VOL. 17 March ISSUE YEAR 2016
in Vol. 17 - March Issue - Year 2016
Nanofeatures Induced by Severe Shot Peening Impede Bacterial Adhesion
Table 1: Shot peening treatment parameters
Figure 1: Cross section optical micrographs showing the microstructure of (a) NP, (b) CSP, and (c) SSP samples
Table 2: Surface roughness of the samples measured by interferometry (micron-scale) and AFM (nano-scale)
Figure 2: Representative AFM topographical images of (a) CSP and (b) SSP samples
Figure 3: (a) Water contact angle measured on the surfaces of samples (DI water droplets of 2 uL). In-depth distribution of (b) residual stresses, (c) FWHM and (d) microhardness; Data = mean +/- St. Dev; N = 3; ***p<0.005
Figure 4: Adhesion of bacteria on the samples' surfaces after 1 and 2 days of incubation (a) Staphylococcus aureus (b) Staphylococcus epidermidis (data represents mean +/- St. Dev, N=3. *p<0.01 compared to NP at the same time point; ^p<0.01 compared to CSP at the same time point); Fluorescent micrographs of Staphylococcus epidermidis cultured for 24-hr incubation on (c) NP, (d) CSP, and (e) SSP samples (green(live) and red (dead) bacteria cells)
The advancement of knowledge about the effect of mechanical cues on cells and bacteria activities has brought great attention to micro-and nano-engineering approaches that are able to modulate the substrate mechanical and physical characteristics to consequently control the response of the biological microenvironment. Among these techniques, severe plastic deformation (SPD) is becoming a vigorously growing area of research with promising technical and biological benefits. SPD methods can be generally categorized as metal-forming techniques that result in grain refinement through high strain rates at relatively low temperatures . Some SPD techniques can control surface topography and grain structure, which are both known to affect bacterial adhesion and cellular functions at the cell-substrate interface . Bacterial infection is one of the most challenging post-surgical complications, which affects biomedical implants and commonly requires long periods of antibiotic administration. Communities of bacteria, known as biofilms, can attach firmly to the surface of implant material and show extreme antibiotic resistance. Therefore, it is exceedingly important to reduce the risk of biofilm formation and thus decrease infection rate and the consequent costly complications including revision surgeries.
In their group, they developed an efficient SPD-based surface treatment, named as severe shot peening (SSP). SSP is performed using the standard shot peening apparatus, but applies a particular set of peening parameters to enhance the impact kinetic energy, introduce dislocations and consequently transform the coarse grained surface structure into a nanostructured one [3, 4]. Contrary to many other SPD techniques, SSP does not involve size and geometrical restrictions for surface nanocrystallization. The characteristics of the affected surface layer in terms of grain size, thickness, surface roughness, and work hardening as well as induced residual stresses obtained by SSP, can be controlled through selecting the right combination of process parameters. Studies performed on SSP in recent years have indicated its ability to significantly improve the mechanical properties of treated materials in terms of hardness, fatigue strength, corrosion, wear, scratch resistance and so on, contributing to enhanced functionality and service lifetime of the material [5, 6].
They performed a study to evaluate the potential application of SSP treatment on bio-implant materials and its effects on bacterial adhesion . 316L stainless steel, as the most widely used stainless steel for biomedical applications, was selected to assess the interaction between SSP treated surfaces and a range of microorganisms, including the gram-positive strains of Staphylococcus aureus and Staphylococcus epidermidis, which are the most prevalent bacteria affecting orthopedic implants. The mechanical and physical properties of the treated samples were characterized by microstructural observation, microhardness measurements, X-ray diffraction (XRD) grain size and residual stress measurements, atomic force microscopy (AFM), interferometric profilometry, and water contact angle measurements. The early adhesion and growth of the gram-positive bacteria was measured by colony count assays. Results showed, for the first time, significant promise to enhance the mechanical and antibacterial properties of 316L stainless steel using SSP treatments without resorting to the use of pharmaceutical agents .
2. Experimental approach and results
Mirror finish stainless steel grade AISI 316L sheets 10 mm thickness were shot peened using cast steel shots of 580 µm diameter with two sets of parameters as presented in Table 1; SSP parameters were chosen based on a numerical model previously developed for surface nanocrystallization in their group . Both shot peened series, were re-peened by glass beads of 120 µm diameter for the purpose of decontamination.
Fig. 1 (a)-(c) show the microstructure of the sample's lateral sections etched after grinding. In the layer just under the shot peened surfaces (Fig.1 (b) and (c)), the microstructural evolution and the presence of multiple slip bands is noted showing a network pattern in the dense deformed grains which indicate dislocation accumulation. SSP affected a thicker layer measured to be around 300 µm thick compared to 150 µm for CSP sample.
The grain sizes on the top-most surface layer of samples were measured: for NP and CSP series, through analyzing multiple images taken from the polished and etched top surface, and for the SSP samples through XRD pattern analysis. The results indicated average grain size of 63±5 µm, 44±4 µm and 25±5 nm for as-received NP, CSP and SSP samples respectively.
Surface topography measurements were performed at two different micron and nano length scales using optical interferometric profilometer and atomic force microscope (AFM). The results indicated multiple peaks and valleys on the peened surfaces and a hierarchical surface topography confirming the presence of nano-roughness on top of micron-sized surface features. The roughness trend was similar at both scale lengths, confirming that the increase in the kinetic energy of the peening process increased surface roughness and that the highest surface roughness parameters were measured on the SSP samples both at micron and nanoscale levels (Table 2). Figure 2 shows a representative AFM image of the peened samples' surfaces to illustrate the evolution of the surface morphologies by shot-peening treatment. The AFM images confirm chaotic surfaces induced by the indentations, while representing a more irregular surface with higher surface roughness induced by SSP treatment.
Surface wettability plays a key role in protein absorption on the surface of bio-implant material that can consequently affect the relevant cellular activities in biological environment. The results of water contact angle measurements indicated higher surface hydrophilicity by increasing the kinetic energy of the process; that is, the SSP samples showed the highest wettability (Fig. 3(a)).
XRD residual stress measurements were also performed on the surface and in depth of the samples using an AST X-Stress 3000 portable X-ray diffractometer. The Full Width at Half the Maximum (FWHM) intensity of the XRD peaks was also measured as an index of work hardening. A higher increase in FWHM and deeper layer affected by compressive residual stresses was observed for the SSP samples compared to the CSP ones (Fig. 3 (b) and (c)). Fig. 3 (d) shows the in-depth profile of microhardness, measured using a diamond Vickers indenter, for all samples. The shot peening treatments increased microhardness values, following the same trend demonstrated for the FWHM; that is, showing maximum values at the surface gradually decreasing to reach the microhardness of the as-received material.
Early adhesion and growth of the most prevalent bacteria species in orthopedic infection was studied on the surface of treated 316L samples following the protocols explained in detail in . The results indicated that SSP significantly decreased the attachment and growth of both gram-positive species (Staphylococcus aureus and Staphylococcus epidermidis) after 1 day of culture (Fig.4 (a) and (b)). The number of bacteria colony forming units (CFU) for these species decreased by surface roughness increase. Bacterial adhesion was less affected after 2 days of culture, although Staphylococcus aureus growth was dramatically inhibited on SSP samples. Representative fluorescence micrographs for Staphylococcus epidermidis are shown in Fig.4 (c)-(e), illustrating the density of live and dead bacteria cells attached to the surface of the samples after 24 hours of incubation. The surface nanofeatures tend to decrease the number of anchoring sites for the small (about 1 µm in diameter) relatively rigid membrane bacteria, reduce their contact surface area, and consequently decrease their adhesion to the surface of the treated 316L samples. The results revealed an inverse correlation between the density of gram-positive bacteria and the surface roughness parameters.
Severe shot peening (SSP), as a mechanical surface treatment based on severe plastic deformation was applied to 316L stainless steel to induce hierarchical surface roughness and surface grain refinement down to nanoscale. SSP is less demanding and more adaptable in inducing grain refinement compared to its mechanical counterparts and can be applied to a wide range of metallic materials without involving geometrical and dimensional restrictions.
The results indicated the notable effect of SSP in creating a deep work-hardened surface layer with high compressive residual stresses. These characteristics can promote the functionality and durability of the material used for load-bearing orthopedic implants under static and dynamic physiological loadings.
Reduced gram-positive bacteria adhesion was observed on SSP samples. Since the gram-positive bacteria account for approximately two-thirds of orthopedic infections, it is of utmost importance to manage to decrease their activity and reduce the risk of infections and consequent long-term implant-associated complications without using antibiotics.
Considering the growing resistance of bacteria to common antibiotics drugs, along with the necessity of using a mechanically robust bone implants to endure physiological stresses, the SSP treatment is concluded to be of high potential for fabrication of multifunctional orthopedic implants.
SB acknowledges funding from Politecnico di Milano International Fellowship (PIF).
 Valiev R. Nanostructuring of metals by severe plastic deformation for advanced properties. Nat Mater. 2004;3:511-6.
 Bagherifard S, Ghelichi R, Khademhosseini A, Guagliano M. Cell response to nanocrystallized metallic substrates obtained through severe plastic deformation. ACS applied materials & interfaces. 2014;6:7963-85.
 Bagheri S, Guagliano M. Review of shot peening processes to obtain nanocrystalline surfaces in metal alloys. Surf Eng. 2009;25:3-14.
 Bagherifard S, Fernandez-Pariente I, Ghelichi R, Guagliano M. Severe Shot Peening to Obtain Nanostructured Surfaces: Process and Properties of the Treated Surfaces. Handbook of Mechanical Nanostructuring. 2015:299-323.
 Bagherifard S, Fernandez-Pariente I, Ghelichi R, Guagliano M. Fatigue behavior of notched steel specimens with nanocrystallized surface obtained by severe shot peening. Materials & Design. 2013;45:497-503.
 Bagherifard S, Guagliano M. Fatigue behavior of a low-alloy steel with nanostructured surface obtained by severe shot peening. Engineering Fracture Mechanics. 2012;81:56-68.
 Bagherifard S, Hickey DJ, de Luca AC, Malheiro VN, Markaki AE, Guagliano M, et al. The influence of nanostructured features on bacterial adhesion and bone cell functions on severely shot peened 316L stainless steel. Biomaterials. 2015;73:185-97.
 Bagherifard S, Ghelichi R, Guagliano M. A numerical model of severe shot peening (SSP) to predict the generation of a nanostructured surface layer of material. Surface and Coatings Technology. 2010;204:4081-90.
Department of Mechanical Engineering
Politecnico di Milano, Milan, Italy