In-situ formation of Zn-MOF coating on MgO/HA composite layer produced by plasma electrolytic oxidation on Mg-Sn-Mn-Ca alloy for orthopedic internal fixation devices

ABSTRACT

A novel ultra-sound assisted method was used to grow zinc-metal organic framework (Zn-MOF) film on magnesium oxide/hydroxyapatite (MgO/HA) composite layer developed using plasma electrolytic oxidation (PEO) on Mg-5Sn-0.2Mn-0.2Ca alloy. The microstructure surface roughness and microhardness tests were performed on the samples to evaluate the mechanical properties of the coatings. The electrochemical corrosion test was performed to evaluate the corrosion resistance of the coated Mg alloy. Further, an in-vitro bioactivity test was performed to study the biocompatibility and bioactivity of the coated alloy, which is an essential aspect of biomedical applications. Results showed that the MgO/HA composite coating was helpful in the in-situ growth of the Zn-MOF particles by mechanically interlocking them. Further, incorporating hydroxyapatite in the coating could also increase the bioactivity. The study also confirmed that the bi-layer coating of Zn-MOF on the MgO/HA composite layer improved the surface roughness, hardness, biocorrosion resistance, and bioactivity making it more likely to be used for orthopedic internal fixation devices.

KEYWORDS:

• Magnesium alloy
• plasma electrolytic oxidation
• metal-organic framework
• immersion
• biocompatibility

1. Introduction

Magnesium (Mg) and its alloys have attracted considerable attention as biodegradable materials for orthopedic internal fixation devices, due to their excellent biocompatibility, mechanical properties, and degradation behavior [1–3]. There has been a lot of research toward choosing suitable alloying elements based on the required properties needed for the designed alloy. Of late, the addition of Sn also was proven to be very effective for enhancing the corrosion resistance of Mg by forming the SnO₂ layer along with the corrosion products [4–6]. The addition of calcium (Ca) and zinc (Zn) would help in decreasing the average grain size and refining the grain structure respectively in the Mg-Mn system [7]. It was also noted that the mechanical properties of Mg-Si alloy have been improved by the addition of 0.18 wt. % Ca and 1.5 wt. % Zn [8]. Mg‒Zn1.0‒Ca0.3 (ZX10) and Mg‒Zn1.5‒Ca0.25 alloys exhibited uniform degradation and no adverse properties on the tissue [9]. However, the rapid and uncontrollable corrosion of Mg alloys in physiological environments may cause adverse effects such as hydrogen gas accumulation, alkalization of the surrounding tissues, and loss of mechanical integrity. Therefore, various surface modification techniques have been developed to improve the corrosion resistance and biocompatibility of Mg alloys, such as anodization, micro-arc oxidation, sol-gel coating, hydrothermal treatment, and biomimetic mineralization [10,11]. Among these techniques, plasma electrolytic oxidation (PEO) is a promising method to produce ceramic coatings on Mg alloys, which can effectively protect the substrate from corrosion and enhance the surface hardness and wear resistance [12]. The PEO coatings on Mg alloys usually consist of MgO as the main phase, and other phases depending on the alloying elements and the electrolyte composition [13]. Ly and Yang reported that PEO coating on Mg–4.71Zn–0.6Ca alloy could greatly improve corrosion resistance and bioactivity [14]. Further, many investigations have focused on adding bioactive ceramic particles in the electrolyte such as Calcium phosphate, hydroxyapatite (HA), ZrO₂, SiO₂, ZnO₂and TiO_2, etc. in the PEO process [15–19] However, the PEO coatings may also have some drawbacks, such as high porosity, low adhesion, and poor bioactivity, which may limit their application in biomedical fields. To overcome these limitations, various strategies have been proposed to modify the PEO coatings, such as incorporating bioactive particles, doping with metal ions, and applying post-treatments. Among them, metal-organic frameworks (MOFs) have emerged as a novel class of materials that can be used to modify the PEO coatings, due to their unique properties such as high surface area, tunable pore size, adjustable chemical functionality, and versatile structural diversity [20]. MOFs are crystalline porous materials composed of metal nodes and organic linkers, which can form various structures and host different guest molecules. MOFs have been widely used in various fields, such as gas storage, catalysis, sensing, drug delivery, and biomineralization. The bio-MOF-1 coating on AZ31B alloy has shown improved corrosion resistance, and biocompatibility suggesting their potential application in orthopedic implants [21]. The bilayer composite dressing composed of Ag-MOF loaded chitosan nanoparticles and polyvinyl alcohol/sodium alginate/chitosan (PACS) accelerated the wound healing process [22]. In this study, we report a novel approach to fabricate a Zn-MOF coating on a MgO/hydroxyapatite (HA) composite layer produced by PEO on a Mg-Sn-Mn-Ca alloy. The PEO process was conducted in a phosphate-based electrolyte containing HA particles, which resulted in the formation of a MgO/HA composite layer with enhanced corrosion resistance and bioactivity. The Zn-MOF coating was synthesized in situ on the PEO layer by immersing the PEO-treated alloy in a solution containing 2-methylimidazole (Hmim) as the organic linker, which acted as a secondary coating to further improve the corrosion resistance, and bioactivity of the Mg alloy. The proposed Zn-MOF/PEO coating is expected to be a promising surface modification technique for Mg alloys used as orthopedic internal fixation devices.

2. Materials and methods

2.1. Preparation of magnesium alloy (mg-5Sn-0.2Mn-0.2Ca)

Mg-5Sn-0.2Mn-0.2Ca was fabricated using a squeeze casting method. In the preparation of the alloy, 50 g of tin (Sn), 2 g of manganese (Mn), and 2 g of calcium (Ca) were added to 956 g of magnesium (Mg) to make 1000 g of alloy. Sn ingots, powdered Ca, and Mn granules were introduced into a preheated crucible containing magnesium melt inside the electrical resistance furnace under the controlled atmosphere of pure argon gas. The molten charge is stirred at 600 rpm for 5 min at 960°C using a stirrer. Subsequently, the molten material was drawn by gravity into the metallic mold preheated to 400°C and then subjected to a squeeze load of 40 tons to facilitate further solidification.

2.2. Synthesis of hydroxyapatite (HA) and zinc metal-organic framework (zn MOF)

Hydroxyapatite was prepared using a wet chemical precipitation process. 1 M of calcium nitrate and 0.6 M of diammonium phosphate are combined with 400 ml of distilled water to maintain the Ca/P ratio of 1.67. pH of the solution was adjusted to 10.4 using ammonium hydroxide. Di-ammonium phosphate was added to the calcium nitrate solution drop by drop at a rate of 2 ml per minute while constantly stirring the solution. The mixture was kept in the microwave for a total of 10 min (30 s – on and 30 s – off) to aid the nucleation process. The precipitate was then filtered, washed, dried, and then powdered using mortar and pestle. To improve the crystallinity and purity of hydroxyapatite before being heat treated, the powder was heated in a hot air oven for 2 h at 200°C and then heat treated in a muffle furnace at 1200°C for 2 h. The preparation of Zn-MOF was carried out by mixing 2.38 g of Zinc Nitrate [Zn(NO₃)₂⋅6 H₂O] and 2.62 g of 2-methylimidazole (2-mIm) and stirred in 30 ml of methanol until a homogeneous mixture is obtained [23].

2.3. Plasma electrolytic oxidation coating on Mg alloy (with the incorporation of HA) and Zn-MOF coating on PEO-coated Mg alloy

Mg alloy samples for PEO coating were prepared by polishing the samples up to 1200 grit emery paper and then cleaning them with ethanol. The PEO process was carried out in a constant current mode with the value fixed at 1 A while the duty cycle and frequency are set at 50% and 50 Hz, respectively. The electrolyte was prepared by mixing 2 g of Potassium Hydroxide (KOH) pellets and 5 g of Sodium phosphate dibasic in 1 l of distilled water. A stainless-steel container was used to hold the electrolyte which also served as cathode, while the Mg alloy sample served as cathode. The process was carried out for 5 min and the final voltage was noted to be 560 V. In the case of MgO/HA composite coating preparation, 2 g of HA was added to the base electrolyte solution along with 10 ml of ethylene glycol and 15 ml of triethanol amine [24]. The process parameters used were current: 10 A, duty cycle: 50%, frequency: 50 Hz and coating time: 5 min. For the preparation of Zn-MOF coating, both the plain PEO coated and MgO/HA composite coated samples were immersed in the already prepared MOF solution for 3 h under continuous stirring using a probe sonicator. The samples were then removed from the solution and cleaned with ethanol and deionized water to remove any excess precursors and impurities. Finally, the prepared coating is dried at 80°C for 8 h in an oven. The sample codes have been assigned to all the processed and bare alloys as given in Table 1. These sample codes will only be referred to after this section of the manuscript.

Table 1. Designation of the samples under this study.

Sl. No	Sample Code	Description
1	M	As-cast Mg alloy of composition Mg-5Sn-0.2Mn-0.2Ca
2	M_P	PEO treated Mg alloy in standard electrolyte (KOH − 2 g/l; Na2PO3.10H2O − 5 g/l)
3	MH_P	PEO-treated Mg alloy in the standard electrolyte containing HA particles suspended in it
4	MF_P	PEO treated Mg alloy in standard electrolyte and then coated with Zn-MOF by probe sonication
5	MHF_P	PEO treated Mg alloy in the standard electrolyte containing HA particles suspended in it and then coated with Zn-MOF by probe sonication

2.4. Microstructural characterization

The morphologies of the samples M, M_P, MH_P and MF_P and MHF_P and the samples after the immersion and bioactivity tests were analyzed by Scanning electron microscopy (SEM-Oxford Instruments). The compound formation in the MHF_P sample was investigated through X-ray diffraction technique (XRD). Fourier transform infrared spectroscopy (FT-IR) was employed to detect the functional groups in the corrosion product layer formed on MHF_P using an infrared spectrometer (SHIMADZU). The pores were examined on the MF_P and MHF_P using image J.

2.5. Surface roughness and microhardness test

The average surface roughness of the as-cast and coated magnesium alloys was measured using a Mitutoyo SJ-210 stylus surface profiler. The device scans the material’s surface and records height variations using a stylus. A 10 mm stretch was analyzed to obtain surface roughness and wear profile data. The roughness parameters, specifically, the 10-point height (R_z) and the arithmetic mean of the sum of the roughness profile values (R_a) were measured. The collected scan results were analyzed to determine the surface roughness.

The micro-hardness test was conducted on as-cast Mg alloy, and all the coated samples by applying the load of 10 g for 15 s on the surface of the samples in accordance with ASTM E384.

2.6. Electrochemical corrosion tests

The electrochemical tests were performed using Gamry Instruments (Interface 1010T) as per the standard ASTM G3–14. The three-electrode setup was used for the electrochemical corrosion tests. The Saturated Calomel Electrode (SCE) served as the reference electrode; the platinum electrode served as the counter electrode and the magnesium samples served as the working electrode with an exposed area of 0.375 cm². The Tafel test was performed in a Simulated Body Fluid (SBF) environment. The SBF solution was prepared by adding 7.9950 g of NaCl, 0.3528 g of NaHCO₃, 0.2239 g of KCl, 0.1742 g of K₂HPO₄, 0.1428 g of MgCl₂, 0.2775 g of CaCl₂, and 0.0710 g of Na₂SO₄ to 1 l of distilled water.

2.7. Immersion & bioactivity test: in-vitro

An immersion test was performed by immersing the samples in SBF for 7 days while maintaining a constant temperature of 37°C using a constant temperature water bath. Weight gain/loss of the sample and pH of the solution were measured, and the solution was changed every 24 h. An in-vitro bioactivity test was performed by placing the samples in the SBF for 14 days while maintaining the temperature at 37°C and changing the solution for every 24 h. After 14 days of immersion, the samples were analyzed for the deposition of apatite using Scanning Electron Microscopy.

3. Results and discussion

3.1. Microstructure analysis

Figure 1a illustrates the SEM image of the M_P sample which revealed a typical PEO coating surface morphology containing microporosity formed during micro-arcing process. The coating seems to be rough and porous along with micro cracks. Meanwhile, the morphology of the sample MH_P [Figure 1b] showed comparatively less porosity, and the surface looks more uniform with no cracks present in the coating. This can be attributed to the HA particles being entrapped in the coating which were electrophoretically drawn onto the surface thereby reducing the porosity and cracks. Figure 1c depicts the surface morphology of the sample MF_P which shows the reduced porosity and granular appearance. This could be due to the presence of Zn-MOF on the surface which was further confirmed by analyzing the EDS elemental mapping shown in Figure 2. It is evident from Figure 2b,c that the Mg and O are densely distributed on the surface as the major phase of the coating is MgO formed from PEO process. Further, the uniform distribution of Zn can also be seen in Figure 2f which confirms the properly adhered coating of Zn-MOF formed on the PEO-treated surface. Figure 1d shows the morphology of the sample MHF_P in which it is very evident that the Zn-MOF was uniformly coated resulting in a porous free surface. It can be attributed to the presence of HA in the coating prior to the in-situ formation of Zn-MOF using the ultra-sound assisted method. It can be hypothesized that the composite coating of MgO/HA layer can be helpful for in-situ formation of the Zn-MOF layer and keeping it intact on the substrate. It was further affirmed by the EDS analysis of the MHF_P sample (Figure 3) which shows the presence of a higher amount of elemental Zn as compared to that of the sample MF_P. The porosity measurements were performed using image J, and it was plotted in Figure 4. Hence, it can be concluded that the composite layer of MgO/HA would be helpful in the in-situ coating of Zn-MOF on the PEO-treated Mg alloy. The grain size of MHF_P was measured as 35.28 nm using XRD by employing Williamson-Hall (W-H) plot method and 36.73 nm using image J from SEM micrographs.

Figure 1. SEM micrographs of (a) M_P, (b) MH_P, (c) MF_P, and (d) MHF_P.

Figure 2. SEM-EDS elemental mapping and spectra of MF_P.

Figure 3. EDS elemental mapping and spectra of MHF_P.

Figure 4. Porosity measurements.

3.2. Surface roughness and microhardness test

Surface roughness of the bare magnesium alloy and all the PEO coated samples are evaluated by the surface roughness measuring instrument, the 2D surface profiles of which are shown in Figure 5. The average values of Ra, Rq, Rsk, and Rku can also be found in Figure 5. It is evident from the results of Ra that all the PEO coated samples have the higher roughness as compared to the bare alloy. It is also interesting to note that the sample MF_P has a higher roughness value as compared to all other samples. It can also be noted that the higher value of Rq which confirms the presence of deeper craters with rough texture. In addition, the negative value of skewness indicates that it has a larger number of valleys. Such textures are more useful for drug delivery applications to retain the drug. Meanwhile, all other samples except MF_P show positive Rsk values indicating a higher count of peaks above the mean line. This provides the better adhesion of cells and their proliferation which in turn helps the faster bone healing process. Surprisingly, the kurtosis values of all the coated samples are found to be closer to 3 suggesting that the tails of the distribution are moderate, with no extreme outliers and have relatively balanced distribution of peaks and valleys. Hence, these tailor-made surfaces are highly potential for biomedical applications where hydrophobicity is a major influential factor.

Figure 5. Individual surface roughness profiles and average roughness parameters of M_P, MH_P, MF_P, MHF_P.

Figure 6 shows the values of the microhardness of all the coated samples in comparison with bare Mg alloy. It can be observed that the hardness of the PEO-treated samples has remarkably increased by ~44% as compared to bare Mg alloy. It is interesting to note that the samples coated with Zn-MOF have shown higher hardness as compared to their PEO-coated counterparts, i.e, the samples MF_P and MHF_P exhibited ~45% increment in hardness as compared to the samples M_P and MH_P, respectively. Hence, it can be understood that the Zn-MOF coating contributes to the significant improvement in the hardness of the coating. Although a little observation by comparing the hardness of the samples M_P and MH_P reveals that the incorporation of HA would also help to the increase in hardness of the coating, the more significant effect of Zn-MOF in increasing the hardness cannot be ruled out.

Figure 6. Microhardness.

3.3. Electrochemical corrosion test

Figure 7 depicts the tafel curves of bare, M_P, MF_P, MH_P and MHF_P samples and the corresponding results obtained from tafel extrapolation are given in Table 2. In comparison with the bare alloy, all the coated samples have shown higher corrosion resistance. However, in comparison with only PEO treated sample (M_P), the HA-incorporated coatings and Zn-MOF coated samples have exhibited much higher corrosion resistance. Out of all the samples, the sample containing HA and coated with Zn-MOF (MHF_P) has shown superior corrosion resistance which can be attributed to the pore-free surface and uniform layer of Zn-MOF on the surface as observed from the SEM micrographs. Zn-MOF layer could act as a barrier to the transportation of corrosive ions thereby protecting the inner layers and the substrate. It also conforms with the kurtosis value of MHF_P which declined as compared to others, indicating a more hydrophobic nature due to deeper craters. The same trend can be observed in the sample MF_P which contains Zn-MOF. Hence, it can be understood that the microporosity of the Zn-MOF coating and the unique structure of the MOF make the surface more hydrophobic, thereby reducing the corrosion rate. Furthermore, the sample containing HA in the coating (MH_P) also showed higher corrosion resistance. This could be due to the HA particles being electrophoretically drawn to the surface of the oxide coating and blocking the pores that were already formed in the oxide coating. This will prevent the corrosive medium from diffusing into the pores thereby preventing degradation of the substrate. At the outset, it can be understood that the Zn-MOF strengthens the barrier oxide film produced by PEO and fosters to stabilize the layer formed by corrosion products during exposure in corrosive media exhibiting better protection stability.

Figure 7. Tafel plots of M, M_P, MH_P, MF_P, and MHF_P.

Table 2. Electrochemical parameters of the samples derived from Tafel extrapolation.

Sample	Corrosion rate (mpy)	icorr (µA)	Ecorr (V)
M	35.44	14.70	−1.520
M_P	3.410	1.410	−1.480
MH_P	34.28e-3	0.5240	−0.684
MF_P	69.56e-3	0.2890	−0.271
MHF_P	126.3e-3	0.1420	−0.532

3.4. Immersion test

Based on the results obtained from surface roughness, hardness test, corrosion test, and immersion test were conducted on MF_P and MHF_P samples alone. Weight loss/gain and pH were measured during the immersion test for 7 days in SBF, and results are presented in Figure 8. It is evident from the results that both the samples had weight gain initially and in the last 3 days there was almost no weight gain observed as shown in Figure 8. Meanwhile, a trend of pH variation for 7 days in the sample MHF_P reveals that the release of OH ions was high, indicating the formation of more Mg(OH)₂. This justifies the reason for the MHF_P sample to show higher corrosion resistance as can be seen from the result of the corrosion test. The more stable and dense formation of the corrosion product layer would arrest the further degradation of the substrate. The compounds on the MHF_P and MHF_P immersed in SBF after 7 days were detected by strong peak intensities on XRD (Figure 9) for Mg on2θ = 36.619°, MgO on 2θ = 42.856◦, Mg₂Sn on 2θ = 37.728°, ZnO on 2θ = 36.337°, MgOH₂ on 2θ = 38.269°, CaMgSn on 2θ = 31.463°, Zn-MOF on 2θ = 5.343° and HA on 2θ = 32.14°, respectively, and also found that there is a difference in the level of intensity. The FT-IR spectrum of MHF_P and MHF_P after 7 days of immersion (Figure 10) in which MHF_P indicates the absorption peaks at 3469.94 cm⁻¹, 3847.99 cm⁻¹, 2908.65 cm⁻¹ and 1408.04 cm⁻¹ due to stretching vibration of OH, band at 1531.48 cm⁻¹ shows the stretching vibration of nitro compound, C-H bending vibrations of methylene groups at 2908.65 cm⁻¹ and 1869.02 cm⁻¹, respectively. The FT-IR spectrum of MHF_P after immersion indicates that the characteristic stretching vibration of C=O at 1855.82 cm⁻¹ and 1936.53 cm⁻¹ exhibits the carboxylate groups. The band at 2950 cm⁻¹ is attributed to the C‒H stretching vibrations of the methylene groups. The vibrational peaks between 3233.70 and 3932.86 cm⁻¹ reveal the presence of OH groups in MgO and Mg(OH)_2. The absorption peaks at 1435.34 cm⁻¹ represented the bending mode of ${\mathrm{C}\mathrm{O}}_3^{2-}$, and the absorption bands between 497.63 and 1000 cm⁻¹ can be attributed to ${\mathrm{P}\mathrm{O}}_4^{-}$ which normally associates with hydroxyapatite.

Figure 8. Weight loss/gain & pH measurements.

Figure 9. XRD of MHF_P and MHF_P after 7 days of immersion in SBF.

Figure 10. FT-IR spectra of MHF_P and MHF_ P after 7 days of immersion in SBF.

3.5. In-vitro bioactivity test

In-vitro bioactivity was also carried out on MF_P and MHF_P samples. The SEM micrographs of these samples after the test are shown in Figure 11. It is observed that both samples showed the formation of an apatite layer over the MOF-coated samples. It suggests that the surface of the alloy is bioactive and has the potential for biomedical applications. The MOF coating on the Mg alloy can serve as a scaffold for the growth of the apatite layer, and the resulting layer can have improved biocompatibility. However, on the surface of the MHF_P sample, the formation of apatite layer was significantly high which implies that the HA particles are helpful for the increase in the bioactivity of the Zn-MOF coated Mg alloy.

Figure 11. SEM micrographs showing bioactivity of (a) MF_P, and (b) MHF_P after 14 days of immersion in SBF.

4. Conclusion

After conducting a comprehensive investigation on the in-situ formation of zinc metal-organic framework on PEO-treated Mg alloy, the following conclusions are drawn:

1. Zn-MOF coating using an ultra-sound assisted technique on the PEO-treated Mg alloy could help seal the pores formed in the plasma electrolytic oxidized Mg alloy.

2. The in-situ growth of Zn-MOF also creates the surface texture which is amicable for the surface hardness and surface hydrophobicity.

3. In particular, the Zn-MOF layer on the HA containing oxide layer is highly useful for improving corrosion resistance and bioactivity as well.

At the outset, it can be concluded that the present work paves the way for surface modification of Mg alloys using the metal-organic framework for biomedical implant applications.

Acknowledgment

The authors thank VIT for providing “VIT RGEMS SEED GRANT” for carrying out this research work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The surface roughness data for some samples are provided in the link below.https://github.com/radha8vit/Experimental-data.git

The remaining data will be available made on request.

Additional information

Funding

The work was supported by the Vellore Institute of Technology, Chennai.