Corrosion in Mg-alloy biomedical implants-the strategies to reduce the impact of the corrosion inflammator reaction and microbial activity

Soumy Sh,Wiy Lstri,Crolin Dini,Murni Nzir Srin,Hnr Hrmwn,Vlntim A.R.Br?o,Cortino Sukotjo,f,*,Christos Tkouis,g,*

aDepartment of Biomedical Engineering,University of Illinois at Chicago,Chicago,United States

b Fundamental Dental and Medical Sciences Department,Kulliyyah of Dentistry,International Islamic University Malaysia,Kuantan,Malaysia

c Department of Prosthodontics and Periodontology,Piracicaba Dental School,University of Campinas(UNICAMP),Piracicaba,Brazil

dInstitute of Systems Biology(INBIOSIS),Universiti Kebangsaan Malaysia,Bandar Baru Bangi,Selangor 43600,Malaysia

eDepartment of Mining,Metallurgical and Materials Engineering,Laval University,Quebec City,QC G1V 0A6,Canada

fDepartment of Restorative Dentistry,University of Illinois at Chicago College of Dentistry,Chicago,United States

g Department of Chemical Engineering,University of Illinois at Chicago,Chicago,United States

Abstract The most common complication of orthopedic surgery is implant failure,which can result in catastrophic injury and a significan financia burden for patients.Implant failure can be caused by a variety of factors,the most common of which are peri-implant infection(or implantrelated infection),excessive inflammator response which caused pain and aseptic loosening.Orthopedic surgeons now have a variety of options for treating these issues,including revision surgery,which has demonstrated to be effective.If excessive inflammator reaction caused by the corrosion and peri-implant infection can be avoided,it will be of enormous social benefits This review will provide a summary of corrosion and the inflammatio reactions due to the corrosion and antimicrobial properties of Mg alloy-based implants covering both in vitro and in vivo studies.The strategies on hindering/overcoming the excessive inflammator response and enhancing the antimicrobial activity are discussed in this review.? 2022 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-alloy implants review;Corrosion and inflammation Microbial infection.

1.Introduction

Biodegradable metal implants are being used for orthopedic and dental applications[1,2].These biomaterials are expected to corrode and be absorbed by host cell machinery without any tissue damage but achieving the ideal corrosion rate for favorable bone growth in still under investigation[3,4].Magnesium(Mg)is investigated as a prospective implant material with biodegradable properties[5].Mg alloys are biodegradable materials with advantageous mechanical and physical properties for cardiovascular and orthopedic applications[1-3,6,7].

However,the major drawback of magnesium in many engineering applications is its low corrosion resistance and lower mechanical strength[8].Higher corrosion rate of magnesium implants can have immunotoxin effects[5]and induce pain and inflammation Most studies have reported the safety levels of immunological response to magnesium implants[9-14],but excess corrosion rates have been associated with increased immune cell infiltratio[15,16].

Foreign body reaction(FBR)is an unavoidable phenomenon for any implanted material.FBR has both acute and chronic inflammator stages[17].It was determined in the early days that FBR depended on the type of materials used for implantation[18].Normal inflammator response is an adaptive response of the innate immunity to initiate the healing and clean up the debris,whereas excessive inflam mation is a severe inflammator response where the foreign body is placed.Excessive inflammator responses prevent initiation of the healing process[19].It was reported thatin vivocorrosion of an Mg alloy-based implant involves the formation of a soluble,nontoxic oxide that is harmlessly excreted in the urine.Besides,due to functional roles and presence in bone tissue,magnesium may have stimulatory effects on the growth of new bone tissue[20].

Biofilm-associate infections can occur in the metal implants,compromising the tissue healing and triggering host immune reactions with the release of inflammator cytokines and phagocytic cells.These biofilm are difficul to be removed with antimicrobials and may cause prolonged inflam mation[21].These infections comprise a wide variety of microorganisms;however,staphylococci such asStaphylococcus aureusare the most involved pathogens[22,23].The bacterial cells also lower the pH of the environment,creating an acidic environment around the implant that accelerates Mg corrosion during the early stages[24].In addition to that,the biofil cells enmeshed in the extracellular matrix can increase microbial virulence and host-tissue damage,hindering the effectiveness of antibiotics[25].

The type and extent of Mg-based bioimplants corrosion were extensively covered in the literature previously[7,8,20,26,27].Conversely,the number of articles covering inflammatio due to the Mg implants is limited in the literature.Studies have found that Mg bioimplants have good antibacterial activities-their degradation produces a highly alkaline environment which is able to hemolyze bacterial cells[28].However,Mg implants often contain added alloying components to control its corrosion rate;this lowers the antimicrobial activity of Mg bioimplants.The use of rare earth(RE)elements such as neodymium,yttrium,cerium,gadolinium,lanthanum as alloying components is a recent practice to lower the corrosion rates[29].However,the toxic effects of these elements are not well understood[30].This review discusses the positive and the negative aspects of Mg and its alloying components that have a role to play in determining the ultimate biocompatibility and applicability of the implants.A summary of corrosion and the inflammatio reactions due to the corrosion and biofil associated products of Mg alloybased implants with an aim to connect inflammatio with corrosion and bacterial attachment at the implant site are extensively elaborated.To tackle the afore mentioned issues,the strategies to control the rate of corrosion of Mg implants and to reduce the impact of the corrosion inflammator reaction and microbial activity are further highlighted.

2.The physiology of magnesium

Magnesium is essential for several enzymes and cell functions,acting as an important modifie of the inflammator and immune response[31-37].It also plays a role in bone tissue and mineral homeostasis and may directly affect the function of bone cells and hydroxyapatite crystal growth[38].Around 99% of magnesium is stored in the body,and less than 1%is present in serum and red blood cells[39].Bone tissue is the largest store of magnesium in the body.Approximately one-third of this is concentrated on the bone surface and is related to serum magnesium concentration[40].

Mg is a co-factor for many enzymes and stabilizes the structures of DNA and RNA[31].The level of Mg in the extracellular flui where homeostasis is maintained by the kidneys and intestine ranges between 0.7 and 1.05 mmol/L;serum magnesium levels exceeding 1.05 mmol/L can lead to muscular paralysis,hypotension and respiratory distress[41],and at severely high serum levels of 6-7 mmol/L,cardiac arrest may occur.However,the incidence of hyper-magnesium is rare due to efficien excretion of the element in the urine[42].

Mg depletion has been associated with decreased osteoblastic and osteoclastic activity,osteopenia,bone fragility[43,44],vitamin D resistance or reduction[44-46]and parathyroid hormone resistance[47]or reduction[46].Studies on animals with different levels of Mg deficien y showed bone loss characterized by a decrease of trabecular bone volume,followed by an increase in the release of proinflammator cytokines and alteration in secretion and action of the parathyroid hormone(PTH),contributing to decrease in bone formation[44,46].

3.The role of Mg on bone-implant interaction

Magnesium is found to be most abundant in cartilage and bone tissue during the initial stages of osteogenesis[44].Mg is an essential micronutrient for the human body and recent studies show a slow release of Mg2+can be osteoconductive and osteopromotive[44].Few studies have reported an increase in the attachment and function of osteoblasts on magnesium surfaces[48].LY He(2016)demonstrated that magnesium ions induced the activity of osteoblasts by enhancing gap junction intracellular communication(GJIC)between cells and influencin bone formation[49].

In a recent study by Zang et al.(2021),the authors designed a complex porous dental implant using biodegradable magnesium alloy.The authors reported significantl faster proliferation rate of cells on the titanium sheet fille with magnesium alloy compared to the unfille titanium sheet,indicating that decomposed magnesium ions were able to reach the surface of the titanium sheet through the porous structure to assist with osteocyte proliferation[50].

Another study reported magnesium strontium(Mg-Sr)alloy deposited with a zoledronic acid calcium phosphate(ZACaP)bilayer coating was able to regulate osteogenesis and osteoclastogenesis through the Estrogen Receptorα(ERα)and NF-κB signaling pathway.The ZA-CaP bilayer coated Mg-Sr alloy regulated osteoblast-osteoclast cross talk and increased the ratio of osteoprotegerin(OPG):Receptor activator of nuclear factor kappa-B ligand(RANKL)in the co-culture system through the OPG/RANKL/RANK(Receptor activator of nuclear factor kappa-B)signaling pathway,which promotes balance in the bone remodeling process.These promising results suggest the potential clinical applications of ZApre-treated Mg-Sr alloys for bone defect repairs and periprosthetical osteolysis due to excessive differentiation and maturation of osteoclasts[51].In vitroandin vivostudies found on bone-Mg implant interactions are listed in Table 1.

4.The influenc of Mg2+on immune,bone,and tissue cells

In biomaterial-regulated reactions,host immune response is divided into the following stages:blood-biomaterial interaction,inflammation foreign body reactions(FBRs)and fi brous capsule formation[62].Among the inflammator cells,macrophages and monocytes play the most important parts in inflammatio and FBR triggered by biomaterial implantation[63-66].In brief,when circulating monocytes derived from committed progenitor cells in bone marrow migrate to peripheral blood,they can differentiate into monocyte-derived macrophages to participate in immune response.

Macrophages are the key cells during inflammatio and can influenc the fate of tissue healing and implant performance.M.D.Costantino in 2019 showed the beneficia and nuancing effects of magnesium and selected alloying elements,silver,and gadolinium on macrophage polarization.Magnesium extracts simultaneously exacerbated the profile of macrophage M1(tumor necrosis factor-alpha(TNFα),interleukin(IL)1βand chemokines such as IL8 and monocyte chemotactic protein 1(MCP1 or chemokine(C-C motif)ligand 2/CCL2)and M2(anti-inflammator IL10 and IL1ra,and chemokines such as osteopontin(OPN)).Meanwhile,Mg-2Ag and Mg-10Gd inhibited M1 differentiation,showing the beneficia and nuancing effects of magnesium and the selected alloying elements on macrophage polarization.Magnesium-based biomaterials could thus resolve inflammatio faster while improving tissue repair[67].Compared to polymer-based biomaterials,magnesium-based biomaterials reduced the formation of foreign body giant cells which function as inducers of chronic inflammatio and can be used to avoid the deleterious effects of an out-of-control mandatory foreign body reaction[68,69].

Magnesium ions(Mg2+)are essential for life;they function as cofactors for ATP and polyphosphates such as DNA and RNA,and as secondary messengers in intracellular signaling[70].Magnesium ions also suppress the production of inflammator cytokines[71].Jiazeng Xia in 2017 observed strong IL-1β-mediated inflammatio and inflammator cell infiltratio on day 1 after rectal anastomoses were stapled with high-purity magnesium or titanium(Ti)in vivo.However,inflammatio and inflammator cell infiltratio decreased more robustly 4-7 days post-operation in tissues stapled with high-purity magnesium.This rapid reduction in inflammatio was confirme by immunohistochemical analysis of IL-6 and TNF-α.Similarly,in vitro,expression of MCP-1,uPAR,and VEGF was high in primary rectal mucosal epithelial cells exposed to extracts from magnesium staples,as measured by antibody array assay.These results suggest that highpurity magnesium staples suppressed inflammator response during rectal anastomoses via TLR4/NF-κB and VEGF signaling[72].

Lei Sun in 2020 also suggested magnesium ions as a potential novel anti-inflammator agent that can be used to suppress excessive inflammator reactions[73].Magnesiumbased biomaterials especially may endow dental implants with anti-inflammator function.Previously,it was reported that a prospective biodegradable implant made from metallic magnesium showed bacterial biofil formationin vivo,despite the material exhibiting antibacterial propertiesin vitro;however,these bacterial biofilm-associate infections were able to induce robust localized and systemic inflammator reactions in a mouse modelin vivo[74].A recentin vivostudy performed on ZX Mg alloy made of Mg,Zn and Ca showed that the alloy could stimulate bone healing in juvenile rats via macrophage stimulation at the implant-bone interface over a period of 10 days.After an initial decrease,the osteoblast activity increased after 10 days of implantation and a good alignment of collagen fibril was observed[75].In another study conducted on osteosynthesis of cranio-osteoplasty with a biodegradable magnesium plate system in miniature pigs reported that bone healing was undisturbed in all cases and that biocompatibility with hard-and soft tissue was sufficient No complications were observed in this study.The study was also suggested that magnesium implants might help avoid longterm complications and secondary removal procedures due to their biodegradable properties[76].Findings onin vivoandin vitrostudies of the interaction between the Mg implants and the immune cells are listed in Table 2.

There is however evidence in literature that pure magnesium can cause higher inflammator reactions as observed in one of thein vivostudies in terms of accumulation of higher neutrophil concentrations[84].The optimal immunomodulator response of an implant should strike a balance between osteogenesis and osteoclastogenesis favoring bone regeneration.Excess degradation of Mg implants causes unfavorable inflammator response damages the surrounding healthy bone and hinders formation of fibrou tissue[85].Faster corrosion rate of Mg implants is also accompanied by increase in pH value which can cause hemolysis[86].Hydrogen gas bubbles formed due to accelerated corrosion can damage the surrounding tissues around the implant[8].

5.Mg particle-related biological complications

Along with other desirable features,Mg implants also havein vitroantibacterial properties[54,87]which is attributed to the large amount of OHˉproduced during degradation,resulting in a strongly alkaline environment[88].However,in a dynamic condition,the high pH can be balanced by the body fluid and hence the antibacterial ability is diminished[89].In the case of increasing the concentration of Mg to maintain an alkaline pH to produce an antibacterial effect,undesirable biological effects can be produced by the extensive formation of hydrogen in the form of gas bubbles and

implant corrosion[90].The excess of Mg ions can induce local inflammatio and infection,besides affecting the osmotic pressure of cells.The inflammator process in the presence of a high concentration of Mg is related to an elevated expression of the inflammator factors IL-8,IL-6,and ICAM-1,while cells damage is produced by swelling and membrane rupture due to the high osmotic pressure[28,91].In addition,the infection is enhanced by the presence of corrosion products,which form a deposition layer and provide a favorable place for bacterial adhesion[89].

Table 1Bone-magnesium implant interaction in vitro and in vivo.

Table 2Immune cell and Mg-based implant reaction in vitro and in vivo.

Fig.1 shows a schematic of Mg implant degradation and the biological complications due to the corrosion products.A previous study demonstrated that Mg implants augmented the bacterial survival,prolonged inflammator cytokines expression,and induced severe neutrophilic inflammator infiltrate surrounding the bacterially infected Mg implants[54].Therefore,considering the available evidence,Mg alloys should show a slow/controlled degradation behavior as an ultimate goal to improve cell biocompatibility.In addition,it can be meaningful to explore metallic elements to compose Mg alloys or produce surface coatings with properties that favor corrosion resistance and improve antimicrobial activity.

6.Strategies applied to control the rate of corrosion of Mg implants

To reach the optimal success level with Mg implants it was highly important to control the corrosion rate of magnesium.Several strategies have been applied over the years to achieve the same.

Fig.1.An overview of fracture-related infections after implant fixatio illustrating material degradation and biological complications.The in vitro antibacterial ability of Mg comes from producing large amount of OHˉduring degradation,resulting in a strongly alkaline environment.However,local inflammatio as well as the osmotic pressure of cells are also affected by the presence of Mg ions.Created with BioRender.com(License number:RS24ELHWBI).

Fig.2.Overview of the localized corrosion types commonly observed on the bone-magnesium implant interface.Due to accelerated corrosion the wear particles can detach from corrosion sites and cause inflammatio in the surrounding tissues.

6.1.Revision of manufacturing processes of Mg and Mg-alloy implants

Magnesium in body flui forms Mg2+ions via the electrochemical reactions:

The rate of corrosion typically increases with increase in immersion time[92]until a stable corrosion stage is reached at the equilibrium of deposition and dissolution of Mg(OH)2on the surface.The types of corrosion observed on the magnesium implants include crevice,pitting,galvanic corrosion which are localized in nature(Fig.2).Stress corrosion cracking and filifor corrosion have also been reported for Mgalloys in the literature.Filiform corrosion usually appears during early stages of immersion because it is controlled by concentration of Cl-present in the solution[93].A very recent study by Hu et al.showed that type and extent of corrosion depended on crystal orientation of the Mg alloy and weak passivation fil formed on the surface allowed hydrogen atom diffusion between the lattice gaps while various defects in the forms of dislocations,microcracks,grain boundaries and voids acted as hydrogen traps[94].They further reported that with extended immersion time,pitting corrosion,which gradually became the main form of corrosion,appeared synchronously alongside filifor corrosion.Fatigue crack growth(FCG)due to stress corrosion cracking was higher during immersion and FCG was influence by combination of hydrogen induced cracking and anodic dissolution[94-96].

The manufacturing process chosen for Mg implant has a direct impact on the degradation rate,mechanical properties,and performance in the biological environment of the implant[97].In ball milling several factors including ball-topowder ratio,milling time,turning speed,preventive atmosphere and pressure can alter key properties such as grain size,secondary phase formation and uniformity of the microstructure of the fina alloy product.Presence of non-uniform microstructure can cause corrosion of the implant[8].

Though presence of interconnected porous structures on Mg implants allows enhanced biological interaction with surrounding bone and host tissue,higher degree of porosity may introduce localized corrosion such as galvanic or pitting corrosion[98].The leftover porosity agents in the Mg matrix can affect the bone scaffolds[97].

Casting and wrought techniques are two methods that are commonly applied to prepare the Mg implant.Casting allows easy mixing of the alloy components[99-101]and wrought technique uses mechanical force to deform bulk metals into desired shapes.Rolling,forging and extrusion are some of the wrought techniques used by research groups for fabrication of Mg implant.Cao et al.reported hot rolling reduced number of secondary phase particles and produced a homogenized microstructure with refine grains in binary Mg alloy[102].For both casting and wrought techniques,the amount of the alloying impurities present,such as Fe,controls the extent of corrosion.High purity casting from a low purity feedstock can be produced by controlling the process parameters[103]which was observed to have reduced rate of corrosion[92].Fabrication of porous Mg scaffolds with good mechanical properties and lower degradation rate using laser additive manufacturing was reported by Li et al.[104].The commercially available WE43 alloy was prepared with customized porosity by Kopp et al.via this technique[105].Mg scaffolds with smaller pore size were reported to have reduced hydrogen evolution over time.Laser additive manufacturing can also produce refine microstructure,smaller grain sizes,regulate phase composition which collectively lower corrosion rates[106].

Based on the published works reviewed in this study,three factors:presence of secondary phases,uniformity of the microstructure and larger grain size/grain boundaries that influenc accelerated localized corrosion of Mg implants were identifie and the percent contributions of each manufacturing method discussed above towards these factors are shown in Fig.3.Out of these factors,larger grain size and grain boundaries increase corrosion as these are high energy sites[107,108].Two-phase or non-uniform microstructure is often caused by presence of impurities like Fe,Ni and Cu over the tolerance limit and cause accelerated galvanic corrosion[92].Mg alloys of the AZ series can haveαandβphases andβphases were found to be more corrosion resistant.However,βphases can also act as galvanic cathodes and accelerate corrosion if present in lower volume fraction and over smaller distribution[109].Furthermore,presence of almost continuous secondary phases across grain boundaries can cause intergranular stress corrosion cracking in Mg alloys.Secondary phase driven micro-galvanic corrosion along with applied stress cause crack which propagates through the alloy[92].

Fig.3.Three essential factors(microstructure non-uniformity(license #:5384,931,165,819),larger grain size(license #:5384,931,453,846)and secondary phase(license #:5387,650,435,903))contributing towards accelerated corrosion of magnesium implants were identifie and the percent contribution of each factor is indicated.

Laser additive manufacturing(LAM)has been employed in recent years for fabrication of Mg alloys[110-113]which can diminish all the three factors mentioned above.Implants of complex shapes may be manufactured using computer aided design via LAM.However,LAM of Mg alloys can be challenging due to poor quality of Mg powder,high reactivity of Mg[104],low vaporization temperature of Mg[112],and poor process control.A recent review by Wu et al.summarizes the challenges faced and remedies applied in LAM of Mg alloys.They also mentioned that LAMed Mg alloys had increased biocompatibility[111].Addition of Cu to LAMed ZK60-Cu imparted antimicrobial properties in the implant[114].Preliminary success in creating porous WE43 scaffolds using LAM was reported by Li et al.[104].Even though LAM is a promising technique to reduce grain size and produce fine microstructure in Mg alloys,it is still in its early stages and studies regarding fatigue test,interaction between process parameters and composition microstructure uniformity are yet to be performed[111].

6.2.Alteration of alloying elements of Mg implants

Magnesium can degrade too quickly in the physiological environment to provide necessary support for bone repair[115].The fast corrosion rate hinders Mg implants from providing the needed mechanical support for bone reconstruction.To lower the rate of corrosion,magnesium is often mixed with other alloying elements for implant applications.However,Mg alloys also suffer from pitting corrosion and corrosion of the surface via galvanic corrosion[115].Pitting corrosion is localized in nature and often results from presence of secondary phase,impurities,and local corrosive environment[116].Though pitting corrosion mechanism usually involves oxygen reduction reactions at cathode,the same for Mg implants mostly depends on hydrogen evolution reactions[117]where oxygen may not have a significan role to play[118,119].In general,pitting corrosion depends on three important factors:microstructure,corrosive media,and the fil on the surface.Presence or absence of secondary phases influenc the microstructure of the implant which in turn influence the pitting corrosion observed.Abundance of chlorine in simulated body fluid moisture and acidic media were observed to drive pitting corrosion.Localized corrosion usually produces circular blackened region on the surface of the commercially pure Mg when placed in concentrated chloride solution accompanied by rapid generation of hydrogen gas bubble[120].Magnesium hydroxide,which is formed as a thin fil on top of the Mg implants can protect the surface from further corrosion[115].However,in chloride solution,such a fil is broken down rapidly and corrosion is accelerated[121].Similar corrosion phenomena were observed in older Mg alloys in which the second phases usually acted as micro-cathode[116].In AZ91 presence of secondary phases were observed to drive the micro-galvanic corrosion[103,122]and homogenization via heat treatment was proposed to control the rate of corrosion[122].In Mg alloys of the AZ series,the alloy is typically made of anα-Mg matrix and intermetallicβ-Mg17Al12[123].Though presence of the intermetallic particles strengthens the alloy and increases the corrosion resistance[123,124],the toughness and ductility of the AZ series was found to be low[125].Reduction of intermetallicβ-Mg17Al12was necessary in AM60 and AM50 series of Mg alloys to improve toughness and ductility[126].Moreover,softening ofβ-Mg17Al12phase beyond 120 °C limited the application of the Mg alloys of the AZ series[27,127].Use of RE elements as alloying components is a recent practice[116].Heavy RE elements such as Gd,Y and Nd have high mechanical strength and improved heat resistance[128].Presence of cerium as an alloying element improve deformity at room temperature[129];neodymium decreases rate of corrosion[130,131]and yttrium can improve ultimate tensile and elongation strength of the Mg alloys[132].Yttrium,gadolinium,and neodymium increase the ignition temperature and make the alloys resistant to inflam mation[133].These alloys,similar to pure magnesium and the other Mg alloys,form a protective surface layer mainly composed of MgO and Mg(OH)2[125].These surface layers were reported to be thinner than that on pure magnesium and on other Mg alloys but more stable and protective due to the presence of RE elements[134-137].However,addition of RE elements can result in formation of second phases which were more cathodic than the Mg matrix and induce micro-galvanic corrosion[138-140],but they could also act as micro-anode such that in the case of WE43 tested in Na2SO4solution[141].In Mg-Gd alloy,the micro-galvanic corrosion by the second phases was accelerated with the increase in Gd content[142].In Mg alloys containing RE elements,the protective oxide layer was found to contain mixed oxides[116]which could improve corrosion resistance[140].The pitting corrosion in Mg alloys containing RE elements was found to follow a different mechanism than that in traditional Mg alloys due to presence of different second phases and surface oxide film[116]which can generate different intermediate and fina byproducts.In Mg-Y alloys,corrosion increased with the increase in Y content and filifor corrosion was introduced[92].Addition of Zr to WE54(Mg-5%Y-4%RE)was reported to provide refinemen to the microstructure with an increase in mechanical strength[128].

7.Outlining the antimicrobial activity of Mg tailored by alloying elements and surface coatings

7.1.Introduction of rare earth(RE)elements as alloying components

The Mg alloys with RE elements do not seem to cause major inflammator reactions[143,144].However,lanthanum and cerium showed higher cytotoxicity than the other RE elements,whereas gadolinium and dysprosium had better compatibility than yttrium[145].Thein vivostudy performed on rabbit tibiae showed that a combination of RE elements lowered the corrosion rate,but a severe clinical reaction was observed involving massive gas formation,bone loss and implant failure when only cerium was used[146].Marukawa et al.reportin vivostudy of WE43 did not show any systemic inflammatio in the tibial fracture model used[147].MgYREZr alloy screws were biocompatible and osteoconductive in the rabbit model and did not show any acute,subacute,or chronic toxicity up to twelve months[148].Another similar study by Schaller et al.(2018)reported that WE43 magnesium alloys exhibited excellent fracture healing properties before full degradation in a long-term porcine model without causing any substantial inflammator reactions[149].The LAE442 alloy in a sheep model showed good biocompatibility and only a minor inflammator infiltratio[150].In rodents implanted with WE21 the phagocytic activity decreased at highest degradation rate of the implant and the reason was believed to be the high release rate of yttrium[151].

7.2.Use of surface coatings and other alloying elements

Data from studies with various Mg alloys surface modification and with different antimicrobial Mg alloying elements were detailed outlined to explore differences between and among the study’s results(Supplemental File,available online).Among the 86 included studies,only 4 had anin vivomicrobiological assessment,out of these 3 evaluated different Mg alloys[152-154],and only 1 investigated a surface treatment using plasma immersion ion implantation treatment on a ZK60 alloy[155].The main bacteria strains used to investigate the antimicrobial effect of Mg alloys onin vivoandin vitrostudies with or without surface modification wereEscherichia coliandStaphylococcus aureus,only 8 studies out of 86 evaluated other bacteria.Regarding the quantitative test for the microbiological assessment,out of 86 included studies,32 studies used the classical colony-forming unit method,20 studies measured the inhibitory zones,and 20 studies evaluated the antibacterial rates(%),while the remaining 14 studies used other methods.Fig.4 shows the performance of Mg implants in-vivo in mouse model.

Fig.4.Overview of Mg biodegradable implants performance in vivo.(A)In vivo antibacterial performance of implanted metal rods that were retrieved from mouse femur at day 5 in post-op time period.Histological slices in a coronal plane image stained with Masson’s Trichrome dye.(B)Characterization of implants volume and the surrounding bones by Micro-CT.(a)Micro-CT 2D(The red arrows refer to the new bone formation)and 3D reconstruction models showing the status of the implants(white in color)and bone(pink in color)response 4,8,and 12 weeks after surgery.Reprinted with permission from Elsevier(License Numbers:5387,621,070,267,5387,620,833,855).

Specificall,the studies with different antimicrobial Mg alloying elements(Table 3)showed that the alloying with multiple elements were the most common Mg alloys studied.The metal elements used in eligible studies to produce biodegradable Mg alloys were Zn,Gd,Al,Sr,Cu,Ca,Sn,Ag,Nd,Zr,Y,Mn,and Ga.Out of 29 studies,only 2 studies evaluated the behavior of pure Mg[154,156],and only 9 studies evaluated the behavior of Mg alloyed with only one element,including MgSr[157],MgAg[101,157],MgCu[100,158-160],MgY[161],and MgZn[191].For the Mg alloys with multiple elements,Zn was the most common element used in the composition of most alloys:MgZnGd[163],MgZnSn[164],MgZnAg[165],MgZnMnCa[166],MgZnCa[167],MgZnSr[168],MgZnYNdAg[169],ZC21[170],ZK30[171],ZK60-Cu[173],ZM31[173],MgCaSrZn[174],MgNdZnZr[176],AZ91E[153],and ZQ,ZQ71,ZQ63[176].Within the studies with multiple elements,only 3 studies used elements other than Zn,evaluating MgAlCu alloys[177],MgYAg[178],and MgGaSr[179].Among the used elements,Zn,Cu,Ag,and Ga are associated with antibacterial effects,whereas the other elements can improve the mechanical properties and corrosion resistance of Mg alloys[88,100,180,181].Most of the studies presented at least one metallic element with an antimicrobial effect and also in combination with other elements that promote corrosion resistance.Fig.4 illustrates a few methods

applied to introduce anti-corrosion resistance in Mg implants and to provide anti-microbial activity.

Table 3Summary of included studies of Mg alloys with different coatings and studies investigating different alloys elements for Mg biodegradable implants.

Table 3(continued)

Regarding the antimicrobial effect of Mg alloys,13 studies out of 29 presented statistically significan differences in the bacteria load for the experimental Mg alloys compared with the control groups,while only 1 study showed no statistically significan differences,and 15 studies did not perform statistical analysis(Supplemental File,available online).Considering the studies with statistically significan differences in bacteria load reduction and the biocompatibility of the surfaces,for most studies,cytotoxicity was not observed,and for some,cell proliferation was even improved due to the presence of other elements in the Mg alloy.Concerning the degradation and corrosion behavior,the results indicate a relationship with the type and amount of the element added to the Mg alloy.The greater the amount of Cu and Ag,for example,the greater the rate of degradation and the worse the corrosion behavior[100,157,158,160,165,171,169],whereas the addition of other elements such as Zn,Ca,Sr,and Ga decreases the degradation rate and favors the corrosion behavior[168,182].

Within the antimicrobial effect of Mg alloys with surface modifications 21 studies out of 58 presented a statistically significan reduction in the bacteria load for the experimental Mg alloys with a surface treatment compared with the untreated surfaces.The remaining 37 studies did not perform statistical analysis,but most articles demonstrated a bacterial reduction in the surface treated group compared to the control group based on quantitative data(Supplemental File,available online).Considering the studies with statistically significan differences in bacteria load reduction and the biocompatibility of the surfaces,17 studies showed improved biocompatibility[183-199,186],2 studies demonstrated similar results between experimental and control surfaces with no cytotoxicity[193,200],and 2 studies did not perform biocompatibility analysis[201,202].Overall,the surface modification effectively improved the degradation and corrosion behavior of Mg alloys.

Improving corrosion resistance of Mg implants via surface modificatio or alteration of alloying components often results in loss of antimicrobial property[203].Functionalized antimicrobial coating may help lowering rate of corrosion while incorporating antimicrobial properties.Fig.5 shows schematics of surface modificatio techniques applied and their mechanism of action.

8.Discussion-the strategies to reduce the impact of the corrosion inflammator reaction and microbial activity

A controlled corrosion of the magnesium implants is beneficia for bone growth but accelerated localized corrosion and wear may cause inflammatio in the surrounding tissues.The alloying elements often introduce secondary phases,nonunform microstructure and larger grain sizes or grain boundaries which influenc faster degradation rates.Along with localized corrosion,filifor corrosion and stress corrosion cracking are also observed on bone-magnesium implant interface.Tribocorrosion was mostly observed in dry conditions and experiments performed in simulated body fluid had lower rate of tribocorrosion.LAMed Mg alloys show promising results in terms of lowering rate of corrosion despite the challenges posed by chemical properties of Mg.Biocompatibility and inflammatio studies on the LAMed Mg alloy implants are very limited.However,a recent study by Xie et al.showed high Mg2+concentrations from additively manufactured Mg alloy promoted M1 macrophage polarization and the excess Mg2+did not accumulate in any of the major organs[175].

Addition of RE elements can stabilize the protective layer forming on the implant surface and increase corrosion resistance as reported in a few studies.It is worth noting that the dual criteria,biodegradability,and biocompatibility,need to be satisfie even with the addition of other metallic elements or surface coatings in Mg alloys for short-term or temporary applications[244].Hereby,the biodegradable implants need to corrode gradually,with an appropriate host response,and within an adequate time for bone repair.According to the included studies in this critical evidence-based review,many of the Mg alloys with other metallic elements and surface modification presented the good potential to be used as biomedical implants due to their good corrosion resistance and antibacterial properties.

A recent study by Yang et al.discussed successful integration of mesoporous bioglass(MBG)coating on Mg based composite via LAM that increased corrosion resistance due to thick layer of apatite formation on the surface[245].Zhang et al.reported mesoporous 45S5 bioactive glassceramic coated Mg alloy,prepared also via sol-gel method,had improved corrosion resistance under low applied pressure.However,the entire coating was lost under applied force of 25 MPa or greater[246].

Concerning the Mg alloys,the main used metallic elements associated with antimicrobial properties were Zn,Ag,Cu,and Ga.The Zn incorporation in Mg alloys is acknowledged for its biocompatibility,osteogenic activity,and antimicrobial activity,besides the natural existence of this element in the human body yielding natural metabolism and release[162,247].It has been demonstrated that Mg alloying with a proper amount of Zn would stimulate antimicrobial and osteogenic properties[162].The ability of Zn to prevent biofil formation is still not clear,but it has been hypothesized that electrostatic interactions of positively charged Zn ions with negatively charged bacterial membranes can lead to bacterial membrane disruption[192,248].In addition,many studies have incorporated Ag into Mg alloys because it has effective antimicrobial properties related to its ability to bind to proteins,destroy the membrane of bacteria,interfere with DNA expression,and create reactive oxygen species(ROS)[249].Liu et al.[157]reported that MgAg alloys obtained good antibacterial properties in harsh and dynamic conditions.Accordingly,Tie et al.(2013)[101]corroborate that MgAg alloys reduce the viability of bacteria with a killing rate exceeding 90%.The combination of Mg and Ag with other elements has also demonstrated that the antimicrobial activity remains for alloys such as MgYAg[181]and MgZnAg[247].However,the addition of Ag proportionally increases the corrosion and degradation of Mg alloys since Ag can form microgalvanic couples with Mg matrix,which can serve as cathodic sites,accelerating the corrosion of Mg[101,157,247].Another used antimicrobial element for Mg alloying is Cu.It is associated with osteogenesis and angiogenesis stimulation and anti-inflammator properties while performing an antibacterial activity.Studies evaluating the Cu incorporation into Mg alloys show promising antibacterial activity againstE.coli,Staphylococcus aureus,Staphylococcus epidermidis,andCandida albicans[158-160].However,Cu also leads to increased corrosion and degradation of Mg alloys,which is an undesirable characteristic when degradation is expected to follow bone repair[158-160].Little attention has been cast on Ga,and only one study investigated alloying Mg with Ga for bacterial control[182].This element acts as an iron mimetic and is supposed to exploit Fe transport systems to enter bacterial cells[250].The antimicrobial mechanism is explained by Ga3+ability to disrupt the irondependent oxidation and reduction,which are essential for the reproduction and metabolism of most bacteria[251].Gao et al.[182]showedin vitroantibacterial activity of MgGaSr for gram-positiveStaphylococcus aureus,Staphylococcus epidermidis,and gram-negativeE.coli.

Regarding the surface modification of Mg alloys,the main antimicrobial strategies used were incorporating metallic ions,graphene,antibiotics,antimicrobial peptides,natural substances,and modificatio in surface hydrophobicity.Within the antimicrobial metallic ions,Ag,Zn,and Cu were the most used elements in surface coatings due to their recognized antimicrobial properties,as previously reported.The incorporation of Ag has shown appropriate degradation characteristics,cytocompatibility,and antimicrobial activities for bone tissue engineering applications through deposition by different treatment techniques such as electrospinning,layer-by-layer,and replication technique[185,187,221,223].Surfaces with Zn deposition also exhibited an improved antimicrobial effect than those of naked Mg alloys,using the solid diffusion technique[192],microwave aqueous synthesis,and heat treatment[211].

Fig.5.Overview of some surface modification strategies to provide antibacterial activity to Mg biodegradable implants.(A)Schematic illustrations of the anti-corrosion mechanism,antibacterial ability and cytocompatibility of the classical HAp and(CIP/PAH)10/CIP-HAp coatings produced via the combination of layer-by-layer assembly and hydrothermal treatment.(B)Schematic of the antimicrobial mechanism of Mg-GNPs composite,in this study Mg-based composite was fabricated by semi powder metallurgy and was reinforced by graphene nano-platelets.(C)Schematic of some possible interactions between the PLLA/GO-Ag fibrous-coate Mg alloys surface and the biological environment,PLLA/GO-AgNP with different concentration of GO-AgNPs were deposited on Mg alloy via electrospinning method for enhancement of corrosion resistance and antibacterial performance.Reprinted with permission from Elsevier(License Numbers:5387,651,493,856,5387,660,358,219,5387,660,550,706).

Modification of Mg alloys with graphene nanoparticles and nano-platelet composites have also been proposed[167,173,197,187].Graphene is a two-dimensional carbon material arranged in a fla honeycomb lattice,which present excellent mechanical strength,antimicrobial potential,and osteogenic properties[252,253].The antibacterial mechanism of graphene is related to its oxygen-containing functional groups and its ability to disrupt and damage the bacteria cell membranes due to its membrane oxidative stress induction[254].The incorporation of graphene into magnesium alloys have been proposed using spark plasma sintering[197],electrospinning method[187],and adding graphene to magnesium alloy in solution and mixing followed by placement in a dry oven[167,173].as a strategy to enhance antibacterial potential,besides promoting greater corrosion resistance.

Another effective way used to reduce implant-associated infections was the local delivery of antibiotics.Doxycycline and gentamicin are among the most used antibiotics on surface coatings.Doxycycline belongs to the class of medicines known as tetracycline antibiotics,while gentamicin belongs to a class known as aminoglycoside antibiotics,and both inhibit bacterial protein synthesis by binding to the 30S ribosomal subunit[255].Doxycycline was loaded on Mg surface through the electrospinning method[186]and demonstrated good biocompatibility and excellent antibacterial performance against gram-positiveStaphylococcus aureusand gram-negativeE.coli.Gentamicin-loaded surfaces were obtained by layer-by-layer technique[158,213,222],hydrothermal synthesis method,and electrostatic adsorption[214].This antibiotic loading promoted effective antibacterial activity with sustained drug release.Ciprofloxaci and Levofloxaci were other used classes of antibiotics,they belong to quinolone antibiotics and inhibit DNA replication by inhibiting bacterial DNA topoisomerase and DNA-gyrase[256].Both antibiotics were loaded on surfaces using a layer-bylayer technique and showed antibacterial effica y with a prolonged release profil[195,219,224,257].Overall,antibiotics showed positive results because of the controlled release preventing infections until complete biodegradation of the implant,which can be mainly attributed to the construction of coatings with layers that allow the antibiotic to be interspersed between them and to be released gradually.

Other strategies used were incorporating antimicrobial peptides and natural compounds on surface coatings as a strategy to overcome antibiotic resistance problems.Antimicrobial peptides present rapid and broad-spectrum antimicrobial activities and have been immobilized on coatings by immersion,covalent technique,and spinning coating process[193,205,206].These studies showed that the antimicrobial peptides loaded on coatings for Mg alloys could be considered an ideal orthopedic implant against infection.Concerning surface loading with natural compounds,chitosan,lysozyme,and lawsome were used in some included studies.Chitosan is a natural polysaccharide extensively used in the biomedical fiel due to its antibacterial properties,biocompatibility,low toxicity,and biodegradability.For biodegradable Mg implants,chitosan deposited via electrophoretic deposition and layer-by-layer has presented a good bacteriostatic effect,reducing the risk of a bacterial infection within the implant system[183,226].Lysozyme was also incorporated by layerby-layer showing an excellent antibacterial activity(＞99.9%againstStaphylococcus aureusandE.coli)due to the mechanism of cleaving the peptidoglycan component of bacterial cell walls,which leads to cell death[232].Lawsome is another natural compound used,it is extracted from the leaves ofLawsonia inermisplant and was used on a Mg alloy coating,showing corrosion inhibition properties and anti-bacterial and anti-biofil activities[217].

In addition to incorporating all these antimicrobial elements,the fabrication of superhydrophobic coatings with water contact angles greater than 150° have attracted widespread attention[258].The properties including anti-wetting ability,anti-corrosion,self-cleaning,anti-bacteria,and water-repellent are considered a novel technique to protect metals from corrosion and bacteria colonization[259].The use of this type of coating on biodegradable Mg alloys has shown antibacterial adhesion effect and corrosion resistance after fabrication through hydrothermal method[212,258].

Surface modification and coatings on magnesium implants have shown promising results with increased antimicrobial potential and greater corrosion resistance,with gradual degradation that follows the bone repair process.However,it is important to consider that as the material degrades,the coating that is superficia will be degraded first In fact,from this point of view,the use of alloying elements that compose the material uniformly throughout its composition would be more viable and favorable for maintaining the desired antimicrobial and corrosion resistance properties.On the other hand,the use of surface modification can eliminate completely the microorganisms before its degradation,inhibiting the development of further infections.However,in cases of major infections,an antimicrobial coating may not be fully effective in eradicating the bacteria.The same reasoning can be considered in relation to corrosion,in the presence of infection and an environment that favors a greater degradation of the material if only a coating was deposited on the surface,these properties will not be guaranteed until the fina degradation of the material,being the alloying of Mg alloys an option with longer effect overtime.

Still,it is important to emphasize that most studies arein vitroand only 4in vivopreclinical studies evaluate the antimicrobial performance of biodegradable Mg implants.Furthermore,three compositions of Mg-based systems have been approved to be conducted for orthopedic applications,but none evaluated the antimicrobial potential of the tested implants:pure Mg[260],MgYREZr alloy[261],and MgCaZn alloy[262].Altogether,the conduction of further preclinical studies to evidence the antimicrobial potential of these materialsin vivois essential before they can be safely deployed in a clinical setting.Especially,coatings and high concentrations of antimicrobial agents must be tested to confir their biocompatibility,anti-corrosion properties,and sustaining of the antimicrobial potentialin vivo.

During implantation and due to presence of implanted material,tissue injury is caused which in turn activates signaling cascades leading to acute and chronic inflammator responses[263].Protein adsorption,neutrophils and type 1 macrophages direct wound cleaning process and wound healing involves polarization of type 1 macrophages to type 2 macrophages and neovascularization.The success of bioimplants depends on effective polarization of the macrophages and stimulation of neovascularization[75].Magnesium was found to suppress excess inflammatio[73]and induce better bone formation[75].One of the suggested pathways for bone stimulation by Mg was by increased expression of the neuropeptide Calcitonin Gene-Related Peptide(CGRP)[264].Between pure Mg and bio-inert titanium,Mg had less host body reactions in mice over 32 weeks of healing[265].Also,Mg porous scaffolds were observed to reduce inflammator responses while stimulating expression of collagen type 1 and osteopontin markers to enhance bone formation[266].However,excess degradation rate of Mg alloys results in hydrogen gas formation causing tissue damage[8].These gas-fille pockets may allow formation of biofilm by shielding the bacterial cells from host immune cells[21,267].In a study performed on transgenic mice it was observed thatP.aeruginosabiofilm could form on Mg implants and induce strong host immune reactions in the surrounding tissue.It was also indicated that the implant material sheltered the planktonic bacterial cells which would be killed by the immune cells in absence of the implant[21].Use of RE elements as alloying components lower the corrosion rate of Mg alloys and thereby reduces the formation of gas bubbles.However,up regulation of host inflammator genes was observed when Mg alloys with RE elements were used[143].Some of the RE elements are toxic to the host body[145]and their use should be properly investigated.

9.Future perspectives

The research on the application of biodegradable Mg implants in orthopedic,oral,and maxillofacial reconstructions still needs further efforts.Specificall,more preclinical studies are required to investigate and confir thein vitroresults of the degradation and antimicrobial activity of Mg alloying with different metal elements and surface coatings in a dynamic environment.Mostin vitrostudies of Mg alloys with different metal elements or surface modification have yielded encouraging results with well-demonstrated antibacterial potential.Although only 4in vivostudies were included in this critical evidence-based review,most showed positive results.Therefore,we believe that this research fiel has great research value and potential for future application.Addition of RE elements as alloying components is a recent trend to lower the corrosion rate of the Mg alloy implants.However,the literature indicates there is a lack of enoughin vivostudies to be performed on such Mg alloys.The methods used for fabrication of such implants played an important role in determining the degradation rate.Current fabrication methods for Mg implants do not guarantee complete removal of impurities and/or secondary phases which contribute towards accelerated corrosion.Pristine chemical vapor deposition techniques such as atomic layer deposition(ALD)may be used to form magnesium oxide as a protective coating layer on top of Mg implants to lower the corrosion rate.ALD,by nature,uniformly deposits conformal,pin-hole free metal or metal oxide layers free from any surface impurities[268].Such film typically have a uniform microstructure free from any secondary phase.ALD is already an established technique for manufacturing antibacterial dental implant materials[269].Controlling bacterial invasion at the implant site can lower the inflammatio in the host body.Finally,for the biodegradable Mg implants design,the mechanical,chemical,physical,and biological properties should be considered and envisioned to achieve the desirable properties.

10.Conclusion

Magnesium and its alloys benefi from biodegradability and mechanical characteristics that are like those of natural bone.The rapid rate of corrosion,however,makes it diffi cult for alloys to collapse and for wounds to heal.A perfect magnesium implant alloy should possess sufficien mechanical qualities,appropriate biocompatibility,and corrosion rate that is compatible with tissue healing rate.To control the degradation rate in accordance with the tissue growth rate,revision manufacturing process and surface treatments,integration with other metal elements,alteration of alloying elements,and introduction of RE elements should be properly designed.As magnesium-based implants show antibacterial function due to the increase of pH value,this function is clinically valuable since infections associated with surgical implants are considered as a serious issue.The strategies to reduce the impact of corrosion inflammator reaction and microbial activity has been discussed such as control the corrosion,integration of other elements,surface modifications effective antibiotic,incorporation of antibacterial peptide and natural compounds,fabrication of superhydrophobic coating and effective polarization and stimulation of neovascularization.Since the metabolic processes of certain elements,such RE metal elements,protein peptide and natural compounds are not well understood,future comprehensive studies are needed to better understand the impacts of some elements on the human body.In short,the development of magnesiumalloys implants is an area wide open for exploration and innovation.

Declaration of Competing Interest

The authors declare that they have no known competing financia interests or personal relationships that could have appeared to influenc the work reported in this paper.

Supplementary materials

Supplementary material associated with this article can be found,in the online version,at doi:10.1016/j.jma.2022.10.025.