteria [71].The antimicrobial activity of fluorid is facilitated by the generation of metal-fluorid complexes, interruption of the proton excretion in the cell and inhibition of bacterial enzymes.The bacterial cell membrane permeability is increased by fluorid ions causing acidificatio of cytoplasm.The fluorid in micromolar concentration is sufficien to inhibit bacterial growth[72].

4.Conclusion

The biodegradability and biocompatibility of Mg and its alloy make them an incredible material for orthopaedics.The establishment of an appropriate barrier between the substrate and the surrounding physiological flui is a demanding challenge.The expeditious deterioration of Mg was depressed by forming a novel anodized layer incorporated with titanates.The Ti-AMg layer is composed of MgO, MgF2, magnesium hydroxyfluorid and magnesium titanate.The periodical corrosion analysis showed the corrosion rates were steadily decreased to a greater extent by incorporation titanate on Ti-AMg sample implying that it could fetch sufficien time for healing without losing mechanical integrity.The localized corrosion studies revealed the resistance offered by the Ti-AMg was meager and uniform.Fluoride and Mg2+ions liberated from the Ti-AMg initiated the deposition of nanospherical structures of carbonated magnesium and fluorid incorporated apatite (bone-like apatite) assuring its bioactivity.

The nanostructures formed on the magnesium alloys directly impact the cell proliferation rate and the expression of the osteogenic markers.The quantity and physicochemical properties of the nanostructures and degraded products on the surface governs the significatio of the peri-implant tissues.The proliferation rate of the bare and the Ti-AMg sample was around 30 and 5%, respectively.Nevertheless, the expression of the osteogenic factors was increased for Ti-AMg sample compared to the bare.The release of Mg2+ions was high in bare that benefite the proliferation and prevented the expression of the osteogenic markers.In the Ti-AMg sample,the dissolution rate of Mg2+and F-ions were controlled by titanate incorporation, which favored the notable proliferation and osteogenic marker expression.The Ti-AMg sample exhibited antibacterial property against both gram-positive and gram-negative bacteria as an added advantage.As a whole,the obtained results have favored the Ti-AMg alloy for orthopedic usage.

Declaration of Competing Interest

There are no conflict of interest.

Acknowledgment

One of the authors K.Saranya is thankful to the Department of Science and Technology (DST), New Delhi, India for financia assistance under women scientist scheme (WOS-A)(Ref.No: SR/WOS-A/ET-17/2017).The authors are thankful to DST-FIST and UGC-DRS for providing instrumentation facilities to carry out this work.