Tuning biomechanical behavior and biocompatibility of Mg–Zn–Ca alloys by Mn 3 O 4 incorporated plasma electrolytic oxidation coatings

In this study, the mechanical behavior and biocompatibility of plasma electrolytic oxidation (PEO)-coated Mg – Zn – Ca alloy specimens were investigated. The coatings were synthesized by incorporating KMnO 4 and Mn 3 O 4 nanoparticles into an electrolytic solution. An indentation test revealed a significant increase in the reduced elastic modulus of the PEO coatings with incorporated Mn 3 O 4 under various loads. This increase was attributed to the higher coating thickness and reduced porosity achieved by the addition of Mn-based additives to the electrolyte. The composite PEO coatings prepared with Mn 3 O 4 nanoparticles exhibited a more pronounced reduction in elastic modulus under pressure. Wettability tests showed that the prepared coatings maintained their hydrophilic nature with water contact angles in the range of 25 – 63 ◦ . The presence of Mn 3 O 4 in the PEO coatings provided a conducive environment for cell viability. The enhanced biocompatibility of the composite coatings achieved by incorporating KMnO 4 into the electrolyte was particularly noteworthy. This improvement was attributed to the controlled release of Mn ions, which generates a microenvironment that favors cellular activities. The study showed that incorporating Mn 3 O 4 into PEO coatings enhances mechanical properties, preserves hydrophilicity, and improves biocompatibility, thus indicating its potential for orthopedic implant applications.


Introduction
Metallic implants play a major role in bone tissue engineering by providing structural support and enhanced stability and promoting integration with surrounding tissues, thereby addressing critical functional and biomechanical requirements for effective bone regeneration [1,2].Due to their remarkable properties such as near-density similarity to natural bone, biodegradability, and biocompatibility, magnesium and its alloys have garnered significant attention in the field of bone tissue engineering [3,4].However, the high corrosion rate of Mg in physiological solutions poses a considerable challenge to its application as a bone implant [5].This accelerated corrosion results in a decline in mechanical performance because of biodegradation and the release of byproducts including hydroxyl ions and hydrogen, which negatively affect the long-term efficacy of Mg-based implants [4,5].
To address this challenge, two primary strategies have been employed to control the corrosion rate of Mg, namely alloying and surface modification [6,7].Alloying with elements such as zinc, calcium, and rare-earth elements has proven to be effective in enhancing the corrosion resistance of Mg.Among these alloys, Mg-Zn-Ca systems have emerged as promising candidates for Mg implants, showcasing biocompatible Zn and Ca elements and demonstrating favorable in vitro and in vivo results [8,9].
Plasma electrolytic oxidation (PEO) is effective in improving the anticorrosive properties of Mg-based biomaterials [10,11].Although PEO coatings offer several advantages such as enhanced corrosion resistance, their inherent limitations, such as low thickness and porous structure, restrict their applicability in long-term implantation scenarios [12][13][14].Recent advancements have sought to overcome these limitations by introducing additives to the electrolyte composition for the purpose of decreasing the porosity and increasing the coating thickness [15][16][17].Moreover, these additives may enhance the biocompatibility of Mg implants by incorporating bioactive elements such as Ca, P, and Ag into the PEO coatings to further optimize the interaction of the implant with the surrounding biological environment [18,19].
The mechanical properties of Mg implants, particularly their elastic modulus, play a pivotal role in their success as bone implants [20,21].The reduced elastic modulus (elastic modulus divided by unity minus the squared Poisson's ratio) is a measure of a material's ability to deform under load [22].Knowledge of the elastic modulus of bone implants enables the mechanical compatibility with bone to be optimized, the biocompatibility to be enhanced, and the overall performance and longevity of the implant in the human body to be improved [23].Studies have shown that the elastic modulus of metallic implants can be improved using PEO coatings [24][25][26][27].However, to the best of our knowledge, the reduced elastic modulus of coated Mg biomaterials under loading has not been explicitly studied.
In our previous study, Mg-Zn-Ca alloys were coated with Mn 3 O 4incorporated PEO using the following different routes: in situ chemical reactions by KMnO 4 addition and physical incorporation of Mn 3 O 4 nanoparticles [28].The findings demonstrated a significant improvement in the corrosion performance when compared with the composite coatings to the basic PEO coatings, which was attributed to the reduced porosity and increased thickness.Through indentation testing, this study further examined the reduced elastic moduli of the basic PEO and Mn 3 O 4 -incorporated PEO coatings.We also present results regarding the cell behavior of uncoated and coated Mg-Zn-Ca alloys, providing insights into how these coatings affect the biomechanical and biocompatibility of Mg implants.

Coating preparation and characterization
The previous study provides a detailed account of the coating preparation and characterization procedures [28].Briefly, PEO coatings were prepared using a direct current Plasma-Pazhouh power supply, where the Mg-2.1Zn-0.6Caalloy was metallurgically produced by the authors to serve as the anode and a stainless-steel thermal regulation tube to serve as the cathode.The PEO coatings were prepared over a 15-min oxidation period under a duty cycle of 50 %, frequency of 1000 Hz, and an adjusted 100 mA cm −2 for the current density [28].The basic PEO coating (BSC-PEO) utilized a water-based electrolyte solution containing tripotassium phosphate trihydrate and potassium hydroxide.The composite coatings (Table 1), that is, MnPR-PEO and MnSL-PEO, were prepared by introducing manganese (II,III) oxide nanoparticles and potassium permanganate salt, respectively, into the base electrolyte.The surface features were observed using scanning electron microscopy (SEM, Zeiss Evo-Ma15), as shown in Fig. 1.The images were analyzed for coating porosity using ImageJ software (version 6.0).The mean surface roughness and coating thickness were assessed in 10 regions of the coatings using PHYNIX TR-100 and PHYNIX FN devices.Table 2 lists the porosities, thicknesses, and roughnesses of the prepared coatings.

Reduced elastic modulus
Reduced elastic modulus (E r ) is a key material property that offers insights into a material's elastic response when subjected to indentation [29].This parameter is typically derived using instrumented indentation-testing techniques.In this method, a sharp indenter is systematically pressed into the material surface under continuous recording of the load and displacement data [29,30].E r is calculated based on the initial unloading phase of the indentation curve, which reflects the elastic deformation of the material.E r provides valuable information regarding the elastic deformation capacity of a material under an applied load during the indentation test.This is particularly beneficial for characterizing small-scale mechanical properties, as observed in applications involving thin films, coatings, and specific regions of interest within bulk materials.In this study, E r values for uncoated and coated samples were measured using an indentation mode of a dynamic mechanical analyzer (DMA 242 E Artemis, Netzsch Gerätebau GmbH, Selb, Germany) equipped with a flat-ended cylindrical indenter of 1.0 mm in diameter.Measurements were conducted at load levels equivalent to applied pressures of 0.2, 0.5, 1.0, 2.0, 4.1, and 8.2 MPa.Data were collected and processed using Proteus 6.1 software (Netzsch Gerätebau GmbH, Selb, Germany).The DMA results were statistically assessed using the estimation protocol described in detail in Ref. [31].

Wettability measurements
The evaluation of wettability involved assessing the contact angles of 5 μL of deionized water droplets using a Theta Flex optical tensiometer (Biolin Scientific) in the sessile-drop configuration.Five contact angles were measured in distinct areas, and the average results and standard deviations were computed for both uncoated and coated specimens.All measurements were conducted under consistent humidity and temperature conditions.

Cell viability assay
Cells with osteoblast-like features (NCBI C-116, sourced from the National Cell Bank of Iran), specifically G292 cells, were cultured in Dulbecco's modified Eagle medium (DMEM).The solution incorporated 10 % (v⋅v −1 ) fetal bovine serum (FBS) and 1 % pen/strep (Gibco), and the cells were kept at a temperature of 37 ± 0.5 • C within an adjusted humid atmosphere containing 5 % CO 2 .Following a 48-h incubation interval, the cells were harvested and distributed at a concentration of 5 × 10 5 cells per well in 96-well plates.The cells were then cultured in DMEM supplemented with 10 % FBS for 24 h.Sterilized, uncoated, and coated Mg samples were added to the wells.A well without a sample served as a negative control with 5 × 10 5 cells.The culture medium was substituted by introducing 50 μL of DMEM enriched with 20 % FBS along with 50 μL of diluted extracts (at concentrations of 0.78, 6.25, and 12.5 mg mL −1 ).A negative control consisting of medium and 10 % FBS devoid of diluted extracts was used.After 1, 4, and 7 d of incubation at 37 ± 0.5 • C and 5 % CO 2 , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) medium (0.5 mg mL −1 ) per well was added.
Following a 4-h incubation, 750 μL of dimethyl sulfoxide per well was added to halt the MTT-cell reaction.The optical density (OD) was assessed using a microplate reader (ELISA) at a wavelength of 570 nm.Cellular morphological changes around the samples were observed under an inverted phase-contrast microscope (Olympus IX51) upon completion of the incubation period.

Coatings morphology
Fig. 1 shows the SEM surface micrographs of the PEO coatings applied to the Mg-Zn-Ca substrate.The coatings exhibited a distinctive porous microstructure reminiscent of a volcanic crater, which is a common characteristic of PEO-coated metals.These pores were generated through local microdischarge occurrences during the microarc oxidation step [10,32].At this stage, the liquefied substrate material flows through the discharge pathways and solidifies upon contact with the cold electrolyte.Because of gas evolution, this process induces the

Table 1
Codes assigned for PEO coatings along with their corresponding electrolyte compositions.

Sample
Electrolyte (g⋅L formation of cracks and pores [10,11].In our study, microcracks emerged on the surface of the PEO-coated specimens because of rapid solidification under heat shock.This shock was caused by the electrolyte's rapid cooling of the material, which had recently melted during discharge [10,11].The incorporation of Mn-based additives into the electrolyte, as detailed in Table 2, contributed to a reduction in the surface porosity as compared with that of pure PEO coatings [28].This suggested that the additives may have affected the dimensions of the pathways for discharges and the quantity of the expelled oxygen [33].A previous study highlighted that the decline in the relative porosity of the MnSL-PEO coating could be ascribed to the diminished plasma discharge energy.This reduction was attributed to the lower breakdown and final voltage resulting from the incorporation of KMnO 4 .The MnSL-PEO samples exhibited a surface particle distribution, distinct from the PEO coatings formed using a simple electrolyte and lacking KMnO 4 .For the MnPR-PEO samples, the incorporation of the nanoparticles resulted in elevated process voltages.Subsequently, these particles adhered closely to the coating surface, effectively occupying defects such as cracks and pores [28]. The results listed in Table 2 indicate a minor increase in coating thickness when KMnO 4 was introduced into the electrolyte.By contrast, the introduction of Mn 3 O 4 nanoparticles contributed to a significantly enhanced thickness as compared with the PEO coatings.The observed differences could be explained by the effects of additives on the gradients in the voltage-time diagram throughout the first stage (anodization) [34].Indeed, nanoparticles within the test electrolyte increased the process voltages, resulting in heightened inserted energy.This in turn caused a greater volume of the substrate material to undergo liquefaction and subsequent solidification during each microdischarge event.The inclusion of KMnO 4 led to lower final and breakdown voltages, indicating a reduced energy input.However, this alteration did not translate into a decrease in the thickness of the composite coatings.The complex interactions of various factors such as modified discharge dynamics, shifts in the ionic concentration within the electrolyte, and potential thermal influences collectively shaped the characteristics of the PEO coatings [28].
The introduction of KMnO 4 additives into the electrolyte was thought to influence the energy of the plasma discharge, which in turn induced changes in the roughness of the synthesized MnSL-PEO coating.As the intensity of the microdischarges diminished due to the presence of KMnO 4 , the discharge channels were reduced in size, resulting in a reduction in the surface irregularities between the peaks and valleys [28].

Indentation test
The variations in E r of the uncoated and coated Mg alloys when different loads were applied are shown in Figs. 2 and 3.The variations in E r among the different PEO-coated Mg-Zn-Ca substrates could be explained by the observed differences in the coating thickness and porosity.Indeed, the higher E r measurements for the MnSL-PEO and MnPR-PEO samples as compared with the BSC-PEO coating at various pressures aligned seamlessly with the corresponding variations in the coating thickness and porosity.As Table 2 shows, the incorporation of KMnO 4 salt and Mn 3 O 4 nanoparticles into the basic electrolyte decreased the porosity and increased the thickness of the MnPR-PEO and MnSL-PEO specimens.In general, as the thickness of the PEO coating increased, the overall stiffness of the coated material tended to increase [35].Thicker coatings often contributed to significant changes in the

Table 2
Mean values and standard deviations of relative surface porosity, coating thickness, and surface roughness.mechanical properties of the implant surface, including an increase in the elastic modulus [36].A critical aspect to be considered is the significant thickness of the coating.If the thickness decreases to less than a critical value, the coating may reflect the properties of the substrate.However, if the thickness exceeds a critical value, the coating asserts its self-identity, thus exhibiting unique properties [37].This transition in thickness can also affect the load-bearing capacity of the implant, thereby affecting stress distribution during loading [35][36][37].The elastic response may have been significantly affected, potentially resulting in a higher E r .
The lower porosity of the Mn 3 O 4 -incorporated PEO coatings indicated a more compact and denser structure [13].This denser structure tends to exhibit improved mechanical properties, including higher hardness and stiffness [38].Compact coatings with lower porosities are likely to provide better load transfer between the coating and underlying Mg-Zn-Ca alloy.This improved load transfer can contribute to a more uniform stress distribution and may result in increased E r .The choice between a high or low E r for Mg implants depends on factors such as the intended application, load-bearing requirements, and the desire to minimize stress-shielding effects.Considering the specific biomechanical and biological context of the implant site and the degradation characteristics of the material is critical to ensure the long-term success of Mg implants in medical applications.

Contact angle measurements
The coatings were subjected to contact angle measurements to examine their wetting properties, with the results shown in Fig. 4. A contact angle of less than 90 • signified hydrophilicity, whereas angles greater than 90 • indicated hydrophobicity [39].In this study, all coatings displayed contact angles of less than 90 • , suggesting the spreading and wetting of the surface by water droplets.MnPR-PEO exhibited the highest contact angle at 63 • , whereas BSC-PEO, because of its notably rough surface, exhibited the lowest contact angle at 25 • .The wetting characteristics of the PEO coatings were mainly determined by their surface phase, topography, and chemical composition [40].The pore size and porosity inherent to PEO coatings affected the water contact angles.Incorporating Mn 3 O 4 into the MnPR-PEO and MnSL-PEO-coated Mg-Zn-Ca alloys reduced the porosity and pore size (Table 2), resulting in increased water contact angles.
As a crucial parameter for adsorption of molecules, the surface free energy (E s ) was calculated using the formula E s = E vl ⋅cosθ, where E vl was 72.8 mJ m −2 (i.e., surface free energy under ambient conditions of water and air) and θ denoted the static contact angle [41].Fig. 3 shows the calculated surface free energy values.MnPR-PEO had the lowest surface energy, whereas BSC-PEO had the highest, which was attributed to its roughness and open pores.The hydrophilic surfaces used in the present study are preferred for protein adsorption, cell migration, and proliferation in biomedical applications.Implants with suitable wettability, influenced by contact angles, play a major role in early osseointegration during interactions with biofluids, thereby ensuring success in the development of materials for biomedical purposes [42].coated sample exhibiting a higher cell density than that of the uncoated Mg alloy.In addition, the OD values for the PEO coatings were significantly higher than those of the uncoated Mg-Zn-Ca alloys.The PEO coatings prevented the infiltration of corrosive media and lowered the degradation rate of the Mg-Zn-Ca alloy, as evidenced in our previous study [28].Moreover, recent research has shown that protective PEO coatings slow the penetration of OH − groups from Mg surfaces into biological media, resulting in a lower increase in pH [43,44].Despite the reported general benefits of Mg ions in cell metabolism and DNA synthesis, the higher corrosion rate of uncoated Mg can lead to increased alkalization of the culture medium, creating a more toxic environment for live cells and negatively affecting cell adhesion and proliferation [45,46].

MTT assay
Notably, all the PEO-coated samples provided an excellent environment for G292 osteoblast cell line growth.MnSL-PEO and MnPR-PEO exhibited higher cell viability than BSC-PEO, suggesting no significant toxicity at any time interval.Despite the higher porosity and wettability of BSC-PEO, its higher corrosion rate may compromise its biocompatibility, particularly over longer periods.The enhanced biocompatibility of the MnPR-PEO and MnSL-PEO specimens was attributed to the presence of Mn.Incorporating Mn into coatings has the potential to promote bone growth, given the role of Mn ions in stimulating osteogenic activity [47].Mn facilitates the differentiation of mesenchymal stem cells into osteoblasts, supporting their bone-forming capacity.In addition, Mn has been reported to contribute to the synthesis of collagen, a crucial component of the bone matrix in providing structural support, strength, and flexibility [48].Adequate Mn levels play a role in proper collagen formation and maintenance in bone tissues.As a cofactor for enzymes such as alkaline phosphatase, Mn positively influences the mineralization and bone formation processes [49].For MnSL-PEO, the low count of dead cells as compared with MnPR-PEO suggests a lower release of Mn ions, which is consistent with the previous study [28].These findings collectively demonstrate that proper PEO treatment of Mg in a K 3 PO 4 + KOH + KMnO 4 electrolyte can enhance cell viability without inducing cytotoxic effects.

Conclusions
The effects of adding Mn 3 O 4 to a PEO-coated Mg-Zn-Ca substrate were investigated in this study using a dual strategy of incorporating both KMnO 4 and Mn 3 O 4 nanoparticles as additives into the electrolyte process.
1) The addition of Mn 3 O 4 significantly increased the reduced elastic modulus of the PEO coatings.This enhancement was attributed to the greater coating thickness and reduced porosity achieved by incorporating Mn-based additives into the electrolyte.2) All coatings were hydrophilic in nature, with water contact angles of less than 90 • .The slight increase in the contact angle observed in the Mn 3 O 4 -incorporated PEO coatings that resulted from the decreased porosity and roughness did not compromise their overall hydrophilic behavior.3) The Mn 3 O 4 -incorporated PEO coatings provided a favorable environment for cell adhesion, growth, and proliferation.The composite coatings prepared by adding KMnO 4 to the electrolyte exhibited the best biocompatibility, which was attributed to the controlled release of Mn ions and thus ensured a conducive microenvironment for cellular activities.

Fig. 1 .
Fig. 1.Schematic of the PEO setup with different electrolyte composition and SEM micrographs of prepared coatings.

25 Fig. 2 .
Fig.2.The reduced elastic modulus by the indentation method was assessed for both uncoated and coated samples under different pressures of (a) 0.25 MPa, (b) 0.5 MPa, (c) 1.0 MPa, (d) 2.0 MPa, (e) 4.1 MPa, and (f) 8.2 MPa.Median differences for these comparisons are illustrated in the corresponding Cumming estimation plot produced according to the method described in Ref.[31].The top axes present the raw data; the bottom axes display each mean difference through a bootstrap sampling distribution.Dots on the graph represent mean differences, with the vertical error bar endpoints indicating the corresponding 95 % confidence intervals.

Fig. 5 Fig. 3 .
Fig. 5 depicts the cell viabilities of the control, uncoated, and coated samples over the three intervals of 1, 4, and 7 d, as assessed by the MTT assay.The OD values associated with cell multiplication densities showed a slight decrease with prolonged culture time.Examination of the cell morphologies after 7 d of incubation, as illustrated in Fig. 6, revealed elongated and spindle-shaped cells for all samples, with the

Fig. 4 .
Fig. 4. Measurements of water contact angles and surface free energy for the coated samples, with water droplet variations shown in the inset.

Fig. 5 .
Fig. 5. MTT assay results, where formazan absorbance serves as an indicator of cell viability.Osteoblast cells were seeded on samples and incubated for 1, 4, and 7 d, with comparisons made to the control well.