Jeho Prk ,Bo-In Prk ,Young Ju Son ,Sun Hee Lee ,Seung-Hoon Um ,Yu-Chn Kim,d ,Myoung-Ryul Ok,Jeong-Yun Sun,Hyung-Seop Hn,*,Hojeong Jeon,d,e,**

a Center for Biomaterials,Korea Institute of Science and Technology (KIST),Seoul 02792,Republic of Korea

b Department of Materials science and Engineering,Seoul National University (SNU),Seoul 08826,Republic of Korea

c Department of Materials Science and Engineering,Korea Advanced Institute of Science and Technology (KAIST),Daejeon 34141,Republic of Korea

dDivision of Bio-Medical Science and Technology,KIST School,Korea University of Science and Technology,Seoul 02792,Republic of Korea

eKU-KIST Graduate School of Converging Science and Technology,Korea University,Seoul 02841,Republic of Korea

Abstract Mg has received much attention as a next-generation implantable material owing to its biocompatibility,bone-like mechanical properties,and biodegradability in physiological environments.The application of various polymer coatings has been conducted in the past to reduce the rapid formation of hydrogen gas and the local change in pH during the initial phase of the chemical reaction with the body fluids Here,we propose femtosecond (fs) laser-mediated Mg surface patterning for significan enhancement of the binding strength of the coating material,which eventually reduces the corrosion rate.Analyses of the structural,physical,crystallographic,and chemical properties of the Mg surface have been conducted in order to understand the mechanism by which the surface adhesion increases between Mg and the polymer coating layer.Depending on the fs laser conditions,the surface structure becomes rough owing to the presence of several microscaled pits and grooves of nanoporous MgO,resulting in a tightly bonded poly(lactic-co-glycolic acid) (PLGA) layer.The corrosion rate of the PLGA-coated,fs laser-treated Mg is considerably slow compared with the non-treated Mg;the treated Mg is also more biocompatible compared with the non-treated Mg.The fs laser-based surface modificatio technique offers a simple and quick method for introducing a rough coating on Mg;further,it does not require any chemical treatment,thereby overcoming a potential obstacle for its clinical use.

Keywords: Femtosecond laser;Biodegradable metal;Polymer coating;Surface modification Surface adhesion.

1.Introduction

Mg-based biodegradable metals have been proven to be promising candidates for next-generation implantable biomaterials with excellent biodegradable,biocompatible,and mechanical properties [1-4].The controlled degradation of biodegradable metals provides sufficien time for tissue healing while eliminating the need for secondary surgery.Furthermore,the release of specifi metallic ions has been shown to promote bioactivities such as angiogenesis and osteogenesis[5].

Several studies have shown the application of Mg-based biodegradable metals in implantable orthopedic devices,such as bone screws,ligament screw plates,and porous scaffolds[6,7].The primary obstacle in developing such devices is the corrosion rate of Mg in body fluids Mg corrosion in body fluid is accompanied by the generation of hydrogen gas and an increase in the local pH owing to the concentrated hydroxide ions,which,if not controlled,form a gas pocket causing tissue necrosis and,eventually,implant failure.One of the most common methods to control the Mg corrosion rate is polymer coating of the metallic surface [8-12].

Biodegradable polymer coatings,such as poly-epsilon caprolactone,poly(lactic-co-glycolic acid) (PLGA),and poly(lactic acid),on Mg alloys have shown effective prevention of early corrosion.They are often used as vehicles for selective loading of drugs to further enhance the benefit of implantable devices [13-16].However,there are several known issues,such as delamination of the polymer layers and pitting of the polymer coating,that often result in faster corrosion of the Mg alloy,causing a potential blockage of blood fl w by the large polymer fil debris.Therefore,high interfacial adhesion with the metal surface is required for effective utilization of the polymer as a coating material.Pretreatment processes,such as plasma treatment and chemical oxidation have been developed to improve the adhesion strength of polymers on metal surfaces and stabilize the coating layer[17-20].

Recently,laser surface treatment has attracted considerable research interest and has been effectively used in modifying the surface properties of target materials.The laser surface modificatio facilitates simple non-contact manufacturing,selective irradiation,and cost-effectiveness through mask-less single-step procedures in a vacuum-less environment [21].Several different surface modification can be achieved by adjusting laser power/pulse energy.Furthermore,the resistance of the magnesium alloy to corrosion can be improved by laser surface remelting and laser cladding,which facilitate the distribution of refine intermetallic compounds or the formation of an oxidation/passivation layer on the surface [22-26].In particular,the laser surface texturing method using a femtosecond (fs) laser has been investigated in detail in order to understand the easy formation of micro-,nano-,and hierarchical structures on material surfaces,leading to changes in the surface properties,such as surface roughness and surface energy.Furthermore,the thermal stress and damage to the substrate could be minimized because the fs laser applied energy for a substantially shorter time period compared with the pulse duration of a conventional laser.The fs laser energy has been applied to the target substrate without interaction between the laser and plasma,resulting in an alteration of the surface structure and chemical (oxidation,hydrophilic,and hydrophobic) properties[27-31].In a previous work,we have utilized an fs laser to create a thin uniformly refine layer on the surface of an Mgbased alloy in order to control galvanic and pitting corrosion[32].This method allowed the reduction of the secondary phase fraction,while retaining the mechanical properties and biocompatibility.

In this study,the surface of Mg was modifie with an fs laser to promote a stable and strong polymer adhesion force at the coating interface.The drastic increase in surface adhesion strength and the improvement of polymer coating ability were facilitated by the formation of micro pits,groove patterns,and oxidized nanostructures on the Mg surface.The optimized fs laser treatment condition revealed the mechanism by which the adhesion to polymer surfaces improved both physically as well as chemically.

2.Materials and methods

2.1.Preparation of treated Mg

Pure Mg(99.9%purity)of a cylindrical shape,8mm diameter,and 1mm thickness,was prepared.The chemical compositions of pure Mg were verifie via inductively coupled plasma atomic emission spectroscopy.The samples were polished with a 2000 grit SiC paper to remove pollutants and obtain a smooth surface before laser treatment.Thereafter,the samples were sequentially cleaned via sonication (SD-300H,Mujiga,Korea) in 99% ethanol and acetone for 10min,individually,and air-dried.

In this study,a ytterbium fs laser (s-Pulse HP,Amplitude,France) was used.The laser system provides a pulsed width beam of 400 fs at a central wavelength of 343nm,as shown in Fig.1b.The repetition rate was fi ed at 1kHz.The laser-spot diameter on the surface was approximately 100μm.All the samples were mounted on an XYZ stage that was translated at 0.5mm/s into grating tracks in order to induce isotropic wetting behavior.A variety of textured surfaces were formed,not only by altering the laser energy,but also by the variety of interval gaps of the laser tracks.To produce different textured surfaces on pure Mg,the pulse energy was varied with 80,120,and 160 μJ,measured by a power detector (Nova 2,OPHIR),resulting in the laser energy fluenc (F) of 1.0,1.5,and 2.0J/cm2,respectively.

PLGA was spin-coated on the textured Mg through laser modificatio and using polished Mg.PLGA (LG 857 S,ResomerR○) was dissolved in 2,2,2-trifluoroethano with 11%(w/w) concentration.In general,the thickness of the coating layer increased as the PLGA concentration increased [32].Therefore,the PLGA concentration was define in order to provide an evenly spreading coating layer with a constant thickness.The PLGA solution was spin-coated on the polished Mg and on the fs laser-treated Mg for 40s at 2000rpm.PLGA-coated Mg was dried in vacuum overnight at 24°C for further characterization.All processes in this study are shown in Fig.1a.

2.2.Physicochemical characterization

The surface was characterized via scanning electron microscopy (SEM;Inspect F50,FEI,USA).X-ray diffractometry (XRD;D/MAX-2500,Rigaku,Japan) using CuKαradiation was performed for crystal orientation in order to characterize the microstructure of the specimens.The 3D structures and surface roughness of the samples were analyzed using a confocal laser scanning microscope (LSM;LEXT OLS4100,Olympus,Japan).To measure the surface wettability change in the laser-treated Mg,contact angle measurement (Smart-Drop,Femtobiomed,Korea) was performed by dropping 1 μL of deionized water.Elemental distributions and images were simultaneously captured via scanning transmission electron microscopy (STEM;FEI Talos F200X,USA),equipped with a Super-X energy dispersive X-ray spectroscopy (EDS)detector.

Fig.1.(a) Schematic illustration of a PLGA coating on a treated Mg surface through the formation of a hierarchical topography via an fs laser.(b) Schematic representation of a laser surface treatment experimental setup.fs:femtosecond;PLGA,poly(lactic-co-glycolic acid);Mg:magnesium.

2.3.Evaluation of corrosion properties

2.3.1.Hydrogen gas release tests

The corrosion property was evaluated by measuring the amount of hydrogen gas generated because it shows the degree of corrosion resistance of the samples [33,34].All the samples exposed to 8Ф were immersed in 200ml of Hank’s balanced salt solution (HBSS;pH 7.4,Welgene,Korea) at 37°C.The hydrogen gas released from the samples was collected at the top of a beaker.A portion of the Hank’s solution was replaced every 24h,considering the diuresis process in the body.

2.3.2.Mg ion release tests

To quantify Mg ions,the PLGA-coated and non-coated Mg substrates were immersed in a serum-free 1×Dulbecco’s modifie Eagle’s medium (DMEM) in the ratio of exposed Mg area of 1 cm2/mL of media.The Mg in media was incubated at 37°C in 5% CO2to mimic the human environment.A portion of media was removed after 1,3,and 7 d,and the Mg ions were assayed using a Quantichrom Metal Ion Assay Kit series (DIMG-250,Bioassay Systems,USA) [35,36].

2.4.Adhesion strength

Tensile tests were performed to measure the adhesion strength between the coating layer and the textured surface.Tensile force tester (Instron 5966,Instron Engineering Corporation,USA) was used at a speed of 1mm/min.On the cylindrical sample,the bottom was non-treated,and the top was area coated with the polymer.The sample was tested using a highly adhesive glue (Loctite 401,Henkel) at both the bottom and top surfaces.The adhesion strength was measured along the surface where the coating had been detached from the substrate.

2.5.Cytocompatibility test

To evaluate the cell viability,cells were prepared based on the following procedure:L929 cells (NCTC clone 929,Korean cell line bank,Korea) were seeded (7×103cells/well)on a Mg substrate (Ctrl,PLGA@Bare,PLGA@FsMg) for 1 d in serum-supplemented DMEM and incubated overnight at 37°C in 5% CO2for cell adhesion.Thereafter,the medium was changed to serum-free DMEM and the cells were incubated for 1,3,and 7 d.The cells were further incubated for 24h,and their viability was measured using the Cell Counting Kit-8 assay.This assay was conducted according to ISO 10993.

For cell morphology observation,the cells were preincubated at 37°C in 5% CO2overnight on an Mg substrate in a serum-supplemented medium that was changed to an Mg ion-containing broth from day 3.The cells were further incubated for 24h.Thereafter,cells on the Mg substrate were dehydrated using ice-cold methanol and observed via SEM.

3.Results and discussion

3.1.Surface analysis after laser surface modificatio

The surface treatment of Mg substrates was performed using fs-laser under various conditions (Table 1).The surface morphology characteristics changed following the fs laser treatment and are shown in Fig.2.The surface appeared noticeably rough as the laser intensity increased in the range of 1.0-2.0.J/cm2;with increased roughness,they transformed into fla (as-polished),embossed (pulse energy:80 μJ),bump(120 μJ),or canyon (160 μJ)-like shapes,as shown in Fig.2a-d.These surface features were scanned and imaged in three dimensions through LSM analysis,as shown in Fig.2e-h.As the intensity of the laser increased,the surface characteristics revealed increased roughness;these results correspond to the results from the SEM analysis.In particular,anincrease in roughness indicates an increase in the surface area of the Mg substrate,affecting the surface wetting properties[36,37].

Table 1 Laser-processing parameters.

Fig.2i shows the wettability of each sample treated with fs laser.The water contact angle decreased sharply from 75.6°(as-polished) to 5.3° (canyon-like),according to the changed surface roughness.The wettability increased as the absolute value of the water contact angle decreased.Similar results have been reported during the change in wettability of the metal surface owing to laser treatment.It can be used as a controlled method for surface structure modificatio by an fs laser to obtain an ultra-small hydrophilic surface [38-40].With the increase in the laser intensity,the surface oxide content also increased,as shown in Fig.2j.It was confirme that the hydrophilic property was maintained for approximately 3 weeks in the canyon-like structure.Overall,a superhydrophilic substrate-surface structure was formed through fs laser surface treatment;a surface sample of canyon-like structure with the lowest water contact angle is expected to show the most promising performance with respect to adhesion of the polymer coating.The laser treatment and coating were performed under these specifi conditions for all the experiments that followed.

In general,laser surface treatment is known to promote the oxidation reaction of Mg by forming oxides such as MgO or Mg(OH)2on the surface [41-44].XRD analysis was performed to observe the phase change by fs laser treatment,as shown in Fig.2k.Following the fs-laser treatment,major peaks of MgO appeared at 42.8° and 62° that were relatively wide and had low strength;it was clearly distinguishable,when compared with the clear and sharp peaks of the Mg substrate.This was indirect evidence of the formation of nanocrystalline MgO.In accordance with the crystallographic features,highly nanoporous structures (inset of Fig.2d) were observed on the sample surface with canyonlike features.The formation of this unusual surface structure can be explained through the nanoscale Kirkendall diffusion effect [45-47].It is considered that the local energy of the fs laser contributes to the thermal energy that leads to the diffusion of Mg atoms.It does not appear to undergo an oxidation reaction wherein the oxygen sourced from the atmosphere would penetrate into the bulk.Instead,the unique nanoporous surface structure was formed by the simultaneous diffusion of Mg atoms onto the surface along with the reaction with oxygen,according to the nanoscale Kirkendall effect.

Fig.2.Characteristics of Mg treated with an fs laser:(a-d) SEM surface images of Mg substrate morphology and (e-h) 3D topography images and depth profil with respect to each laser parameter.(i) The mean contact angle of a laser-treated Mg surface.(j) Weight percent (wt.%) change of Mg (black square)and oxygen (blue triangle) of each laser condition.(k) XRD patterns of as-received (black line;bare) and laser-treated alloy (red line;canyon).fs:femtosecond;SEM:scanning electron microscopy;XRD:X-ray diffractometry;Mg:magnesium.

The results of TEM-EDS analysis clearly showed that the abovementioned nanoporous surface was composed of MgO (Fig.3).The cross-sectional image that was analyzed in greater detail via TEM clearly showed different boundaries separating the Mg substrate and the oxidized nanoporous MgO region.Selected area (electron) diffraction pattern analysis was conducted to investigate and confir the difference in crystal properties at the boundaries(Fig.5e-h).The MgO region close to the surface showed a ringed pattern with polycrystalline properties;the d-spacing values of 2.15 °A,1.515 °A,and 1.21 °A corresponded to (200),(220),and (311) of MgO (JCPDS 89-7746),respectively.Additionally,a high resolution-TEM image analysis verifie this region to be polycrystalline (Fig.3j).The corresponding d-spacing signals are denoted by red circles (g)in Fig.3e,indicating the interface between the Mg substrate and MgO (Fig.5g).The Mg substrate region marked as red circles (h) showed the d-spacing values of 2.48,1.61,and 1.89,corresponding to the (101),(102),and (110)planes of Mg,respectively,consistent with the XRD results(JCPDS 04-0770).

3.2.PLGA coating on fs laser-treated Mg

Fs laser-treated Mg was spin-coated with PLGA and the coating layer stability was evaluated (Fig.4).The cross-section images of PLGA coated on fs laser-treated Mg (PLGA@FsMg) and the PLGA coated on bare Mg(PLGA@Bare) are shown in Fig.4a.The PLGA layers were well coated on the Mg surface,while the PLGA layers completely fille the groove areas of PLGA@FsMg.The average thickness of the PLGA coating was 64.21±2.3μm and 52.1±6.51μm for PLGA@Bare and PLGA@FsMg,respectively.Well-smeared PLGA polymers on the fs laser-treated surfaces are expected to have super-hydrophilic properties,as can be observed in Fig.2,because the solvent for PLGA dissolution is water-miscible.This is confirme by the Wenzel’s model that supports liquid spreading on a super-hydrophilic rough surface [37,38].Hydrophilic properties make the surface favorable for polymer deposition because the coating materials are quickly and widely spread on the textured metal surfaces,facilitating good adhesion [31,39].

Fig.3.(a-d) TEM-EDS of cross-section mapping on the canyon surface,(e-h) SAED pattern at the boundary between MgO and Mg.(i) EDAX line profil from the surface to the bottom.(j) HR investigation at the boundary between MgO and Mg.

The polymer adhesion force of the surface was measured with the increase in roughness of the laser-treated Mg surface,as shown in Fig.4b.As a result,PLGA@FsMg endured 382.84±73.14N of load that had 77% higher adhesion strength compared with that of PLGA@BareMg which endured 215.91±51.98N of the load.A rough surface structure resulting from the laser treatment hooked the polymer coating layers,influencin the strong adhesion force.A similar effect was observed with the plasma treatment of Mg,wherein the surface-coated PLGA layers were strongly bound [20,40,41].In addition,the bonding with the polymer layer became more stable as the oxide surface area increased [39,42].These results indicate that the oxide nanostructures fabricated by fs lasers had improved coating ability.Protein absorption also showed a similar trend:the absorption of elongated fibrino gen was improved on a nanoporous surface as compared to that of globular bovine serum albumin [43].Therefore,we suggest a fs laser-treated surface rather than a fla Mg surface because the former adds hooked layers that physically attach the linear biomolecules with improved adhesion strength.

3.3.Corrosion property

Fig.4.(a) SEM images of cross-sections of samples after spin-coating;PLGA@Bare (left) and PLGA@FsMg (right).(b) Adhesion strength curve of PLGA coating layer and Mg substrates.(c) Amount of hydrogen gas and (d) concentration of Mg ions released by Control (Ctrl),PLGA@Bare,and PLGA@FsMg.(e) Behavior of the coated layer on substrates during an immersion test after 7 d (left image:PLGA@Bare;right image:PLGA@FsMg);scale bar=4mm.(f) SEM images of cross-sections after corrosion test (left image:PLGA@Bare;right image:PLGA@FsMg);scale bar=20 μm.

PLGA-coated Mg was immersed in Hank’s solution and tested for corrosion,as shown in Fig.4c.As PLGA was coated on Mg,hydrogen gas release was delayed for the firs 3 d in comparison with the case of bare Mg.Thereafter,PLGA@BareMg abruptly released hydrogen gas;only PLGA@FsMg maintained a stable hydrogen gas release(Fig.4c).The PLGA coating layer of PLGA@BareMg was damaged,exposing Mg to the buffer and resulting in rapid corrosion.However,the PLGA layers on PLGA@FsMg were stable up to 162h.The anti-corrosion effect of the PLGA coating was also confirme by the Mg ion concentration profil in Fig.4d.PLGA@FsMg with 15.7mM concentration showed effective control over Mg ion release,similar to the hydrogen gas release profile compared with PLGA@BareMg and Bare Mg with 32.6 and 40.8mM concentrations,respectively.Additionally,the Mg ion concentration increase slowed down between days 3 and 7 in the case of PLGA@FsMg but accelerated in the case of PLGA@BareMg.The PLGA-coated samples were removed and the coating layer was confirme after the corrosion test.PLGA@BareMg demonstrated the delamination of the entire coating layer from Mg.In contrast,PLGA@FsMg showed slight damage in the coating layer along the edge of the Mg substrate,as shown in Fig.4e.The cross-sectional morphology of the corroded Mg sample obtained via focused ion beam-SEM is shown in Fig.4f.There was no coating layer on the specimens for the control group as it had been already peeled off from the substrate.In contrast,the coating layer adhered onto the laser-treated Mg surface after the corrosion test was confirme via SEM imaging.In addition,PLGA is considered semi-permeable to solution,which would eventually lead to a corrosion reaction at the interface of the coating layer and Mg substrate [32].If the substrate presented poor resistance to corrosion,the rapid generation of hydrogen gas bubbles at the interface could cause premature delaminating of the coating layer.Therefore,it is clear that the laser treatment not only increased the adhesion ability but also improved the overall resistance to corrosion.

3.4.Cell test

Fig.5.(a) Cell viability of Control (Ctrl),PLGA@Bare and coating on PLGA@FsMg after immersion test in DMEM without serum for 1,3,and 7 d.(b)Cell morphology on surface of Ctrl (upper images) and PLGA@FsMg (below images) after corrosion tests for 3 d.

The Mg samples coated with PLGA after fs laser treatment were incubated with cells to evaluate the stability of the PLGA coating layer under cell culture conditions,as shown in Fig.5.To evaluate cell viability,the L929 cells were directly seeded on the PLGA-coated Mg substrates,cultured for 7 d,demonstrating normalized cell viability as shown in Fig.5a.PLGA@FsMg showed stable cell proliferation for 7 d However,the viability in the case of PLGA@BareMg decreased to 20% on day 3 because the cells on the PLGA layers detached from Mg in the media,similar to how it proceeded in the corrosion test (Fig.4e,f).

Although the cells on bare Mg show stable cell attachment for 3 d,there is a continuous release of hydrogen gas as the number of hydroxide ions increases,posing a problem for implantable devices as they cause pain owing to the presence of gas pockets and local tissue necrosis.Therefore,efficien control of Mg corrosion is required for biomedical applications.The cell distribution on the PLGA-coated Mg was visualized via SEM;it was observed that a small number of cells were distributed on PLGA@BareMg.In contrast,a dense cell distribution on PLGA@FsMg was observed for 3 d (Fig.5b).

4.Conclusion

In this study,we developed a quick and simple method to obtain a physically and chemically modifie Mg surface with extremely hydrophilic properties to improve the coating ability using Fs laser.Through an Fs laser texturing technique,we physically modifie the Mg surface into a textured surface and chemically,into a super-hydrophilic state,such that the coating material effectively penetrated,embedded,and interlocked into micro-and nano-complex structures without any primers or chemical pretreatment.The enhanced coating quality led to increased adhesion strength and minimized the delamination from the substrate,resulting in improved corrosion properties and biocompatibility.

The use of this Fs laser surface treatment technique on Mg to improve the polymer coating ability could open doors for the future application of completely biodegradable metallic devices with drug-eluting polymer coatings.Furthermore,it is especially beneficia when a stable organic coating without chemical pretreatment is required.

Declaration of Competing Interest

Authors declare no competing interests.

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.2020R1A2C2010413) and the KIST project (2E30341).