Investigation of in vitro behavior of composite coating hydroxyapatite-nano silver on 316L stainless steel substrate by electrophoretic technic for biomedical tools

Nabaa Sattar Radhi; Awham Jumah Salman; Zainab Al-Khafaji

doi:10.1515/eng-2024-0017

Article Open Access

Investigation of in vitro behavior of composite coating hydroxyapatite-nano silver on 316L stainless steel substrate by electrophoretic technic for biomedical tools

Nabaa Sattar Radhi , Awham Jumah Salman and Zainab Al-Khafaji

Published/Copyright: September 26, 2024

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From the journal Open Engineering Volume 14 Issue 1

Abstract

Surface changes of biomaterials are essential for aligning with the biological system’s dynamics and enhancing the effectiveness of bioimplants. Customized surface alterations based on the material’s bonding ability, biocompatibility, and interactions with host cells may have a substantial impact. This investigation uses hydroxyapatite (HAp) with 0, 10, 20, 30, and 40 wt% silver as a thin coating layer on stainless steel (SST) 316 L by electrophoretic deposition preparation at 30 V and 30 min coating duration. The coating’s crystallinity, morphology, and microstructure have been investigated using structural characterization methods such as X-ray diffraction, Scanning electron microscopy, and energy dispersive spectroscopy. The resistance to corrosion of uncoated and coated SST substrates has been assessed using potentiodynamic polarization experiments. The results show that the HAp-nanosilver coating layer increases the SST’s resistance to corrosion in Ringer solution. The HAp 10% silver-coated SST displays a reduction in corrosion current density. These further demonstrate the potential for using HAp and silver-coated stainless steel as a surgical instrument to increase biocompatibility and resistance to corrosion.

Keywords: biomaterial; surface modification; bioimplant; SST-316L; electrophoretic deposition; biocompatibility

1 Introduction

Metals and alloys are utilized widely in many applications, such as dental amalgams, traction devices, braces, splints, joint replacements, and fracture fixation devices [1,2,3]. While they usually display excellent strength and hardness, they are vulnerable to electrochemical and chemical deterioration. Implant materials can corrode or wear down, producing small particles that might trigger biological reactions locally and across the body [3,4,5]. 316L stainless steel (SST-316L) is often utilized to make implants in orthopedic applications. It has good inherent mechanical characteristics, biocompatibility, adequate resistance to corrosion, high tensile strength, and appropriate density for supporting loads [6,7,8].

However, many alloys such as SST-316L [9,10,11,12,13,14,15,16,17], Ti-alloys, and cobalt-chromium (CoCr) alloys seem to be the bio-metallic inert metals that are most often utilized for bone remodeling, angioplasty, and fracture repair [18], which is a significant factor since it has better mechanical qualities and long-term stability under highly active in vivo environments [18]. While it is believed that these materials have a low corrosion rate, it is essential to bear in mind that material deterioration may be caused by wear, friction, and a very hostile microenvironment [11], which might result in the release of metallic ions that are undesirable. Some potential outcomes include inflammatory reactions, local tissue damage, progressive osteolysis of adjacent tissues, systemic injury, and metal hypersensitivity. Osteolysis can affect the fixation of the implant and its loading and force transmission in the long run, which might cause implant failure, corrective surgery, or issues following the treatment [19]. A common bioceramic utilized as a biomaterial for bone replacement is hydroxyapatite (HA), which is the calcium phosphate with the chemical formula Ca 10 ( PO 4 ) 6 ( OH ) 2 . HA has many applications, such as bone fillers, scaffolds for bone tissue creation, implant coatings, soft tissue healing, and drug delivery systems [20]. It is also intriguing because of its biocompatibility, osteoconductivity, non-inflammatory qualities, and mechanical properties. About 60–70% of the inorganic material in bone tissue is HAp [21]. Because of its bio-mimicking qualities, this bioceramic is compatible with natural bone. For the manufacture of HA, several synthetic approaches have been widely described [22]. Although several synthetic techniques have been devised to produce HA with specific properties, it is still difficult since harmful intermediates might arise. The sol–gel technique, hydrothermal, co-precipitation, and mechano-chemical approaches may synthesize HA [23].

Electrophoretic deposition (EPD) is a coating technique widely utilized in many applications, including intricate microstructures and functionally graded nanostructures. The benefits derived from the EPD were significant, leading to widespread adoption of the approach due to its simplicity, ease of use, and scalability. Significant benefits include cost-effectiveness, adaptable microstructure, precise control over intricate forms, and even dispersion of particles [24]. The technique involves the movement of charged particles toward an electrode in an electrical field to accomplish deposition by particle agglomeration. The EPD process often requires Adequate heat treatment to improve mechanical characteristics. The factors, including voltage and duration, are crucial in the EPD process and are typically maintained consistently.

Nanostructures such as nanotubes and nanowires may be created utilizing EPD processes. Boccaccini et al. [25] examined the production of carbon nanotubes using silicon dioxide, titanium dioxide, manganese dioxide, iron oxide, and other composite materials. This technology may also be utilized to manipulate the structures or layers of carbon nanotubes in order to get thin coatings. Kwok et al. [26] previously analyzed HAp produced using EPD on Ti-6Al-4V for its uniformity throughout the deposition process. The optimum coating thickness was 10μm, and they were observed to be crack-free, with strong adhesion strength ranging from 6.8 to 10.7 MPa. Boron-doped HAp has been utilized to coat implantation material to enhance corrosion resistance, interaction at the implant-tissue interface, and cell adhesion. The improved characteristics of the coatings may be attributed to the morphology seen following EPD [27].

Drevet et al. [28] suggested creating nano-HAp on Ti-6Al-4V utilizing EPD. They underwent different tests involving nanoindentation, scratch, and mechanical tests to demonstrate their enhanced qualities. Chellappa and Vijayalakshmi [29] utilized the EPD technique to study several composite coatings involving SiO 2 / ZrO 2 , SiO 2 / TiO 2 , and ZrO 2 / TiO 2 on Ti-6Al4V. They varied duration lengths and voltage to enhance the mechanical coatings’ strength. All EPD-derived composite coatings exhibited improved corrosion resistance and increased scratch resistance. Furthermore, EPD is integrated with another coating method to enhance its effectiveness. Nawaz et al. [30] integrated electrostatic powder deposition (EPD) with radiofrequency sputtering methods to produce high anti-bacterial effectiveness and biocompatibility coatings.

Araghi and Hadianfard [31] assessed the adhesion strength of HAp/TiO₂ by applying a layer of HAp and TiO₂ onto Ti alloy implants using the EPD method. The sintering temperature, among other parameters, has a significant impact and requires us to choose the most suitable temperature for the process. Alternatively, a high sintering temperature results in high-density coatings but may lead to HAp breakdown, whereas a low temperature may result in poorly bound HAp. During EPD coating, many fractures were observed under high voltage conditions.

In the last published research, most of the researchers focused on using either HAp or nanosilver (nAg) as a coating for steel substrate; the novelty of the current study is affected as composite coating consisting of both HAp and nAg is used. In addition to that, using composite coating consisting of ceramic-metal materials is considered a new trend in bio-composite materials due to improvement in both selected materials’ properties, such as the use of nAg reducing anti-bacterial infection produced when implementing this in the live body, while at the same time HAP leads to accelerating the osteointegration process of live bone. Therefore, applying composite coating on SST-316L substrate improved corrosion resistance and enhanced the biocompatibility of the selected base materials.

The current study aims to improve SST-316L substrate’s corrosion resistance and in vitro behavior by applying a composite coating consisting of HAP- nAg on a steel substrate using an electrophoretic composition coating technique. The electrophoretic technique is considered a low-cost process, as well as this process could allow a comprehensive materials range to be deposited on the steel substrate, such as insulator materials (HAP could be easily deposited).

2 Experimental part

Nano HAP (Shanghai Hualan Chemical Technology Co., Ltd; China; mean particle size 40 nm) and nAg particles (Hongwu International Group Ltd; China; mean particle size 20 nm) were utilized to produce coatings on stainless steel plates as shown in Figure 1, and the chemical composition of stainless steel 316L substrate are listed in Table 1. Ethanol (Scharlab S.L., Spain) was applied as the suspension medium. Acetic acid (Hopkin and Williams, Britain) has been added to the suspensions to enhance particle charging as a dispersion material.

Figure 1

Working methodology flowchart.

Table 1

Chemical composition of SST Plate

Elements	C	Si	Mn	Cr	Ni	Mo	Al	Co	Fe
Percent (%)	0.03	0.34	0.54	18.47	7.74	0.22	0.17	0.03	Rem.

Suspensions for electrophoretic experiments have been produced by ultrasonic agitation of HA powders (synthesized in our laboratory) in ethanol. A minimal amount of 1 M HCl and HNO 3 was added to the ethanol alcohol to conduct stable suspensions and gain the desired electrophoretic mobility level. The suspensions were sealed in glass bottles and aged for different periods ranging from 1 to 30 days.

The TP120-5S power supply was utilized as a DC source in the EPD process. The electrophoretic cell (shown in Figure 2) consisted of a 50 ml cylindrical glass beaker, and stainless-steel plates measuring 2.5 × 1 × 0.1 cm were utilized as electrodes. The separation between the two electrodes was 1.5 cm. one weight percent solid was added to the ethanol solution. Five groups of HAPs coated on stainless steel plates were prepared with 0, 10, 20, 30, and 40 wt% silver addition. During the 30 min procedure, a voltage of 30 V is supplied, resulting in the deposition of particles on the cathode electrode. The coated specimens were sintered at 450°C for 1 h and then heated to 800°C for 3 h in a vacuum furnace using Argon gas. X-ray diffraction (XRD) and potentiodynamic polarization experiments were conducted on both uncoated and HAP-nAg coated specimens in simulated bodily fluid (Ringer’s solution) at a pH of 7.4 and a temperature of 37 ± 1°C. The breakdown potential (E _b) and corrosion potential (E _corr) were determined from the polarization curves.

Figure 2

Electrophoretic deposition cell.

2.1 Tests

2.1.1 XRD

XRD techniques define the presence of phases in SST-316L alloy before coating and after EPD with HAP 0, 10, 20, 30, and 40 wt% silver. The XRD device model “SHIMADZU Lab X XRD-6000,” Japan, with a nickel filter and generator copper Kα radiation (λ = 1.5406) has been used. The scanning speed of the diffract meter was adjusted to 60 per min with a diffraction angle range 2θ° of 0–80°. The resulting peaks are then compared to standard peaks for each phase appearing in the 316L stainless steel alloy and phases appearing due to surface modifications.

2.1.2 Scanning electron microscopy/energy dispersive spectroscopy (SEM/EDS)

SEM offers topographical and elemental data at magnifications ranging from 10× to 300,000× with an almost infinite depth of focus. Field emission SEM provides higher-quality pictures with less distortion and improved spatial resolution of up to 1.5 nm, 3–6 times superior to conventional SEM. This test utilized specimens of stainless steel coated with HAP with varying percentages of silver (0, 10, 20, 30, and 40 wt%).

2.1.3 Atomic force microscopy (AFM)

The deposition layer’s surface image feature and nano roughness are observed using the AFM (SPM AA3000, Angstrom Advanced Inc., USA). AFM is a beneficial tool for detecting the high-resolution topography of coated films. This technique is very effective for characterizing the material on the nanoscale. Stainless-steel coating samples with HAP with 0, 10, 20, 30, and 40 wt% silver were tested.

2.1.4 Electrochemical test

Studying the electrochemical corrosion tests for the specimens involved conducting polarization tests (potentiodynamic) before and after surface modifications. These tests are conducted in “Ringer’s solution” with the chemical composition provided in Table 2. The test uses potentiostats/Galvanostats from MLab Bank Elektronik in Germany. There are three electrodes in the corrosion cells:

The electrode can work with “316L specimen or coated specimens.”
The counter electrode (platinum rod).
The saturated calomel electrode (SCE). The surface area subjected was 1.767 cm² for the electrode utilized for every specimen.

Table 2

Tyrode’s solution chemical ingredients

Components	Amount (g/L)
NaCl	8
KCl	0.2
CaCl₂	0.2
MgCl₂	0.1
NaH₂PO₄	0.05
NaHCO₃	1
Glucose	1
pH	7.4

The corrosion rate is calculated using the equation below [32]:

(1) CR ( mpy ) = 0.13 i corr ( E .W . ) A ⋅ ρ ,

where, E.W. is the equivalent weights (g/eq), A is the area (cm²), ρ = density (g/cm³), 0.13 = metric and duration conversion parameter, and i _corr is the current density (μA/cm²).

Potentiodynamic polarization, the corrosion behavior of 316L stainless steel, was studied here both before and after the surface changes (samples of stainless-steel coating with HAP of 0, 10, 20, 30, and 40 wt% silver) were made by using an electrochemical cell (three-electrode Polarization experiments were carried out using a potentiate of the kind “Winking M Lab 200.” The potentiodynamic polarization curves were drawn. Tafel plots were utilized to quantify the potential of corrosion (E _corr) and the current density of corrosion (I _corr) using both anodic and cathodic polarization branches. The experiments were performed at 37 ± 1°C in “Ringer’s solution.”

2.1.5 Anti-bacterial test

Microbial colonization and bacterial assault may occur on the surface of biomaterial. An investigation was conducted to assess the anti-bacterial properties of the surface layer of modified 316L to determine its anti-bacterial efficacy. The inhibition zone approach assesses anti-bacterial activity. An anti-bacterial kinetic test is conducted using the bacterial strain “E. coli” (Escherichia coli, American Kind Culture Collection 5922), a gram-negative bacterium, at the College of Girls Science, University of Babylon. E. coli bacteria were cultured on nutrient agar and incubated for 24 h. The bacteria were then distributed on a Petri dish and kept at 37°C for another 24 h. Subsequently, specimens were put in three distinct places on the Petri dish, and the inhibitory zones were detected.

3 Results and discussion

3.1 XRD analyses

The level of crystallinity affects the dissolution and biological response of HAP coatings. Prior research indicates that a covering with a highly crystalline structure results in reduced dissolving [33]. Figure 3 displays the XRD pattern for a 316L stainless steel specimen. The displayed patterns are compared to the standard patterns for the specified alloy. The specimens were coated electrophoretically at 30 V for 30 min with HAP containing varying percentages of silver (0, 10, 20, 30, and 40%). The specimens were then sintered in a vacuum furnace at 450°C for 1 h, followed by an increase to 800°C for 3 h in a vacuum furnace with Argon gas, as depicted in Figure 3(b)–(f). These patterns exhibit similarities in terms of the coating crystallographic structure. The XRD patterns include diffraction peaks with minimum line broadening and high intensities, reflecting the highly crystalline and stoichiometric HAp. No further Ca₃(PO₄)₂ phases were detected. The prominent peaks in the XRD patterns are attributed to reflections from certain crystallographic planes of HAp, as identified by JCPDS file #09-0432: (002), (211), (112), (300), (202), (222), and (213). A strong peak at around 26° indicates the preferred crystal orientation of HAp along the [002] direction, as described in the prior studies on HAp coatings. Other crystalline phases, such as tricalcium phosphate, are not found [34].

Figure 3

XRD pattern for utilized samples: (a) SST; (b) SST coated by HAP; (c) SST coated by HAP-10% nAg; (d) SST coated by HAP-20% nAg; (e) SST coated by HAP-30% nAg; and (f) SST coated by HAP-40% nAg.

3.2 SEM results

Figure 4 shows the surface morphologies of the coated specimens produced using EPD. Figure 4a–e displays a homogeneous distribution of particles, suggesting that the coatings are compact and microporous. No cracks in the coating indicate that there was little shrinking of the coating. This finding validates the convenience of using the electrophoretic processing method to produce dense specimens post-sintering. The coating comprises a network of evenly distributed big granules, each made of smaller equiaxed nanometric crystals as seen using atomic force microscopy, showing uniformity and absence of fissures. The findings confirm the prior XRD data, showing solely the presence of stoichiometric HAP phase and silver. The EDS measurements of the HAP align well with the food and drug administration (FDA) recommendations for a Ca/P ratio of 1.67–1.76 and a max of 50 ppm heavy metals in the coating – measurements of open circuit potential (OCP) over duration. Figure 4a displays the OCP – duration plots for both uncoated and HAP-coated SST-316L samples, which were prepared using EPD with 30 V for 30 min. The uncoated specimen’s OCP decreases in the active direction until it almost reaches a stable state potential.

Figure 4

SEM and EDS for utilized samples: (a) SST coated by HAP-10% nAg; (b) SST coated by HAP-20% nAg; (c) SST coated by HAP-30% nAg; and (d) SST coated by HAP-40% nAg.

On the other hand, the OCP of all HAP-coated specimens gradually shifts toward a more noble potential over duration, reaching a stable condition rather quickly, suggesting that the coatings remained undamaged. The specimen coated for 30 min reached a steady state in a much shorter duration [26], which may result from a rise in the weight of the covering and its porous characteristics. The exemplary conduct of the specimen coated at 30 V for 30 min might be ascribed to the consistent behavior of the coated surface.

3.3 AFM

Figure 5 shows the surface roughness analysis of the 316 stainless steels coated by HAP only and HAP with 10, 20, 30, and 40 wt% nAg. The surface morphologies of silver-substituted films indicate high-quality thick films with a relatively large surface area, which is attributed to the more significant amount of silver.

Figure 5

AFM for utilized samples: (a) SST coated by HAP; (b) SST coated by HAP-10% nAg; (c) SST coated by HAP-20% nAg; (d) SST coated by HAP-30% nAg; and (e) SST coated by HAP-40% nAg.

3.4 Linear polarization results

To sum up, achieving stoichiometric HAP coatings is possible through EPD processes. When electrochemical deposition is utilized to create HAP coatings on 316L stainless steel, the choice of applied potential and deposition duration plays a crucial role in the development of the coatings. The coating weight and thickness rose as the applied potential and deposition period increased [35]. Increasing the weight of the coating on the HAP may not be ideal, as it can lead to more noticeable delamination from the substrate. Lab tests indicate a change in the OCP and pitting potential magnitudes favoring the HAP-coated specimens over the uncoated SST-316L. Redpenning et al. have documented a process for electrodepositing HAP that consists of two main steps: the initial Ca₃(PO₄)₂ apatite mineral formation on the surface of the metal, followed by the continuous growth of the mineral layer through precipitation with constant composition. Throughout this procedure, water electrolysis causes a localized rise in pH at the cathode surface since the creation of OH⁻ and H₂. When present in a stable Ca₃(PO₄)₂ solution supersaturated with apatite, the pH increases, resulting in higher (PO₄)³⁻ concentration, causing further Ca₃(PO₄)₂ salt supersaturation and resulting in uniform precipitation. It is unlikely that the Ca₃(PO₄)₂ is nucleated on the metallic surface. On the other hand, precipitation could occur in the solution, and the mineral particles move to the metal surface through gravity and electrophoretic attraction. This process will lead to a relatively consistent mean spherules size over duration, as demonstrated in this study.

Nevertheless, as the bulk solution remained clear throughout the deposition, it can be inferred that the Ca₃(PO₄)₂ precipitation did not occur far from the metal’s surface. To improve the quality of HAP coatings and optimize their formation, it is crucial to better understand the electrochemical reactions occurring at different pH levels and varying magnitudes. The findings suggest that coatings obtained by EPD could be a promising option for enhancing implant device corrosion resistance and biocompatibility.

The polarization diagrams of all samples and the E _corr (mV) and I _corr (κA) are shown in Figure 6. Table 3 presents the corrosion rates (mpy). The findings suggest that the Hap-nAg coating layer exhibits a consistent and reliable performance. This coating not only enhances corrosion resistance but also alters the surface properties of the materials without impacting the bulk characteristics [9]. In addition, the application of coatings may enhance the ability of the coated specimens to resist corrosion [36,37,38].

Figure 6

Linear polarization findings of samples in Ringer solution: (a) Uncoated SST; (b) SST coated by HAP; (c) SST coated by HAP-10% nAg; (d) SST coated by HAP-20% nAg; (e) SST coated by HAP-30% nAg; and (f) SST coated by HAP-40% nAg.

Table 3

Corrosion results

Sample composite	I _{corr (nm)}	E _{corr (mV)}	Corrosion rate (mpy)	Improvement (%)
SST	1,430	−241.4	0.3357	—
SST coated by HAP	437.65	−56.7	0.1025	69.4
SST coated by HAP-10nAg	66.99	−271.6	0.0154	95.4
SST coated by HAP-20nAg	72.29	−477.8	0.0169	94.9
SST coated by HAP-30nAg	130.06	−76.2	0.0305	90.9
SST coated by HAP-40nAg	263.56	−244.3	0.0617	81.6

The addition of nano-sized silver particles leads to a reduction in porosity. The assertion is substantiated by the observation that the presence of nano-sized particles leads to a substantial reduction in both porosity and corrosion current density. This is because these little particles have the potential to occupy the pores. The addition of silver particles to the HAP solution resulted in a significant decrease in the corrosion current density, reaching its minimum value as seen in Table 3.

3.5 Anti-bacterial results

The impact of anti-bacterial properties on SST-316L alloy coated with HAP specimens containing varying weight percentages against E. coli culture was examined, as illustrated in Table 4 and Figure 7. When a precise region forms around the disc, it is known as the bacterial inhibition zone. After 24 h of incubation, the coating layer showed a potent anti-bacterial impact. Thus, the inclusion of HAP and nAg successfully inhibited bacterial attachment on the surface, consequently hindering bacterial proliferation and enhancing anti-bacterial properties. Coated specimens have demonstrated a significant antimicrobial activity attributed to the silver coating’s ability to eliminate bacterial strains [39] effectively.

Table 4

Anti-bacterial results

Sample composite	Results
SST	−
SST coated by HAP	−
SST coated by HAP-10nAg	−
SST coated by HAP-20nAg	−
SST coated by HAP-30nAg	+
SST coated by HAP-40nAg	−

Figure 7

Anti-bacterial results.

4 Conclusion

SST-316L was effectively coated with bioactive HAP utilizing EPD.
XRD investigation verified the HAP coatings’ phase purity and stoichiometric properties, whereas SEM revealed a consistent distribution of deposits.
The HAP and HAP-nAg coating morphology is uniform and without cracks when the voltage is set at 30 V, and the coating period is 30 min.
Electrochemical analysis in Ringer’s solution showed that samples coated with HAP had greater corrosion resistance than the uncoated SST-316L, shown by their nobler OCP magnitudes and greater breakdown and protection potentials. The SST-316L without a coating was shown to be very prone to corrosion.
The results of the antibacterial analysis have confirmed that the HAP coating by Ag reveals good antibacterial activity against gram-negative E. coli. This antibacterial activity increases when the amount of silver increases, making these coatings safe and able to be utilized for surgical instrument applications.

Funding information: Authors state no funding involved.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and consented to its submission to the journal, reviewed all the results and approved the final version of the manuscript. NSR prepared the discussion on the obtained results from experimental work, AJS conducted the experimental study, ZA-K prepared the manuscript-writing the introduction and experimental work.
Conflict of interest: Authors state no conflict of interest.
Data availability statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Received: 2024-02-29

Revised: 2024-03-21

Accepted: 2024-03-27

Published Online: 2024-09-26

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/eng-2024-0017

Keywords for this article

biomaterial; surface modification; bioimplant; SST-316L; electrophoretic deposition; biocompatibility

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