2.1. Roughness and wettability

Sharp edges and burrs can cause thrombus formation and neointimal hyperplasia on stents once implanted. Bhuyan et al. [21] identified optimal electropolishing conditions for real stents able to produce a surface with improved mechanical properties (minimized thickness reduction and pitting corrosion) at the desired surface roughness (100 nm). The effect of different surface treatments (polishing, aluminum oxide blasting, and hydroxyapatite(HA) coating) on osteoblast-like cells was recently evaluated by Zhang et al. [3]. This study revealed that rough surfaces were better than polished ones in terms of promoting cell morphology phenotype (flattened shape and complete spreading of cells) and adhesion. Among three treatments used, hydroxyapatite coating had superior results in supporting cell adhesion, but cell viability was reduced in the long-term (7 days) on HA-coated samples (reduced absorbance in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay). This is probably due to the change of the microenvironment for cell proliferation. Plasma spray of HA results in the formation of calcium phosphate and metastable compound with reduced crystallinity. These properties are thought to promote adhesion of osteoblast cells. However, excessive dissolution of calcium phosphate and metastable compound affect the long-term stability of the HA coating. Lower crystallinity may reduce pH of the medium and thus increase cytotoxicity of the coating in the long run. Another reason for a reduced long-term viability of cells on the surface might be due to an increased confluency of the cells. This study was important in demonstrating that different surface modification techniques can produce similar surface morphology, but with a difference in chemical composition, or similar chemical composition, and varied surface morphology [3]. In the case of vascular implants, the extent of surface roughness was found to affect the expression of three genes of human umbilical vein endothelial cells (HUVECs) as indicators of cell injury and activation. McLucas et al. [25] tested three types of surfaces: roughened (by sand blasting; details were not shown), polished (600, 800 and 1200 grit silicon carbide paper followed by polishing cloths (3 μm) and sol-gel alumina suspension (0.06 μm)) and as received SS (surface roughness average root mean square (rms) 95.8 ± 5.7 nm). Surface roughness (rms) was 40.9 ± 1.7 nm for polished samples and 671.8 ± 27.8 nm for roughened samples, as estimated by atomic force microscopy (AFM). Cells were shown to be injured and activated when roughened (upregulation of three genes) and polished (upregulation of two genes) as opposed to as received SS and control sample (no SS). This may be an indication that polishing (at least resulting in this surface smoothness) may not be necessary for stents given their roughness is similar to the as received sample. This study is important in establishing a new way of initial testing of biomaterials for cell response. However, having more samples with varying surface roughness would give more valuable information in choosing a target surface roughness (and hence surface treatment).

A study by Hilbert et al. [22] found no influence of surface roughness on bacterial adhesion on SS, while other studies indicated minimal bacterial adhesion at Ra = 0.16 μm that increased when the surface was smoother or rougher than this value [15]. Although both works studied similar Ra values, the contradictory results could possibly be explained by the different SS types (304 and 316L; latter having lower content of carbon and chromium [26]) used, bacteria tested (the common one being only Pseudomonas aeruginosa), and method for measurement of roughness (optical and numerical assessment). Schlisselberg et al. [27] showed that surface roughness alone was not the only factor of increased or decreased biofilm formation, but it was a combination of the chemical composition and surface treatment used. Recent work by Bohinc et al. [28] demonstrated that bacteria adherence increased with respect to increasing roughness, independent of the technique used to change the surface roughness (3D polishing, brushing, grinding, and electropolishing). An increase in effective surface area led to increased bacterial adhesion. Based on these results, it seems plausible that both surface roughness and surface chemistry have an effect on bacterial adhesion, and it depends on the method used to obtain the specific surface and also on the type of bacteria tested.

In addition to the previously mentioned methods, plasma cleaning and blowing with gas are also used to modify SS surface. For example, radio-frequency plasma irradiation under pure oxygen atmosphere was used to enrich oxides on SS surface before spraying an inorganic polymer, silicone rubber, resulting in a uniform and strong layer. In comparison to electropolished and electropolished plus acid-etched surfaces, plasma treated surface had higher concentrations of surface oxides. Surface roughness was the highest in electropolished samples and the lowest in electropolished plus acid etched ones [23].

Distribution of surface irregularities on the surface is also critical in biomaterials, especially those interacting with cells [29]. Hence, creating a micro- and nanostructured surface is another important method in surface modification of SS and novel techniques are being developed to create surfaces with controlled topographies. For example, laser treatment has been employed in a number of studies to modify the surface of SS. Using femtosecond laser, Oberringer et al. [30] were able to engineer a SS surface that reduced the differentiation of myofibroblasts, being this beneficial in the prevention of restenosis. Micro- and nanofeatures produced by this method had no effect on proliferation of endothelial cells (ECs), but the effect of other interfering factors, such as hydrophilicity and oxygen content, were not fully studied. Through a technique using high repetition rate and low pulse energy femtosecond laser, Kam et al. [31] controlled the wettability of SS making it either hydrophilic or hydrophobic (with contact angle of water varied from 0° to 113°). By varying the scan speed, various micro-conical structures with different morphology, size and density, were created. Femtosecond pulse makes the surface rough and the irregularity formed on the surface results in the local variation of absorbance which leads to the formation of micro-cones. Nucleation and growth process of micro-cones depends on the dynamic balance between redeposition and ablation process. Further studies on the effect of these structures on protein adsorption or cell adhesion on the modified surfaces are needed for an optimized use of this technology. Severe shot peening is another mechanical method that can be used to form a nanoscale layer with increased surface roughness and wettability [24]. This technique was claimed to be a promising low-cost technique but it still requires a specialized equipment which may not be available in most of the surface modification laboratories. This method might also be difficult for real implants due to their complex geometrical structures without adapting rotation of the samples during surface modification.

2.2. Corrosion resistance

Chemical composition and the presence of surface oxide layers on SS play an important role in their corrosion resistance. 316L SS is not susceptible to intergranular corrosion due to its low carbon content. It is protected against corrosion by a spontaneously formed oxide layer; this layer enhances properties of metals including increased corrosion resistance and inertness in biological fluids, passivation, improved wear, and adhesion characteristics [33,34]. Different acid treatment methods (Piranha [13,14,[35], [36], [37]], sulfochromic acid [38,39] and nitric acid [40]) have been widely used to obtain SS surface rich in hydroxyl groups. Although no study comparing these pretreatments was found, one can assume that acid treatment conditions, shown in Table S1, would lead to the formation of hydroxylated surfaces due to their previous effectiveness in various applications.

One of the early studies on the electropolishing of SS for corrosion resistance was first performed in the 1980s, and continues to be used widely in SS modification [41]. Two methods were compared by Latifi et al. [10]: (i) electropolishing (phosphoric and sulfuric acids as electrolyte solutions) and (ii) electropolishing with acid dipping (nitric and hydrofluoric acids). Both treatments resulted in corrosion resistant surfaces. However, more chromium oxides were formed after electropolishing and acid dipping in hydrofluoric acid, while after electropolishing alone hydroxide layers were increased [10]. Direct current anodization in the presence of sulfuric acid with hydrogen peroxide was performed on SS to obtain nanoporous chromium-rich oxide films with the size range of 5–20 nm. The modified surface had improved hydroxyapatite deposition [42].

Ceramic materials, especially hydroxyapatite, have also been widely used for having anti-corrosive properties together with its osteostimulative properties, excellent biocompatibility and similarity (in composition and structure) to bone [52]. Hydroxyapatite substituted with Sr or Sr/Mg has been electrodeposited on SS previously electropolymerized with conducting polymers of polypyrrole and poly(3,4-ethylenedioxythiophene), respectively [52,53]. Both of these bilayers significantly improved anti-corrosive properties of the modified surface as opposed to one layer only. Another ceramic coating, composed of fluorapatite(hydroxyapatite with incorporated fluoride ions) and niobum filler, was plasma sprayed on SS and led to improved corrosion resistance and a claimed improvement in biocompatibility [12]. However, biocompatibility in this case was only indicated by the corrosion resistance test and no cytotoxicity or in vivo studies were performed. Ceramic coatings can also be prepared by sol-gel method which produces homogenous coatings and can be exploited for complex-shaped surfaces. Double layer thin film, consisting of inorganic (ZrTiO4) and organic-inorganic (ZrTiO4-polymethyl methacrylate), were spin-coated on SS and showed excellent corrosion resistance. Inorganic layer provided a good adhesion to the surface, while the upper one reduced physical defects of the bottom layer resulting in a less porous upper layer resistant to corrosion [51]. Sol-gel spin coating was also exploited to introduce bioactive glass/zirconium titranate anti-corrosion coating [54]. Although not intended for an increase of corrosion resistance per se but for formation of a bioactive surface, SS modified with nanostructured forsterite [55] made it more resistant to Cl− ion attack in the SBF. Potentiodynamic polarization corrosion test (Fig. 1a) of the coating showed a reduced corrosion current density for the modified surface implying an improvement of uniform corrosion resistance due to coating. Scanning electron microscopy (SEM) analysis of the two surfaces shows (Fig. 1b, c) deep pits on the untreated samples and a milder and more uniform attack of the forsterite coating serving as a barrier against corrosive medium.

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Fig. 1. Corrosion resistance of uncoated 316L SS and forsterite coated 316L SS samples in simulated body fluid: (a) the results of potentiodynamic polarization test; corrosion morphology of (b) uncoated 316L SS (obvious dip pits implying localized severe corrosion) and (c) forsterite coated 316L SS samples (few microcracks implying a milder and uniform corrosion attack).

Reprinted from [55] with permission from Elsevier.

Silane and composite silane can also be used for improving corrosion resistance. Silane coupling agents (SCAs) (see section Silane-based agents) have reactive terminal functional groups, which enable them to improve adhesive strengthbetween metals and polymers [56]. Hosseinalipour et al. [57] developed a crack-free hybrid coating on SS with improved corrosion resistance. The coating was composed of tetraethylorthosilicate and 3-methacryloxy-propyltrimethoxysilane with their ratio being the most important factor in forming of this highly adhesive film. Although the authors do not show SEM images and FTIR (Fourier transform infrared) spectroscopy results for the untreated sample, the results for the silane-coated samples proved a successful coating. SEM images reveal the presence of a hybrid coating on the surface, while FTIR analysis showed the presence of groups attributed to both organic and inorganic components of the hybrid coating. The coating served as a good barrier (crack-free and adherent) between electrolyte and SS surface and no cytotoxic effect was observed on L929 cells (mouse fibroblasts). Trimethylsilane coating was deposited using both radio-frequency glow discharge (RFGD) and direct current (DC) thus combining the advantages of stability of RFGD with the ability of DC to form metal-adherent coatings. This coating had improved corrosion resistance and could be a promising way to block the release of ions into the bloodstream [47]. One might also consider using composite silanes for improving SS corrosion resistance. Carbon nanotubes applied on silanized SS surface had a better corrosion resistance compared to silane alone [58].

Apart from ceramic materials or SCAs, other materials (including composite ones) have been explored to improve corrosion resistance of SS. Composite coating consisting of a polymer (polyaniline) and graphene oxide [48] was electrodeposited on SS and showed enhanced corrosion resistance (higher corrosion inhibition efficiency and protection efficiency) compared to each of them alone.

Another way to increase the concentration of surface oxides consists in depositing metal oxides (other than SS). This is done, for example, by depositing tantalum oxide on SS implants with physical vapor deposition (PVD) to make it more resistant to corrosion [59]. Park et al. [49] coated titanium nitride on the metal surface deposited by plasma assisted chemical vapor deposition, and this resulted in improved mechanical properties, such as surface hardness, and corrosion resistance, and was shown to be cytocompatible according to standard cytotoxicity test (ISO10993-5) and namely an elution test was used [49]. Titanium ethylene glycol-coated nanoparticles were plasma sprayed on SS. The functionalized surface showed improved properties, such as increased hydrophilicity and corrosion resistance [60]. Sirconium carbonitride magnetron sputtering of SS [45] improved its corrosion resistance and hemacompatibility. Electrochemical tests showed corrosion resistance of the formed nanocrystalline coating in phosphate buffered saline at body temperature.

Comparing different surface modification techniques for improving the corrosion resistance of SS (Table 2), most of the studies used potentiodynamic polarization (PDP) to measure the corrosion resistance of the resulting surfaces (Fig. 1a), while a few used more elaborate techniques (electrochemical impedance spectroscopy or EIS). PDP is shown as a current density (in logarithmic scale) dependent on the applied potential measured in the corroding media [61]. Lower current densities in comparison to the untreated samples are usually the evidence of the improved corrosion resistance of the modified surface as shown by [55,57]. PDP is a good technique to study the effect of an inorganic layer or organic compound on corrosion behavior of the surface (relative susceptibility to localized corrosion) [47]. However, it gives a snapshot of corrosion behavior rather than an average value, and only a single scan can be made due to the destructive nature of the test [62]. EIS, on the other hand, is non-destructive and powerful technique used to test barrier property and corrosion resistance of the coatings on metals. It allows to test corrosion behavior over a longer period of time [47]. Combining the two techniques can provide a better understanding on the anti-corrosion properties of the modified surface. The medium used in these tests depend on the final application of the modified surface. Unmodified 316L SS has different corrosion resistance in various solutions: it is higher in NaCl than in SBF or Hank's solution [63]. Acid treatment offers an easy way of increasing corrosion resistance when other methods requiring special instruments are not available. Some of the chemical methods such as electropolishing and electrodeposition might be more advantageous compared to some physical methods, which require more sophisticated instrumentation as in plasma assisted chemical vapor deposition. Most of these studies used SS substrate but not real implants or stents (except [47]) thus needing an additional study to assess variables as deformation (e.g. expansion for stents) or shape itself (differential coating forming on different shapes). Only a limited number from the above studies tested in vitro cytotoxicity of the functionalized surfaces. Gopi et al. [53] incubated autoclaved surface modified samples (at 121 °C) with human osteosarcoma cells but did not mention how this treatment could affect the cytotoxicity; also uncoated samples were not used or not mentioned; Hosseinalipour et al. [57] used mouse fibroblasts to analyze cytotoxicity of the modified surfaces but used coverslips covered with a sol-gel and an untreated cover slip as a control surface instead of real SS covered with hydrogel. None of these works tested the modified surface in vivo as a substrate implying the need for further studies.

2.3. Functional groups

There are a number of studies exploiting native oxide layers on the SS surface to introduce new functionalities such as biomolecules. However, coupling biomolecules on a metal stent is not straightforward due to the need of incorporating linker molecules [64]. Typically, the metal surface requires an ad-layer of functional groups, such as amines, carboxyls, or quinones [65]. In the case of SS, various functional groups have been incorporated either by silanization or coating with dopamine or via self-assembled monolayers (SAMs).