Arash Fattah-alhosseini, Maryam Molaei, Meisam Nouri, Kazem Babaei

Department of Materials Engineering, Bu-Ali Sina University, Hamedan 65178-38695, Iran

Abstract Mg and its alloys are suitable choices for implant materials due to their biodegradability and biocompatibility features.However, the high electrochemical activity of this family of biomaterials results in their fast degradation and severe corrosion in the physiological environment,producing hydrogen (H2) gas, and therefore increasing the pH of the environment.To meet the clinical requirements, the degradation rate of Mg biomaterials needs to be reduced.Nevertheless,higher corrosion resistance of Mg results in a low alkaline pH,weakening the antibacterial activity.Therefore, while the rapid degradation problem of Mg-based biomaterials needs to be addressed, good antibacterial properties are also necessary.By using the plasma electrolytic oxidation (PEO) surface modification technique, the antibacterial activity of Mg and its alloys can be enhanced while maintaining their corrosion protection properties at a high level.Throughout the PEO process, introducing antibacterial agents into solutions results in a major increase in antibacterial activity of the coatings.Moreover, post- or pre-processing on PEO coatings can provide better protection against bacteria.In this review, the antibacterial activity of PEO coatings applied on Mg and also its alloys will be discussed in more detail.

Keywords: Antibacterial activity; Magnesium; Plasma electrolytic oxidation (PEO); Gram-positive bacteria; Gram-negative bacteria.

1.Introduction

Being biocompatible and biodegradable, Mg and its alloys are very attractive to the industry of implants including cardiovascular stents and musculoskeletal systems [1-5].After implantation, Mg-based implants degrade in the human body gradually and are finally substituted by newly grown bone tissue.This eliminates the need for extra surgery to remove the implants and thus decreases the medical expenses and pains of the patients [6,7].Mg implants have low densities, high ratios of strength-to-weight, and also the most similar elastic modulus to that of human bone (3-20 GPa) in comparison to the other metallic implants, e.g., Co-based alloys, stainless steel, and Ti alloys [8-12].The Mg elastic modulus is about 45 GPa that prevents the effects of stress shielding while implanting into the human body [13-15].

Mg with an electrochemical potential of ?2.36 V versus a standard hydrogen electrode is a very reactive element in electrolytic solutions.Due to the high reactivity of Mg, hydrogen gas (H2) evolves as the corrosion by-product and, therefore,rapid degradation in the human body occurs (in the period of one to four months) [16-21].The corrosion process of Mg in physiological environments is complicated and the production of H2increases the pH of the environment.By increasing the concentration of the hydroxide (OH?) near to the surface of the Mg implant to a certain extent, a layer of calcium or magnesium phosphates forms on the surface, leading to a changed interface between the in vivo bio-environment and the implant and a fast degradation.This dynamic interface, shown in Fig.1, is distinct from the interfaces on other biomedical metals and alloys such as Ti and stainless steels.Although Mg corrosion products of in vivo biodegradation can be simply absorbed and metabolized by the human body[22,23], the quick degradation results in loss of mechanical integrity before the healing of the bone [24-26].Besides, H2gas evolution within Mg corrosion can produce subcutaneous gas bubbles.These gas bubbles can cause tissue layer separation near the implant [27].Therefore, the surface of the Mgbased implants should be modified to control the evolution of H2, localized basification, in addition to surface degradation[28].In addition, the spontaneous oxide film (MgO) formed on Mg does not cover the entire underlying metallic surface because of a shrink in the volume; the production of MgO from pure metallic Mg leads to a volume reduction of about 20% [29-31].The weak corrosion resistance of Mg limits its clinical applications [32-36].On the other hand, improving the corrosion performance of Mg leads to a low alkaline pH which may compromise its anti-inflammatory or antibacterial activities [37,38].The low pH of the human body environment causes Mg implants to exhibit weak antibacterial activity [39].The weak antibacterial property of the implants is an important issue and the infections caused by bacteria on the implants are responsible for diseases and mortality in humans[40-43].

Fig.1.The schematic of the dynamic interface between the bio-environment and the Mg-based biomaterial within the degradation of the surface [28].(Reproduced with permission from Ref.[28]; License Number: 4,982,840,371,495, License date: Jan 05, 2021).

Post-treatment, surface coating, and alloying are proper techniques to control the rate of degradation in the Mg and also its alloys as well as enhance their antibacterial activity [44-49].The plasma electrolytic oxidation (PEO)process, derived from conventional anodizing, is an advanced environmental-friendly and efficient coating method that can deposit corrosion protective and antibacterial ceramic layers on the surface of Mg and its alloys [50-55].Anodic and thermal oxidation, plasma chemical reactions, and thermolysis procedures at the discharge regions lead to a very complicated formation mechanism of PEO coatings having a double-layer structure [56-58].From a biological point of view, within the primary period of implantation, the inner dense layer of the PEO coatings can delay the corrosion attack rate, decline the evolution of H2, and boost the production of new bone tissue near the implants.Besides, the porous nature of the outer layer is worthy as a depot for bioactive constituents including bone morphogenic proteins or growth factors [50].Many attempts have been made to test the in vitro [59-67], in vivo[68-80], and antibacterial [39,44,49,53,81-98]performance of PEO coated Mg surfaces, however, there is a lack of a review paper to discuss and report the current achievements and research trends of antibacterial behavior of PEO coatings on Mg and its alloys.The current review aims to fill this gap and provide the authorship with a comprehensive discussion on the present results reported by the researchers active in this field.The details of the works regarding the antibacterial behavior of PEO coatings on Mg substrates are listed in Table 1 [39,44,49,53,81-98].

In the experiments performed in the research works,both types of gram-positive [99-105]and gram-negative[103,106,107]bacteria have been used to study the antibacterial behavior of PEO coatings.Tables 2 and 3 summarize the shape and characteristics of each gram-positive and gram-negative bacteria, respectively [108].For Mg substrate cases, onlyStaphylococcus aureus(S.aureus) [44,81-84,87-98],Escherichia coli(E.coli) [44,49,53,82,85-95,98], and

Porphyromonas gingivalis(P.gingivalis) [39]bacteria have been used.The aerobicS.aureusbacterium is a significant human pathogen that causes a great extent of clinical infections.It is a major reason for bacteremia and infective endocarditis in addition to osteoarticular, soft tissue, pleuropulmonary, and device-related infections [109,110].E.colitypically is discovered as a commensal within the human microbiota.Nevertheless, the plasticity of its genome has resulted in the organism’s evolution into pathogenic strains that can make syndromes and diseases of public health significance in animals and humans.E.colican grow quickly under suitable conditions of growth, replicating in ?20 min[111,112].P.gingivalisis a popular oral anaerobic bacteria and has been acknowledged as the main etiological factor for peri-implantitis [39].

2.The phase compositions of PEO coatings on Mg substrates

Typically, PEO coatings contain predominant substrate oxides and complex oxides originated from the components of the solution [113].According to the phase compositions characterization results in literature (Table 1), due to chemical oxidation reactions between the anions solution and thedissolving Mg2+ions of the substrate within the discharge channels created by the sparks under the electrical field[84,91,95], phases of MgO [39,44,53,81,83-85,87,89,91-97], MgAl2O4[85], Mg2SiO4[39,44,53,85,91,95], MgSiO3[53,93], Mg3(PO4) [83], Mg3(PO4)2[49,97], Mg2PO4(OH)[49], MgF2[49,87], and Ca3(PO4)2[84,97]were detected in PEO coatings on Mg substrates.Besides, including the additives in the electrolytes and post treatment of the PEO coatings may result in the formation of other phases such Ca(OH)2[86], Ca0.7OSr0.3[86], Cu3(PO4)2[97], Ag2CO3[49], Ag2O [49], TiO2[49,53], ZnO [92], Ca10(PO4)6(OH)2[53,92], and Si [95].

Table 1Details of PEO experiments.

Table 2The gram-positive bacteria shapes and characteristics [108].

Table 3The gram-negative bacteria shapes and characteristics [108].

By adding Ag-doped hydroxyapatite (Ag-HA) nanopowders to the electrolytes, no peak for HA was detected on Xray diffraction patterns (Fig.2), however, the FTIR spectrum verified the presence of HA in the coating layer.Also, the broad FTIR peaks which are between 1000 cm?1and 1030 cm?1may show the nanocrystalline or amorphous structure of the HA due to the rapid cooling of the oxide melt on the coating surface [86].The XRD peaks corresponded tothe electrolyte additives of sodium phytate (Na12Phy) [89],silver nitrate (AgNO3) [91], or CuO particles [39]have not been detected.However, the presence of Mg6Phy, as an organic substance, was confirmed by FTIR and XPS spectra[89].In the research by Aktug and his coworkers, they did not identify Ag or its compounds in the XRD pattern of PEO coating post-treated with a physical vapor deposition(PVD) deposited layer of Ag.Due to the physical bonding created by the PVD process, Ag could not react with bioceramic structures on plain PEO coating surface [95].In the case of the PEO/polycaprolactone (PCL) composite coating,due to the sealing effect of the PCL top layer, the characteristic peaks of Mg, MgO, and MgF2were hardly detected in the XRD analysis, while the characteristic peak of PCL was observed.However, in the case of PEO/PCL/PDAM composite coating, the polydopamine (PDAM) was not detected by XRD due to the very thin nature of the PDAM layer [87].

3.Morphology and microstructure of PEO coatings on Mg substrates

Generally, PEO coatings applied on Mg substrates have a porous structure due to the bubbles and spark discharges produced in the discharge channels accompanied by the avalanche of the electrons at the interface of the solution and oxide coating [44,49,53,81-88,90-93,95,96,98].In addition, on account of thermal stresses produced within the process, some microcracks are seen on the surface of coatings[49,82,84,86,90,92,94,95,98].The porous structure is readily observed in micrograph images of the PEO coatings with and without CuO nanoparticles (NPs) in Fig.3.The figure shows no significant difference in thickness between the two coatings, both within the range of 8-11 μm.However, the mean diameter of the micropores (5 μm) for the coating containing CuO NPs was larger than that of the coating with no CuO(2 μm) [97].The coating surface with AgNO3was more uni-form and the density of the micropores was lower compared to coating with no AgNO3[83].Doping AgNO3into the solution led to a fewer number of large and irregular pores and a thicker layer due to the higher primary current density and therefore, more intense anodizing reactions during the process [91].Adding Tannic acid into the PEO solution resulted in smaller micropores, no microcracks, and a thicker coating[82].The high amount of Ag-HA in the solution lowered the breakdown voltage and as a result augmented the number of discharge channels and declined the discharge channels opening size [86].After the addition of 2 g/l CH3COOAg into the electrolyte, the number and diameter of the micropores decreased (from 2.44 ± 0.57 μm to 1.56 ± 0.49 μm) since the pores were filled by nano-sized Ag-containing particles [49].

Fig.2.(a) XRD patterns and (b) FTIR spectra obtained from the PEO coated surfaces with different amounts of Ag-HA nanopowder (AgHA-0: without Ag-HA, AgHA-1: with 1 g/l Ag-HA, and AgHA10: with 10 g/l Ag-HA) [86].(With permission from Ref.[86]; License Number: 5,038,300,536,248, License date: Mar 29, 2021).

Fig.3.(a, c) Cross-section and (b, d) surface morphologies of coatings with and without CuO NPs, respectively [97].(With permission from Ref.[97];License Number: 5,038,300,892,217, License date: Mar 29, 2021).

Fig.4.Surface SEM micrographs of (a) bare Mg alloy, (b) PEO coating, (c) PEO/SA-Van composite coating, and (d) PEO/cross-linked SA-Van composite coating [93].(With permission from Ref.[93]; License Number:4,982,840,658,118, License date: Jan 05, 2021).

The porous morphology of the PEO coatings vanishes partially or completely after post-treatment processes such as conversion coating [94], dip- and spin-coating [84,88,98],electrophoretic deposition (EPD) [44,53], and hydrothermal[81].Based on the surface morphology micrographs shown in Fig.4, the surface of Van-loaded sodium alginate (SA)hydrogel film applied on the surface of the PEO coating was uniform.This was ascribed to the SA solution viscosity.After cross-linking by calcium ions, the micropores were revealed again since the hydrogel lost its fluidity [93].ThePEO/polycaprolactone (PCL) composite coating showed a much smoother and denser morphology than the simple PEO coating.After functionalizing by polydopamine (PDAM),the morphology of the composite coating did not change evidently and only some PDAM particles appeared [87].Nanostructured coating of zirconia (ZrO2) exhibited fewer voids, pinholes, and microcracks compared with simple PEO coating.This case was mostly associated with the nanostructure compactness [85].According to Fig.5, although a very small amount of Ag was uniformly spread over the surface of PEO coating, Ag PVD deposited coating did not fill the pores and did not alter the morphology of the porous surface of the PEO coating [95].The micropores average size on the surface of PEO coated as-casted Mg alloy (4 μm) was larger than the one on the as-solution Mg (2 μm).This showed that the Mg solution treatment affected the intensity of the PEO process reaction [96].

Fig.5.Surface images of PEO and Ag-deposited PEO coatings morphologies[95].(With permission from Ref.[95]; License Number: 4,982,840,828,374,License date: Jan 05, 2021).

4.Antibacterial activity mechanism of PEO coatings on Mg substrates

Infection is one of the most important issues after Mg implants surgery that causes damages to the tissue, raises the infection susceptibility, activates host defenses, and stimulates the generation of inflammatory mediators [114,115].Bacteria grow on the implant surface and form a biofilm, a group of multiple or single bacteria species surrounded by an extracellular polymeric substance (EPS), that can be difficult to treat with antibiotics [116-118].The formation of biofilm can be explained in three steps: attachment, maturation, and dispersion (Fig.6) [119].As bacterial cells attach and then adhere to the surface [120-122], biofilm starts to grow and then mature from a thin film to a shape structure of ‘mushroom’ or‘tower’.After maturation, the dispersal step that is vital for the life cycle of biofilm initiates [119].Infections made by biofilm-forming bacteria are hard to treat and it is necessary to seek new biofilm inhibitors [118].

The PEO process is a promising way to improve the antibacterial activity of Mg implants using an antibacterial agent-doped electrolyte [39,49,82,83,86,91,97].Adding Ag or Cu-based additives to the electrolytes leads to the incorporation of these elements into the coatings and the release of Ag+[49,86,91]and Cu2+[97]ions during the culturing process.This gives PEO coatings an inhibitory effect against bacteria.The released Ag+ions attach to the cell membrane and kill the bacteria.The Ag+ions would break the bacteria wall, bind with organic groups and thiols of enzymes,causing disorder of normal bacteria permeability and respiration function, even changing DNA of bacteria by reacting with sulfur- or phosphorus-containing compounds [49,123].Cu kills the bacteria through a series of reactions.First,electrostatic forces make Cu ions to be adsorbed to the cell membrane of bacteria and limit the activity of bacteria which causes metabolic disorders and cell death.Secondly, Cu ions penetrate the bacteria membrane and destroy the integrity of the membrane.This leads to the leakage of the cytoplasm and the death of the cell.Thirdly, Cu ions can disrupt the activity of respiratory chains and disturb the gene replication process of the bacteria.Finally, the toxicity of Cu2+ions is due to the displacement of iron atoms from iron-sulfur clusters and leads to the damage of central catabolic and biosynthetic pathways [39,124].It should be noted that excessive amounts of Cu and Ag ions can lead to cytotoxicity [125-127].Fig.7 presents the morphologies ofS.aureuson surfaces of the samples after co-culturing for 24 h.For the PEO coating without CuO NPs and the uncoated Mg alloy, bacteria were identified on the surfaces.In the case of the PEO coating containing CuO NPs, no bacterium was observed on the surface that exhibits the excellent antibacterial influence of Cu [97].From the inhibition zone (diameter) images (Fig.8)and the procedure of the disk diffusion technique (resistant area (R) ≈0-5 mm, medium-sensitive area (M) ≈6-15 mm,and sensitive area (S) ≥16 mm), it would be possible to say that the prohibition zone of the coated specimen in the solution containing AgNO3was 40 mm and the coating was bacterial resistant.However, the antibacterial activity of the coated specimen in the AgNO3free solution was identified as medium-sensitive (zone diameter ?15 mm) [83].Therefore, AgNO3which is used extensively in preventing eye infections and treating burns, suppurating superficial wounds, tropical ulcers, and lesions caused by pemphigus and other skin diseases can be considered as a promising antibacterial additive in PEO processes [128,129].The phenolic hydroxyl groups of Tannic acid moieties are able to damage the bacteria and destroy them by interacting with their wall and membrane.On the other hand, Tannic acid can form chelates with metal ions and reduce the activity of metalloenzymes.Metalloenzymes are vital for bacterial growth and lacking them results in destroying the adherent bacteria.Thus, Tannic acid is known as a suitable antibacterial additive [82].

Fig.6.The schematic of the biofilm formation process [40].(Reproduced with permission from Ref.[40]; License Number: 4,982,870,348,746, License date:Jan 05, 2021).

Fig.7.SEM morphologies of S.aureus on surfaces of different samples after co-culture for 24 h with (a) PEO coated alloy, (b) Cu+PEO coated alloy,and (c) uncoated alloy [97].(Reproduced with permission from Ref.[97];License Number: 6, License date: Jan 05, 2021).

Large surface area, high reactivity, and easy penetrability into cell membranes of nanoscale additives help them to intercept the growth of biofilm and successfully disrupt the bacteria activity [130,131].Even though the detailed antibacterial performance mechanism of nanoscale materials (NMs) is not still clear, some mechanisms such as activities of photocatalyst, electrostatic interactions, the release of metal ions, generation of reactive oxygen species (ROS), damage of membrane, and cellular internalization are considered to play a role in the antibacterial performance.Among them, the generation of ROS has been believed as the dominant mechanismfor antibacterial behavior [132].Interactions between biofilms and NMs (Fig.9) can be explained by three vitally ordinal mechanisms: transport of NMs to the interface of the biofilmfluid; attachment to the surface of biofilm (outer region); and migration inside the biofilm.The physicochemical behavior of the NMs (shape, surface charge, size, hydrophobicity, and functional groups) specifies their interaction with components of the biofilm [133].It has been reported that nanoscale additives including Ag-HA [86], Ag [134-136], Cu [137,138],CuO[97],Cu2O[139],and ZnO[140]can give PEO coatings high protection against bacteria.

Fig.8.Inhibition zone (diameter) for samples coated in electrolytes (a) without AgNO3 and (b) with AgNO3 [83].

Fig.9.The stages schematic representation (transport, initial deposition,and migration) including phenomena of NMs transport in bacterial biofilms[40].(Reproduced with permission from Ref.[40]; License Number:4,982,870,348,746, License date: Jan 05, 2021).

Post-processing of PEO coatings by applying layers having antibacterial components can further improve the antibacterial activity of PEO coatings on Mg substrates[44,53,81,84,85,87,88,92-95,98].According to the inhibition zones observed in Fig.10 (a, b, c), bare Mg alloy demonstrated a smaller prohibition area in comparison to the duplex PEO/zinc-doped hydroxyapatite (PEO/ZH) and simple PEO coatings, showing the higher antibacterial capability of the coated specimens againstE.coli.In this regard, the prohibition area diameter of uncoated Mg alloy and PEO and PEO/ZH coatings was in the range of 1.1, 3.2, and 4.8 mm,respectively.As can be seen in Fig.10(e), a bigger number ofE.colicolonies were seen for the bare Mg alloy in comparison to the coated specimens (Fig.10 (f and g).The number ofE.colicolonies for PEO/ZH and PEO coatings declined about 92 and 65%, respectively, proposing good antibacterial behavior of these specimens.Considering the mechanism of high antibacterial activity for the PEO/ZH coating, it was implied that metal ions including Zn2+embedment into the coating create strong bonds with amino, thiol, and membrane carboxyl groups in proteins of microorganisms, resulting in alterations of structure.A membrane of a microorganism having structural alterations shows a remarkable rise in permeability, making the cells of the microorganism to be unable to significantly monitor proteins transport by the plasma membrane and finally, resulting in the death of the cells[53,141].The Zn-incorporated hydrothermal layers on PEO coatings can potently inhibit the proliferation ofS.aureusbacteria.This was due to the Zn2+antibacterial activity [81].Based on the colony growth images in Fig.11, PEO coating and PEO coating post-treated with phytic acid conversion coating (Cu-0) possessed no and low antibacterial property against bothS.aureusandE.colibacteria, respectively.In Cu-loaded PEO/phytic acid composite coatings, the number of alive bacteria slightly declined with the prolongation of the Cu loading, and the antibacterial effect was improved.This was due to the introduction of Cu2+ions in the Cu-loaded coatings which impressively prohibited the growth and propagation of the bacteria.The contact of the negatively charged surface of the bacteria with Cu2+produces a micro-motive force.This force causes the release of Cu2+from the coating that can break the bacterial cell and destroy the bacteria.Higher content of Cu2+on the surface of the coating results in more reactions of Cu2+with the bacteria per unit of time and thus, stronger antibacterial activity [94].The weak bacterial colony formation of the Ag-deposited PEO coating surface can be explained by the inhibitor effect of Ag on the bacteria.However, stronger antibacterial activity against the gram-negativeE.colithan the gram-positiveS.aureuswas observed.This may be ascribed to the thick-structured peptidoglycan film of gram-positive bacteria avoiding the movement of the Ag+ions through the cell wall of bacteria,and therefore declining the Ag inhibitory effects [95].Based on the SEM images in Fig.12, bothE.coliandS.aureusbacteria proliferated well on PEO/polycaprolactone (PCL)composite coating, showing no antibacterial capability of this coating.Polydopamine (PDAM) modification did not devote the PEO/PCL composite coating antibacterial protection against eitherS.aureusorE.coli.Nevertheless, after immobilizing polyhexamethylene biguanidine (PHMB) in the composite coating through the PDAM assistance, significant antibacterial abilities against bothE.coliandS.aureuswere seen with almost no living bacteria on the surface.The relative surface potential was augmented by immobilizing PHMB on the composite coating that simply drew the membrane of anionic microbe into the cationic bio-interface.Then, the force of coulombic interrupted the bacterial membrane and finally destroyed the bacteria [87].The main contribution of antibacterial property of assembled polyelectrolyte layer on PEO coating ascribed to the lysozyme polycationic solution.As the Lysozyme and the negatively charged phospholipid bilayer of the cell membrane come together, the hydrophobic tail of lysozyme is accommodated into the lipid bilayer and makes the membrane integrity annihilate, resulting in the prohibition of the growth of bacteria [98].The higher antibacterial activity of PEO/sodium alginate-vancomycin and PEO/cross-linked sodium alginate-vancomycin composite coatings againstS.aureuswas due to the high activity of vancomycin released in the sodium alginate hydrogel coatings against various gram-positive bacteria [93].The deposited monticellite (CaMgSiO4) coating loaded with tetracycline onPEO coating significantly inhibited the growth of bacterial cells due to the release of tetracycline.The influence of tetracycline hydrochloride was more againstS.aureusthanE.colibacteria.This was attributable to the dichotomy of cellular wall structures betweenE.coliandS.aureus[44].

Fig.10.Inhibition zones of (a) bare Mg alloy, (b) PEO coating, and (c) PEO/ZH coating and (d) antibacterial test photograph of the (d) control, (e)uncoated Mg alloy, (f) PEO coating, and (g) PEO/ZH coating against E.coli bacteria for one day [53].(With permission from Ref.[53]; License Number:4,982,841,473,345, License date: Jan 05, 2021).

Fig.11.Growth of colonies of S.aureus and E.coli on the surfaces of coatings [94].(With permission from Ref.[94]; License Number: 4,982,850,594,194,License date: Jan 05, 2021).

Fig.12.Morphologies of (a, b, c) S.aureus and (d, e, f) E.coli cultured on (a, d) PEO/PCL, (b, e) PEO/PCL/PDAM and (c, f) PEO/PCL/PDAM/P composite coatings [87].(With permission from Ref.[87]; License Number: 4,982,850,091,769, License date: Jan 05, 2021).

Fig.13.Pictures of the agar plates incubated under a condition in the samples of (a) blank, (b) uncoated Mg alloy, (c) PEO coating, (d) PEO/CS composite coating, and the number of S.aureus colonies [84].(With permission from Ref.[84]; License Number: 4,982,850,341,257, License date: Jan 05, 2021).

Fig.14.(a) Bare Mg alloy, (b) PEO coating, and (c) composite coating of PEO/nanostructured zirconia inhibition zones, and (d) growth inhibition zones against E.coli bacteria for uncoated and coated specimens after 1 day [85].(With permission from Ref.[85]; License Number: 4,982,841,349,840, License date: Jan 05, 2021).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Apart from the chemical composition of the coatings, the antibacterial performance of PEO coatings on Mg substrates depends on the pH value of the bacterial suspension.As mentioned earlier, a higher value of pH results in a stronger antibacterial activity.Ren et al.observed that the PEO coated Mg alloy maintained its antibacterial functionality against bothE.coliandS.aureuseven after 24 h of co-culturing with the bacteria suspension because of the change of the pH value from 7.4 to 8.0 in the first 6 h, then to 8.5 at 12 h,and finally a slower increase to 8.8 until 24 h.However, the change was not as much as that caused by the pure Mg (up to 10).As PEO coating had a high content of cracks and pores,prolonging the co-culturing period of bacteria from 6 to 24 h gave the electrolyte the chance of more penetration into the Mg substrate and this would result in more degradation of the substrate and greater levels of the pH[90].In another research study, while at the early immersion time (6 h) in the bacterial suspension, the PEO coated Mg alloy antibacterial property was not obvious, with the increase in the co-cultured time,the antibacterial property was increased due to the increase in the pH value.This was because the proliferation ofS.au-reusis inhibited after pH exceeds 8.5?9 and a higher alkalinity level causes the bacteria to have a fewer chance of survival.The PEO coating containing CuO particles had low degradation behavior, however, due to the continuous release of Cu2+ions, this coating exhibited the highest antibacterial property[97].It can be inferred from Fig.13 that the PEO coating possessed a higher CFU count (253) than uncoated Mg alloy(13) which can be attributed to the very high pH (?9.3) of Mg alloy that would prohibit the growth ofS.aureus.The duplex coating of PEO/chitosan (CS) had a higher antibacterial activity (CFUs: 89) than simple PEO coating.This was attributed to the pH value of about 8.4 and also the CS coating based on the contact-killing strategy which created a suitable environment for cell proliferation or growth [84].Based on the antibacterial test results in Fig.14, a smaller considerable prohibition zone was discovered under contact with uncoated Mg alloy (1.2 mm).The diameter of the prohibition zone was augmented for PEO (1.6 mm) and duplex PEO/nano-zirconia(2.5 mm)coatings that demonstrated the high antibacterial activity towardE.coli.The principal purpose of the proper antibacterial function of PEO/nano-zirconia duplex coating was related to the existence of zirconia NPs.The zirconia NPs escalateE.colimembrane permeability, then deposit in the areas of cytoplasmic in the cells and bacterial membrane and so decelerateE.coligrowth [85].The PEO coated specimen processed in the Na12Phy containing solution exhibited a great antibacterial capability versusE.coliandS.aureusdespite having no phytates in its composition.This was because the corrosion reaction of the coated sample in the tested bacterial suspensions resulted in an increase in the pH level of the environment and the death of bacteria [89].Based on Fig.15 (a-f), representative photos of viable colonies ofS.aureuscultured for 6 h, while there were some colonies on the plates of the uncoated as-cast and as-solution Mg-0.06Cu alloy (AC and AS, respectively) and pure Mg specimens, no bacteria were seen on the PEO coated as-cast (AC-MAO) and the PEO coated as-solution(AS-MAO)samples.According to CFU/ml ofS.aureusresulted from incubation for 3 and 6 h(Fig.15(g)), theS.aureuscolony counts declined from about 3 × 105to about 1 × 105, 4 × 104, and 6 × 104CFU/ml for pure Mg, and AC and AS alloys, respectively.The bacterial colony counts on the PEO coated samples declined intensely to about 2 × 104CFU/ml after a while of 3 h, and by doubling the incubation time to 6 h no bacteria was detected.The antibacterial rate (Fig.15(h)) for both AC and AS Mg alloys presented a higher rate than pure Mg.The AC-MAO and ASMAO specimens indicated the same rates (95% after 3 h and 100% after 6 h).The degradation of PEO coated Mg alloy was slow in Hank’s solution which resulted in the production of a few hydroxyl ions and therefore,a lower pH value(about 9.5).However, the coated alloy still possessed good antibacterial property because of the higher quantity of copper ions and short diffusion distance influenced by the contact-killing strategy [96].

Fig.15.Photos of the colonies growth on agar plates cultured with S.aureus for 6 h, (a) Ti6Al4V control; (b) pure Mg; (c) as-cast Mg alloy; (d) solution treated Mg alloy; (e) PEO treated as-cast Mg alloy; (f) PEO treated as-solution Mg alloy, and CFU (mL) and antibacterial rate of S.aureus after incubation on distinct specimens for 3 and 6 h [96].(With permission from Ref.[96]; License Number: 5,038,301,079,460, License date: Mar 29, 2021).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Conclusions

Bacterial infections are one of the most important clinical problems in Mg-based biomaterials.To prevent these infections, one solution is treating the surface of Mg biomaterials with antibacterial coatings.PEO coatings prepared via antibacterial agents doped solutions are regarded as a proper candidate to overcome the low antibacterial protection of Mg-based biomaterials.The release of antibacterial agents such as Ag+and Cu2+ions incorporated into PEO coatings during the culturing process provides the coatings with an inhibitory effect against bacteria.Having a large surface area,high reactivity, and easy penetrability into cell membranes,nanoscale additives can improve the antibacterial efficiency of PEO coatings by destroying the biofilm.The mechanism for this antibacterial behavior includes moving to the interface of biofilm-fluid, attachment to the surface of the biofilm, and then migrating to it.Post-treated PEO coatings with layers having antibacterial elements or compounds such as zinc,copper, silver, zirconia, lysozyme, vancomycin, and tetracycline can further enhance the antibacterial behavior.The level of degradation of PEO-coated Mg-based biomaterials while immersed in bacterial suspension can influence the antibacterial activity of these coatings by changing the pH value.Increasing the pH weakens the chance for bacteria survival and improves the antibacterial behavior.Moreover, the porous nature of PEO coatings provides the penetrating paths for the bacterial suspension solution into the Mg substrate which can result in a higher risk of corrosion of the Mg substrate,an increase in pH value, and therefore, a higher antibacterial performance.