Characterization and In Vitro Studies of Low Reﬂective Magnetite (Fe 3 O 4 ) Thin Film on Stainless Steel 420A Developed by Chemical Method

: Stainless steel has been the most demanded material for surgical utensil manufacture due to superior mechanical properties, sufﬁcient wear, and corrosion resistance. Surgical grade 420A stainless steel is extensively used for producing sophisticated surgical instruments. Since these instruments are used under bright light conditions prevalent in operation theatres, the reﬂection from the material is signiﬁcant which causes considerable strain to the eye of the surgeon. Surgical instruments with lower reﬂectance will be more efﬁcient under these conditions. A low reﬂective thin -ﬁlm coating has often been suggested to alleviate this inadmissible difﬁculty. This paper reports the development of an optimum parametric low reﬂective magnetite coating on the surface of SS 420A with a black color using chemical hot alkaline conversion coating technique and its bioactivity studies. Coating process parameters such as coating time, bath temperature, and chemical composition of bath are optimized using Taguchi optimization techniques. X-ray photoelectron spectroscopy (XPS) analysis was used to identify the composition of elements and the chemical condition of the developed coating. Surface morphological studies were accomplished with a scanning electron microscope (SEM). When coupled with an energy-dispersive X-ray analysis (EDAX), compositional information can also be collected simultaneously. Invitro cytotoxicity tests, corrosion behavior, the effect of sterilization temperature on adhesion property, and average percentage reﬂectance (R) of the developed coating have also been evaluated. These results suggest adopting the procedure for producing low reﬂective conversion coatings on minimally invasive surgical instruments produced from medical grade 420A stainless steel.


Introduction
Martensitic stainless steel of grade 410, 420A, 420B, and 420C has been widely used for the manufacturing of cutting and non-cutting surgical instruments [1,2]. In addition to biocompatibility, martensitic stainless steel has high strength, hardness, stiffness, rigidity, resilient, and non-corrosive nature [3]. Titanium (Ti) and its alloys are also used in various biomedical applications. Conventional as well as modern surface modification technologies have emerged in the recent decades. The mechanical, chemical, and biological properties are improved by adopting various surface modification methods. Conventional methods like sand blasting, alkali treatment, and plasma treatment have minor enhancements in the surface properties due to the intricate geometry of the work piece. To overcome these kinds of limitations, many modern surface modification methodologies such as laser surface modification, physical vapor deposition (PVD), and plasma spray-PVD show better performance in surface properties [4]. Key-hole or laparoscopic surgeries have been the focus of the emerging medical advancements. A detailed review of different laparoscopic surgical staplers and mechanical components such as gears, links, pivots, and sliders used in performing their required functions is available. This aids in the identification of individual mechanisms used for certain tasks. It will make it easier to grasp the interrelationships between each sub-component that is required or will be used to complete difficult tasks [5]. Minimally invasive surgical instruments are generally made of 420A grade stainless steel. High intensity bright lights are used during minimally invasive or key-hole surgeries to improve the efficacy of surgeon by enhancing proper visualization of the operating field. But the reflection of incident light from the top layers of instruments hinders surgeon's visual field and leads to lack of precision in key-hole surgeries [6]. Low reflective top layers are key to reducing the disturbances caused due to reflection from bright surgical surfaces [7]. Conversion coating techniques are a widely used method for blackening ferrous materials. Hot aqueous alkaline process is a chemical conversion coating process. In such a process, the substrate surface is converted to black color due to chemical reaction between metal and molten salts, salt solution having an alkaline nature or with air at elevated temperatures [8]. The oxide layers developed on the substrate surface does not change the chemical state of the underlying surface.
In recent years various techniques have been adopted for blackening materials for different applications. Solar absorber made of AISI 316L has been deposited with a black coating using electrochemical deposition technique to reduce reflection [9]. Grey cast iron used for manufacturing electric stove was blackened by using a mixture of sodium hydroxide and sodium nitrate. The major component of the black oxide layer was magnetite and this oxide layer protects the hot plates of the electric stove [10]. The chemical conversion coating process has also been utilized to develop black formation on copper and its alloys. These coatings enhance bonding strength between copper and various polymers [11,12]. The anodizing method has been adopted to modify AZ31 Mg alloys to enhance corrosion resistance and biocompatibility, especially used for biomedical devices [13]. Electroless nickel coating technique has also been adopted to produce coating on the substrate surface to improve various properties of the base material [14]. A black film of titanium aluminum nitride and titanium carbon nitride has been produced on the stainless steel by physical vapor deposition which reduced the undesired light reflection, especially for minimally invasive surgical instruments [3,6]. Diamond-like carbon (DLC) coating has been developed on surgical instruments to enhance the intracorporeal instrument surface and durability of devices by using ionized evaporation method [15]. Despite the fact that many methods for coating metals with black oxide have been established, the scope of reducing reflection from surgical grade 420A stainless steel, which is extensively used for surgical instruments, has not been studied at length. This paper reports the development of a black iron oxide (magnetite, Fe 3 O 4 ) coating on the SS 420A surface using a hot aqueous alkaline treatment technique having a black color. The coating parameters such as coating duration, chemical composition of bath, temperature of salt solution, pickling effect, and pH value influence the anti-reflection characteristics of the coating developed [11,[16][17][18][19]. Taguchi optimization technique was used to obtain optimal parameter low-reflective coating on the substrate surface. The XPS analysis, SEM analysis invitro toxicity, corrosion behavior, and sterilization effect of the developed coating were also studied extensively as a part of this research work.

Sample Preparation and Coating Procedure
Surgical grade 420A stainless steel sample with the size of 25 mm × 25 mm × 3 mm has been produced from the ingot. The impurity from the surface was removed by blasting process. Glass beads with 60-80 screen size and 0.250-0.180 mm grain size were employed for an efficient blasting process. Further, it was degreased using an alkaline solution. The sample was pickled by using 20% dilute hydrochloric acid and cleaned in running water. For each experimental trial, laboratory-grade sodium hydroxide (NaOH), sodium nitrate (NaNO 3 ), and sodium dichromate (Na 2 Cr 2 O 7 )-(Nice Chemicals Ltd.,Kerala, India) were dissolved in distilled water to prepare alkaline salt solution. The prepared alkaline mixture is poured into a stainless steel bath. The temperature of the bath can be controlled by using a regulator circuit together with a heating coil capable of heating 200 • C. Further, the samples were immersed inside the alkaline bath for the coating process. The Equations (1)-(3) describe the chemical reactions between stainless steel 420A and as prepared alkaline salt solution which causes the formation of magnetite. Further, the samples were cleaned in de-ionized water and allowed to dry in the air.
The ranges of coating parameters of the hot alkaline conversion treatment process were fixed by conducting numerous trials. The development of a homogeneous coating on the SS 420A surface is reliant on different coating variables, such as coating time, bath temperature, and chemical composition of salt solution. The alkaline salt solution is prepared by varying the weight percentage (wt.%) of sodium dichromate in the composition. Three different wt.% of sodium dichromate were considered. Wt.% of 250, 300, and 350 g/L were fixed after conducting the number of trial experiments. It was seen that no uniform black color coating was formed at less than 250 g/L and the coating changed into golden yellow color when we used more than 350 g/L. The coating time was adjusted between 50 and 60 min and the bath temperature was regulated between115 and 125 • C. Experimental trials showed that lower bath temperature and duration in alkaline solution produced non-uniform black traces on the substrate surface. The duration and bath temperature above 60 min. and 125 • C lead to a change in color, which is undesirable. The wt.% of sodium hydroxide, sodium nitrate, and the pH value of the bath are maintained at constant values of 400 g/L, 320 g/L, and 8 respectively. The images of non-coated and coated stainless steel 420A substrate is as shown in Figure 1. The average percentage reflection (R) is a measurement of the amount of light reflected in the visible region from the substrate surface. A Varian Cary 5000 UV-VIS-NIR spectrophotometer (spectral range of 175-3300 nm, Agilent Technologies, Santa Clara, CA, USA) was used to measure the reflection of incident light (visible range) from the coated surface.

Taguchi Design of Experiments
To determine the optimal parameter combination for minimizing the average percentage reflection (R), Taguchi orthogonal array (OA) was utilized. The coating time (D), bath temperature (T), and weight % of sodium dichromate (C) are selected as control elements and their levels are as shown in Table 1. The signal-to-noise (S/N) ratio was performed using Minitab-17 software. It is mainly categorized according to the required output char-acteristics, that are lower-the-better, higher-the-better, or nominal-the-better [20]. Average percentage reflectance (R) should follow lower-the-better criteria, since lower R value is appreciable to obtain a low-reflective coating on SS 420A substrate surface. Higher-the-better condition is adopted to obtain the optimum combination of control factors from the main effect plot [21]. Since a high S/N ratio value indicates the optimum coating parameters in order to avoid the experimental noises as much as possible. Analysis of variance (ANOVA) and regression analysis were also conducted. Contour plots were used to investigate the relationship of output characteristics and two different control variables by analyzing discrete contours [22]. A conventional L9 OA for a 3 parameters 3 levels are as given in Table 2. XPS was used to identify the compositional and chemical states of the coating produced on the SS 420A substrate.Spectrum were plotted using a XPS, PHI 5000Versa Probe II, (ULVAC-PHI Inc, Hagisono, Japan) having test parameters with beam spot of 200 µm, power of 15 KV monochromatic X-ray stimulation source Al-Kα (hv=1486.6 eV). Survey scans were recorded with an X-ray source power of 50 W and pass energy of 187.85 eV. High-resolution spectra of the major elements were recorded at 46.95 eV pass energy. The morphological studies and elemental composition of the coating were also analyzed using a scanning electron microscope (JEOL Model JSM-6390LV, JEOL, Tokyo, Japan) equipped with an energy dispersive X-ray analysis (EDAX, Oxford XMX N, Wiesbaden, Germany).

Cytotoxity Assessment
Direct extract method according to ISO 10993-5 standard was used for invitro cytotoxicity assessment [23,24]. The L-929 cell line was used for the study. The coated and non-coated substrates were disinfected by autoclaving before conducting the assessment. The cultures were incubated in a minimum essential medium (MEM) supplemented with fetal bovine serum (FBS) for 24-26 h at 37 • C. For extract preparation, the disinfected specimens were submerged in the culture medium for 72 ± 2 h and kept at 50 ± 2 • C. Positive and negative controls were prepared by diluting phenol solution and incubating ultra-high molecular weight polyethylene (UHMWPE) for 72 ± 2 h at 50 ± 2 • C with culture medium. The prepared extract was adjusted to 100, 50, and 25 percentages respectively by mixing the very similar cell culture. To determine the cytotoxic effects, various concentrations of test samples as well as positive and negative controls were seeded on the L-929 cell line and kept at 37 ± 1 • C for 24-26 h. The cellular reaction was investigated by analyzing the incubated cells cultured with positive, negative, and test controls using a microscope.

Corrosion Analysis
Quantitative assessment of corrosion was investigated using potentiodynamic polarization (PDP) tests according to ASTM standard G5-94 [25]. A standard three-electrode with 250 mL capacity cell was used for corrosion analysis. The electrochemical workstation was fitted with saturated calomel electrode (SCE) as the reference electrode, and platinum was employed as auxiliary electrode respectively. Coated, non-coated (control) SS 420A samples were used as working electrode during corrosion test. The electrolyte used for performing corrosion analysis was simulated body fluid (SBF) having a pH of 7.4 to create a natural tissue environment. The methodology adopted to create SBF was Kokubo method [26,27]. The SBF electrolyte was regulated at a temperature of 37 ± 1 • C.
A 1-cm 2 area of the working electrode was kept in contact with the prepared electrolyte. The specimens were allowed to remain in contact with the solution for duration of 3600 s until a constant open circuit potential (OCP) was achieved. The OCP was increased from an initial value of −1000 mV to1000 mV. The sweep rate was set at 0.0005 mV/s. After conducting the potentiodynamic test on both the coated and non-coated SS 420A, Tafel extrapolation was conducted on polarization curves to calculate the corrosion current density (I corr ). The other corrosion parameters were measured to verify the corrosion resistance properties of non-coated and coated samples.
The ASTM B117 standard was followed for performing the salt spray test [28]. The reliability of the test is extremely influenced by the specimen type, evaluation criteria selected, and operating variables. The solution was formed by dissolving 5 ± 1 parts by mass of NaCl in 95 parts of water. The specific gravity and pH value was kept between 1.0268-1.0413 and 6.5-7.2 at 35 • C. During the testing, samples were exposed to a temperature of 35 ± 2 • C in the salt spray chamber for 24 h.

Repeated Sterilization and Morphology Studies
A repeated sterilization test was performed as per ISO 17665-1 standard for assessing the effect of sterilization temperature on the adhesion property of the black oxide coating [29]. In the conventional method, the temperature of the steam varies from 121-134 • C for a period of 15−21 min to ensure adequate sterilization. In rapid cycle, the samples were held at 134 • C for 3 min in an autoclave. To complete the sterilization process, samples were later allowed to cool. The entire sterilization procedure requires 45 min to one hour. Moreover, surface morphological studies of optimal parameter combination black oxide (Fe 3 O 4 )-coated SS 420A sample were accomplished with a scanning electron microscope (JEOL Model JSM-6390LV, JEOL, Tokyo, Japan). Table 2 shows the observed responses for different combinations of trials as well as the computed S/N ratios corresponding to avg. percentage reflectance (R). The obtained avg. S/N ratios for each level of input parameters are tabulated in Table 3. The ranks are assigned based on the statistical delta values. The parameter corresponding to the highest rank has a major influence on the coating process. From Figure 2, the highest mean S/N ratio for avg. percentage reflectance correspond to 60 min. coating time, 125 • C bath temperature, and 350 g/L wt.% of sodium dichromate. Therefore, the predicted optimal parameter combination for getting low avg. reflectance using Taguchi method were found as D = 60 min., T = 125 • C, and C = 350 g/L and it is represented as D3-T3-C3.   Table 4 shows the conformation test results for verifying Taguchi's estimated avg. reflectance parameter combination. When comparing S/N ratios of estimated and optimum process parameters, it was found that they were almost identical. On comparing the S/N ratio value of the preliminary parameter combination, an improvement of 2.217 dB was noticed for the Taguchi estimated parameter combination. Additionally, the %R value decreased by 29.21% with respect to the preliminary parameter combination. Hence, the estimated ideal combination was adopted to produce an excellent low-reflective coating on stainless steel 420A with the least R value. Analysis of variance (ANOVA) results were obtained corresponding to the output characteristics and summarized in Table 5. The p values corresponding to input parameters clearly depict the considerable influence of parameters during magnetite formation on SS 420A. The contributions of each parameter in terms of percentage were calculated and wt.% of sodium dichromate has remarkable influence in the formation of magnetite. Equation (4) shows the mathematical model was formulated between output characteristics and input variables using regression analysis. The corresponding R-sq value is 85.41%, which denotes the effectiveness of the developed linear regression equation in predicting R within the given input range. The confirmation tests were also conducted to verify the validity of the developed mathematical model. Taguchi predicated optimum combination was considered as input values and the obtained results are summarized in Table 6. From the table, it is observed that, the R value predicted by the formulated mathematical model is nearer to the experimental results, which is desirable.

Optimal Parameter Combination Predicted Value Experimental Value
Reflectance (R) D3-T3-C3 (60 min,125 • C, 350 g/L) 7.296% 7.546% Figure 3A-C shows the contour plots that illustrate the desirable average percentage reflectance and corresponding control factors. It is seen that higher bath temperature, longer coating duration, and larger wt.% of sodium dichromate lead to generate coating with low reflectance.

XPS Analysis of Coating
The surface survey according to XPS indicated the presence of C 1s (85.3%), O 1s (14.1%), Fe 2p3 (0.4%), and Cr 2p3 (0.17%) as in Figure 4. Figure 5A,B represents the high resolution spectrum of C 1s and O 1s. The Fe 2p3/2 spectrum was fitted with major and minor peaks of Fe 3+ and Fe 2+ corresponding to the binding energy of 712.4 eV and 709.9 eV as shown in Figure 5C and it is well in accordance with the values of magnetite found. The details about FeO and γ-Fe 2 O 3 can be provided easily by separating the corresponding satellite structure [30]. Therefore, the developed black oxide coating is magnetite (Fe 3 O 4 ) [31,32]. The high-resolution spectrum of Cr 2p3/2 displays metallic species of Cr and formation of Cr 2 O 3 corresponding to 573.6 eV and 576.1 eV respectively as in Figure 5D. The multiplet splitting at Cr 2p3/2 peak is only because of the presence of chromic oxide (Cr 3+ ) since only trivalent chromium out of the possible oxides of Cr shows pronounced multiplet splitting [33]. To ensure the presence of chromium, the elemental composition of the coating is also analyzed by SEM-EDAX. From Figure 6, the presence of chromium is ensured in the developed coating.

In Vitro Cytotoxicity Study
Cellular response of L-929 cell line cultured with extracts of different samples was graded and evaluated. The cytotoxicity was evaluated by comparing the cell response grade of test samples with negative and positive controls. Negative and positive controls gave grades 0 and 4 as expected. The three diluted extracts, 100%, 50%, and 25% of noncoated (control) and coated SS 420A samples have given a grade of 2, which is acceptable by ISO 10993-5 [23,24]. Figure 7A-F are the microscopic images of the L-929 cell line exposed for a duration of 24 h with 100%, 50% and 25% proportions of extracts of non-coated and coated SS 420A. From the figure, it has been shown that up to 50% of cells are round, devoid of cytoplasmic fine particles, showing no cellular lysis, with vacant spaces between cells. These findings suggest that the developed low reflective chemically formed magnetite on stainless steel 420A is non-toxic.

Potentiodynamic Polarization and Salt Spray Studies
The corrosion behavior of control and coated SS 420A was evaluated using an electrochemical workstation together with simulated body fluid (SBF) environment and repeated at least three times. The polarization curves for different samples are given in Figure 8. The measured electrochemical parameters from PDP curves are tabulated in Table 7. From the table, it was noticed that corrosion current density (I corr ) having a strict linear relationship with corrosion rate (CR). Thus, the lower the I corr value the greater the resistance to corrosion [34]. Hence the magnetite over-layered SS 420A sample has slight improvement in resistance to accelerated corrosion. Moreover, the higher E corr value of magnetite layered SS 420A, indicates chemical inertness and the lowest corrosion tendency. The measured R p value also indicates that the coated substrate shows slight improvement against applied accelerated corrosion compared to the non-coated SS 420A. The corrosion resistance of coated samples was also verified by the salt spray test. Samples were visually inspected and no rust was formed after 24 h test and it shows the developed coating can withstand corrosion.

Effect of Repeated Sterilization on Coating
The SEM images (20 kV and 1000× magnification) of coated samples before and after conducting 100 cycles of repeated sterilization as per ISO standards are shown in Figure 9. No damages like discoloration peel off were observed after the repeated autoclaving process. From Figure 9B, minor stress relief cracks were observed. This is due to sudden cooling of the coating and the substrate from treatment temperature to room temperature [35]. If the developed coating detaches due to the sterilization temperature from the instrument during the surgery, it can lead to serious damages to surrounding tissues. The average percentage reflectance (R) value was measured to analyze the effect of repeated sterilization on coatings. The average percentage reflectance graph of coated SS420A before and after steam sterilization is given in Figure 10A,B. The measured average percentage reflectance value of coated substrate before steam sterilization is 7.546%. From Figure 10B, the coating exhibits excellent antireflective property with a minimum R value in the visible region of 8.32% after undergoing100 cycles of repeated sterilization on uniform black magnetite forming SS 420A. There is smaller variation in the R value of coated sample before and after repeated sterilization procedure. Even though the reflection of light comparatively is much lesser than not coated SS 420A. This indicates, repeated sterilization does not adversely affect the antireflective property of the developed coating.

Conclusions
A low reflective black iron oxide (magnetite) coating was produced on stainless steel 420A using chemical hot aqueous alkaline conversion coating technique. The coating parameter values of alkaline conversion coating process were optimized using the Taguchi optimization technique. The optimum coating parameter combination for minimum average percentage reflection (R) value is obtained as D3-T3-C3 (D = 60 min., T = 125 • C and C = 350 g/L). The R value of uniform black coated SS 420A is reduced to 7.546% from a value of 56.14% of non-coated samples. It is seen that the chemical composition of bath and bath temperature has greatest impact in magnetite formation on SS 420A. This coating reduces the undesired difficulties of surgeons due to scattering and reflection of incident light in the visible spectrum from the exteriors of surgical equipment manufactured by SS 420A. The developed coating was characterized using XPS analysis and the formation of magnetite (Fe 3 O 4 ) was identified. An invitro cytotoxicity test was performed using the L-929 cell line and confirmed the non-toxic behavior of the developed coating. Corrosion studies showed that magnetite layered SS 420A has better corrosion resistance properties than the non-coated substrate. A repeated sterilization test was conducted to measure the ability of the developed coating to withstand the sterilization process. The average percentage reflectance (R) value slightly increased to 8.32% after 100 cycles of autoclaving, which is desirable. These results suggest that the developed coating may be used for producing a biocompatible low-reflective black coating for surgical equipment composed of medical grade 420A stainless steel.
Author Contributions: R.A.R., contributed to the experimental and analytical work of the research; K.K.S., contributed to the validation and evaluation of the findings reported in the paper; R.A., contributed to evaluation of the findings. All authors have read and agreed to the published version of manuscript.

Funding:
We did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.