Enhanced corrosion resistance, antibacterial activity and biocompatibility of gentamicin-montmorillonite coating on Mg alloy-in vitro and in vivo studies

doi:10.1016/j.jmst.2021.08.089

Abstract

Abstract:

Implant-associate infection (IAI) is a major cause of failure of bone implant materials, and one of the significant challenges in clinical managements. A synergistic coating strategy combining montmorillonite (MMT) sustained release, adsorption of bacteria and gentamicin (GS) bactericidal is proposed herein to tackle infection issues. Surface morphology, microstructure and chemical composition of the samples were investigated using SEM, XRD, FT-IR and XPS. Electrochemical experiments and immersion experiments reveal that corrosion resistance of Mg samples with GS/MMT coatings was higher than that of bare Mg alloy substrate in DMEM solution. In vitro studies demonstrated that the GS/MMT coating had a significant inhibitory effect on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The viability of MC3T3-E1 cells was 92.7% after a co-culturing for 72 h. After a subcutaneous transplantation of 90 days, the survival rate was 100% for GS/MMT-coated Mg alloy specimens with no infection at the implantation sites and no toxic damage to liver, kidney and local muscles pathological sections. This study provides a novel method for the preparation of sustained-release antimicrobial coatings on biodegradable Mg alloys as promising candidates for orthopedic implant materials.

Key words: Magnesium alloy, Montmorillonite, Gentamicin, Antibacterial activity, Coating

Lijun Fan, Wenxin Sun, Yuhong Zou, Qian-qian Xu, Rong-Chang Zeng, Jingrui Tian. Enhanced corrosion resistance, antibacterial activity and biocompatibility of gentamicin-montmorillonite coating on Mg alloy-in vitro and in vivo studies[J]. J. Mater. Sci. Technol., 2022, 111: 167-180.

Figures/Tables 15

Fig. 1. Process of preparing GS/MMT coating on the surface of Mg alloy AZ31 via hydrothermal method and dipping method.

Fig. 2. SEM morphologies, corresponding EDS spectra, cross-sectional micro-graphs of the MMT (a, c, e) and GS/MMT (b, d, f) coating and FT-IR spectra (g) and XRD patterns (h).

Fig. 3. (a) XPS broad survey of MMT coating and high-resolution of (b) Na, (c) Mg, (d) O, (e) Al and (f) Si, (g) XPS broad survey of GS/MMT coating and high-resolution of (h) S, (i) Mg, (j) O, (k) Al and (l) Si.

Fig. 4. SEM images and the corresponding EDS spectra of the AZ31(a-c, a1-c1), MMT (d-f, d1-f1) and GS/MMT (g-i, g1-i1) coated Mg specimens after immersion in DMEM; the cross-sectional micro-graphs of MMT coating (j), GS/MMT coating (k) after immersion for 5 days.

Fig. 5. HEA curves of AZ31, MMT and GS/MMT coatings immersed for 120 h (a), variation in pH values for AZ31, MMT and GS/MMT coatings in DMEM for 24 h (b), FT-IR spectra (c) and XRD patterns (d) of the MMT coating and GS/MMT coating immersed in DMEM for 5 days.

Fig. 6. Potentiodynamic polarization (PDP) curves (a), EIS and the fitted results for Mg alloy, MMT and GS/MMT coatings: Nyquist plots (b); Bode plots of phase angle vs. frequency (c); Bode plots of |Z| vs. frequency(d) in DMEM; equivalent circuits of the Mg alloy (e); MMT coating and the GS/MMT coating (f).

Table 1. Electrochemical parameters of the polarization curves.

Samples	E_corr (V_SCE)	I_corr (A cm^-2)	β_a (mV dec^-1)	-β_c (mV dec^-1)	R_p (Ω cm²)
Mg alloy	-1.44	1.98 × 10^-5	79.8	159.4	3.49 × 10⁶
MMT coating	-1.43	1.78 × 10^-6	66.3	84.6	7.49 × 10⁷
GS/MMT coating	-1.35	1.67 × 10^-6	49	90.4	2.78 × 10⁷

Table 2. Electrochemical data obtained via equivalent circuit fitting of the EIS curves.

Samples	R_s (Ω cm²)	R_ct (Ω cm²)	CPE₁ (Ω^-1 cm^-2 sⁿ)	n₁	CPE₂ (Ω^-1 cm^-2 sⁿ)	n₂	R_c (Ω cm²)	R_L (Ω cm²)	L (H cm²)
Mg alloy	98.3	153	9.54 × 10^-5	0.89	-	-	-	1.73 × 10³	1.4 × 10³
MMT coating	107.5	1.78 × 10⁴	4.8 × 10^-6	0.77	1.35 × 10^-6	0.67	8.2 × 10³	-	-
GS/MMT coating	102.8	1.71 × 10⁴	1.3 × 10^-6	0.78	1.12 × 10^-6	0.81	1.9 × 10³	-	-

Fig. 7. Inhibition zone of specimens against E. coli (a) and S. aureus (b), inhibition zone of two kinds of bacteria under different immersion concentrations (c), cumulative release of GS from GS/MMT coating in DMEM for 120 h (d), the growth curves of E. coli (e, e1) and S. aureus (f, f1).

Table 3. Comparison of GS release duration of other coatings and GS/MMT coating.

Substrate	Carrier	Time (h)	GS release (μg/mL)	Refs.
AZ31	MMT	120	25.67	This study
Ti alloy	PAA	264	24.1	[49]
AZ31	HA	360	19.98	[50]
AZ31	PMTMS/(PAA)	408	20.4	[41]

Fig. 8. Viability of MC3T3-E1 cells incubated for 24 h and 72 h (a) and images of live/dead staining incubated for 24 h (b1-b4) and 72 h (c1-c4) (*p ≤ 0.05).

Fig. 9. Liver, kidney and muscles sections of mice 90 days after samples implantation.

Fig. 10. PDP curves of Zn-MMT coating immersed in DMEM + 10% CS solution (a) and MMT-BSA coating in Hank's solution (b).

Table 4. Comparison of corrosion resistance of other biological coatings and GS/MMT coating on Mg alloys.

Materials	Coating	Solution	E_corr (V vs. SCE)	I_corr (A cm^-2)	R_ct (Ω cm²)	Refs.
AZ31	Zn-MMT	DMEM+10% CS	-1.38	2.86 × 10^-7	2.94 × 10⁴	[38]
AZ31	MMT-BSA	Hank's	-1.40	9.56 × 10^-7	1.57 × 10⁴	[44]
AZ31	(CIP/PAH/SiO₂/PAH)₂₀/Mg	HBSS	-1.38	1.47 × 10^-7	6.91 × 10⁵	[53]
AZ31	DT/Mg(OH)₂	3.5 wt%NaCl	-1.38	1.68 × 10^-6	5.69 × 10⁴	[54]
Mg-Li-Ca	MAO	Hank's	-1.55	4.73 × 10^-7	878.10	[55]
Mg-Li-Ca	MAO/CS	Hank's	-1.93	6.71 × 10^-6	1.56 × 10⁴	[56]
Pure Mg	Ca-P	Hank's	-1.50	6.79 × 10^-6	2390	[57]

Fig. 11. Antibacterial mechanism of GS/MMT coating.

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