Jafar
Hasan
and
Kaushik
Chatterjee
*
Department of Materials Engineering, Indian Institute of Science, Bangalore 560012, India. E-mail: kchatterjee@materials.iisc.ernet.in; Tel: +91-80-22933408
First published on 27th August 2015
The tendency of bacterial cells to adhere and colonize a material surface leading to biofilm formation is a fundamental challenge underlying many different applications including microbial infections associated with biomedical devices and products. Although, bacterial attachment to surfaces has been extensively studied in the past, the effect of surface topography on bacteria–material interactions has received little attention until more recently. We review the recent progress in surface topography based approaches for engineering antibacterial surfaces. Biomimicry of antibacterial surfaces in nature is a popular strategy. Whereas earlier endeavors in the field aimed at minimizing cell attachment, more recent efforts have focused on developing bactericidal surfaces. However, not all such topography mediated bactericidal surfaces are necessarily cytocompatible thus underscoring the need for continued efforts for research in this area for developing antibacterial and yet cytocompatible surfaces for use in implantable biomedical applications. This mini-review provides a brief overview of the current strategies and challenges in the emerging field of topography mediated antibacterial surfaces.
Jafar Hasan is an Early Career Fellow of the Wellcome-Trust/DBT India Alliance working in the Department of Materials Engineering at the Indian Institute of Science, Bangalore (India). He completed his PhD from Swinburne University of Technology, Melbourne (Australia) in 2013. This was followed by a period of post-doctoral position at the Indian Institute of Technology, Bombay (India). He also has a Bachelor of Technology degree in Biotechnology from Uttar Pradesh Technical University, Lucknow (India). His current research focuses on fabrication of nanostructured materials, bionanotechnology, biomaterials, microbiology and tissue engineering which has resulted in >15 publications. |
Kaushik Chatterjee is an Assistant Professor in the Department of Materials Engineering of the Indian Institute of Science in Bangalore (India). He received his PhD in Bioengineering from Pennsylvania State University (USA) and completed a post-doctoral fellowship jointly at the National Institute of Standards and Technology and the National Institutes of Health supported by the US National Research Council Research Associateship Program. Currently he leads a research group working on a wide variety of biomaterials intended for use in biomedical devices and tissue scaffolds. |
Despite the vast literature on the interactions of bacteria with material surfaces, only a handful of reports have investigated the effect of surface topography on bacterial adhesion. Otherwise, much of the literature in this field has focused on the intrinsic chemical behavior of the surface that retards the attachment and growth of bacterial cells. The antibacterial surfaces are of mainly two kinds, (i) anti-biofouling or bacteria-resistant surfaces and (ii) bactericidal or bacteria-killing surfaces. For information on antibacterial surfaces based on chemical functionalities, readers are directed to some wide-ranging recent literature.18–21
The summary of the recent literature on bacterial attachment on patterned surfaces is presented in Table 1 with some representative results compiled in Fig. 2. In most of the studies it has been shown that sub-micron and micro-sized features can minimize bacterial attachment although the topography does not have any direct influence on the viability of the adhered cells. In these studies, it is typically demonstrated that the bacterial cells prefer to settle in the regions between the micron sized pillars and more cells attach on the smoother surfaces than those attached on the patterned surfaces.31–35 These kinds of antibiofouling patterned surfaces were first explored on shark skin and lotus leaves and due acknowledgment must be given to these early discoveries.22,36 In a recent report, shark-inspired micro-patterns on a polyurethane catheter were shown to result in reduced bacterial colonization of the surface.37
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Fig. 2 Bacterial attachment on micron and sub-micron patterned surfaces. (A) Bacteria are attached on the micropatterned surface which mimics the riblet-like patterns of the shark skin (left) and the cross-patterns (right), reproduced with permission from ref. 34. (B) S. aureus cells adhered on microstructured pillar and lamella structures, reproduced with permission from ref. 33. (C) The bactericidal activity of the cicada wing nanopillars is depicted by the AFM measurement of the sinking of the cell with height vs. time curve, reproduced with permission from ref. 42. (D) Coccoid shaped cells hang with air bubbles between the micropillar regions on the titanium surface which mimics the lotus like pattern, reproduced with permission from ref. 23. (E) Antibacterial activity of the gecko skin, reproduced with permission from ref. 24. (F) The ruptured morphology of P. aeruginosa cell is visible on the black silicon surface which renders the cell dead, reproduced with permission from ref. 43. |
Bacteria and their shape | Incubation time | Surface type | Surface features | Height | Width | Spacing | Observation |
---|---|---|---|---|---|---|---|
Staphylococcus aureus | Incubated for 1–4 hours | (a) Polystyrene, (b) polystyrene-b-poly(acrylic acid) (PS-b-PAA), (c) polystyrene-b-poly(L-glutamic acid) (PS-b-PGA) and (d) polystyrene-b-poly[poly(ethylene glycol)methyl ether methacrylate] (PS-b-PEGMA) | Open boxes | 200 | 20 | 5 | Bacterial immobilization is favored by a PAA block copolymer. Different polymer blends provide insight into bacterial isolation and positioning.38 |
Spherical | Square shaped | 120 | 7.5, 19, 37 | 5, 6, 25 | |||
Crosshatched | 140 | 20 | — | ||||
Pillars | 175 | 5 | 20 | ||||
Lines | — | 22 | 40 | ||||
Hexagon | — (nm) | 41(μm) | 26(μm) | ||||
Staphylococcus aureus | Incubated for 0.5, 5.5 and 24 hours | PEG microgel and silanized glass slide | Circular pillars | 90 | α = 1, 2, 3, 5 | β = α/2, α, 2α | Attachment of an order of magnitude less than on the control; suggests diameter of 2–5 μm and spacing of 1–2 times of the diameter for optimum biofilm inhibition and promoting tissue growth.35 |
Spherical | (nm) | (μm) | (μm) | ||||
Enterobacter cloacae | Incubated for 48 hours | PDMS | Cross pillars | 23, 9 | 21, 4 | 5, 2 | Bacterial cells attached on the walls and recessed regions between the patterns. Confirmed less attachment than the smooth PDMS control surface due to the less area fraction on patterned surfaces.34 |
Rod shaped | Hexagonal pillars | 11 | 3 | 2 | |||
Hexagonal pits | 7 | 3 | 5 | ||||
Sinusoidal Sharklet™ | 3 (μm) | 4, 8, 2, 16 (μm) | 2 (μm) | ||||
Escherichia coli | Incubated for 12 hours | Spinach leaves, PDMS and AGAR | Spatial symmetry of a natural surface | — | — | — | Bacterial cells aggregated in the valleys of the random topographical surfaces even after biocide treatment.31 |
Rod shaped | |||||||
Escherichia coli | Tested under real-time flow conditions | PDMS | Wells | 5(μm) | 10(μm) | 7(μm) | Dynamic stability of the bacterial cells depends on the surface topography and flow parameters. The cells swimming on patterned substrates experience a differential and complex environment.39 |
Rod shaped | |||||||
Staphylococcus aureus | Incubated for 2 and 6 hours | Polystyrene | Line-like | 1.6 | 1, 3, 5 | — | In line- and pillar-like surfaces, spatial period of 1 μm had greater degree of bacterial attachment than on spatial periods of 5 μm. Although, cells on lamella-like patterns were significantly reduced compared to smooth control surfaces.33 |
Spherical | Pillar-like | 1.8 | 1, 3, 5 | ||||
Complex lamella | 0.471, 4.3(μm) | 2, 5(μm) | |||||
Staphylococcus aureus and Escherichia coli | Incubated for 12 and 24 hours | Silicon wafer | Circular and square pillars | 3(μm) | 0.6, 0.8, 1, 1.2, 1.4, 2, 5, 10, 20(μm) | 0.6, 0.8, 1, 1.2, 1.4, 2, 5, 10, 20(μm) | The microtopography patterned surface with equal width and spacing caused bacterial retention in comparison with smooth controls. E. coli adhered more on the 1.4–2 μm patterned surface while S. aureus adhered more on smooth controls.32 |
Spherical and rod shaped |
In addition to biomimetic surfaces, patterned surfaces with micron and sub-micron features have also been tested against bacterial attachment.31–35,38,39 On such patterned substrates, the bacteria are enclosed and surrounded by walls, wells, slopes, slants or other geometric curves. The confined surface structures with pillars of defined geometric shapes limit the attachment such that bacteria have less contact area between the pillars on the surface when compared with the smooth control substrates. Xu and Siedlecki reported that patterned arrays of micron and sub-micron sized ordered pillars on polyurethane significantly reduced the attachment and subsequent biofilm formation of S. epidermidis in buffer and blood plasma under shear flow.40 Interestingly, in a subsequent study they demonstrated that the wettability of the surface also affects the bacterial adhesion.41 When the polymer surfaces were made hydrophilic by plasma treatment, they noted that the sub-micron patterned surfaces but not the micro-patterned surfaces reduced bacterial attachment.
With the recent discovery of bactericidal insect wings exhibiting a nanostructured array of pillars,42 the study of surface topography mediated bactericidal surfaces has undergone a paradigm shift by redefining the old notion about topography based antibacterial surfaces. A number of reports have now focused their attention to designing and producing such topography based surfaces that actually kill the bacteria and not merely resist their attachment, which was earlier thought possible only through chemical routes. Thus, there is rapidly growing interest in fabricating anti-bacterial surfaces with nanoscale topography. Few studies on nano-structured bactericidal surfaces have been reported recently.43–45 Techniques such as deep reactive ion etching (DRIE) and nanoimprint lithography (NIL) have emerged as potent means for fabrication of such high aspect ratio nanostructured bactericidal surfaces.43–45 Black silicon consisting of anisotropic nano-structured pillars prepared by DRIE was the first study to mimic the topography of insect wings on a silicon surface that exhibited a bactericidal activity.43 We have recently engineered a “super surface” that exhibits superhydrophobic and self-cleaning properties in addition to bactericidal properties inspired by the insect wings.44 The DRIE technique which was used to produce the “super surface”44 was similar to the one which was used to fabricate the bactericidal black silicon43 but with slight variations in the processing parameters. The geometry and the architecture of the nanopillars of the “super surface” were rather different, as such the pillars were taller from the previously studied bactericidal insect wings and black silicon surfaces. The differences in nanopillar geometry can alter the killing mechanism and require further investigation for comprehensive understanding. Despite effectively killing the bacteria attached on the nanostructured “super surface” produced by DRIE, the surface was not compatible with eukaryotic cells thereby rendering it unsuitable for use on biomedical implants.44 Nevertheless the self-cleaning surfaces offer great potential to be used in surgical instruments, lenses and biosensors.
In another study, the patterned gecko skin was observed to be superhydrophobic, self-cleaning, antibacterial and yet cytocompatible.24 The tiny hairs (spinules) present on the gecko skin were able to lyse the bacterial cells incubated up to a period of 7 days. Nevertheless, it was shown that such gecko hairs are compatible with human dental pulp stem cells. The reason for the compatibility of the eukaryotic cells in this case is likely due to the presence of softer gecko hairs in contrast to our study. Although the stiffness was not reported in either of the two studies, the reported literature values of elastic modulus of the structured silicon surfaces(≈100 GPa) are greater by 6 orders of magnitude when compared to the elastic modulus of the gecko setal arrays (≈100 kPa).46–48 The elastic modulus of the substrate affects many cell functions such as morphology, migration, polarization, mechanotransduction, differentiation, regeneration, contractile forces and cell–cell signaling.49,50 In addition, the stiffness and geometry of the nanostructures will affect the ability of these pillars to mechanically rupture the cell membrane and thereby affect the target application of the material surface.51 For example, the surface with sharp nanopillars and high elastic moduli can be used for biomedical devices such as surgical instruments but may not be ideal for use as surfaces of implants where integration with the surrounding tissue is desired for the success of the device.
Plasma generated nanostructures have drawn significant attention for the modification of medical implants and instruments.52,53 In a recent study, plasma treated commercial sutures were etched for different time intervals to induce topographical changes and then tested for antibacterial activity.54 The plasma treatment induced a nanostructured lamellar pattern on the suture surface and upon bacterial attachment, only absorbable suture surfaces exhibited an antibiofouling effect where a one fold reduction in the attachment of E. coli cells was observed post 20 minutes of oxygen plasma exposure. Though the bacterial cells were not killed on contact with the sutures, however the low-cost plasma treatment offers a simple strategy for the fabrication and modification of nanostructured biocompatible and antibacterial surfaces.
In a recent intriguing study, diblock copolymers of poly(sulfobetaine methacrylate) (poly(SBMA)) and poly(propylene oxide) (PPO) were adsorbed on three kinds of surfaces consisting of smooth, convex and indented surface topographies.60 The self-assembled zwitterionic moieties exhibited considerable bactericidal properties as expected but the interesting observation was that on curved and indented surfaces, the differential orientation of the similar diblock copolymers increased the antibiofouling performance by limiting the interactions of the bacterial cells with these surfaces. The indented surface showed better antibiofouling ability than the curved surface as the bacterial cells were unable to adhere between the indents.
In another statistical study, multifractal analysis was utilized to define the attachment pattern of S. aureus and S. epidermidis cells on substrates of stainless steel and various bactericidal ceramic based (Ti–ZrN/Ag) silver coatings on the substrates of stainless steel.61 It was found that the clustering of S. aureus cells was more influenced by the chemistry on ceramic substrates and that of S. epidermidis cells was more influenced by topography on silver substrates. Similar comprehensive reports on the combinatorial effect of surface chemistry and topography using rigorous quantification of cellular behavior in differentially confined locations by statistical and mathematical analyses are needed.
Aizenberg and colleagues have produced polymer based nano/microstructured surfaces that swell and contract based on the change in pH.69,70 Such pH responsive polymers can be used to tune the topography and thus produce antifouling surfaces. Other stimuli responsive antibacterial surfaces have also been actively studied in the recent past such as the renewable sacrificial skin based on the pilot whale, the self-assembly of peptide sequences and self-replenishing oil infused surfaces.71–73
Recently, topographical changes triggered by external physical stimuli have also been explored as a plausible strategy to affect the bacterial retention. The surface area and topography of the elastomeric silicone surface were changed in response to two kinds of external stimuli, namely voltage-induced and stretch-induced.74 The surface topography of the elastomer was changed from a smooth surface in the absence of an electric field to a surface with a crater-like pattern when an electric field was applied. It was observed that during the voltage-induced deformation of polymer surfaces, almost 95% of the adherent Cobetia marina biofilms were removed. During the applied voltage deformation, the effect of surface deformation in order to remove the biofilms was more dominant than the actual applied voltage itself. Also, the primary effect of surface deformation by stretching the elastomer uniaxially was studied and it was found that more than 80% of the Cobetia marina and E. coli biofilms were removed. In a more recent similar study, a hydraulic and pneumatic induced elastomeric surface released 90% of Proteus mirabilis biofilms from the strained surface.75 These kinds of biofilm debonding studies suggest a good application platform for biomedical implants along with some other biofilm removal mechanisms that may be triggered on demand.
Herein we have reviewed recent efforts on developing micron and sub-micron sized pillars to inhibit bacterial attachment in comparison with the smooth surfaces but such pillar topography is ineffective in totally controlling the bacterial attachment as cells find sufficient interpillar regions for attachment. Further optimization of the spacing to minimize attachment in the interpillar gaps could be utilized to design superior antibiofouling surfaces. The nanoscale spacing in the patterns of the cicada and dragonfly wings could serve as inspiration. Optimization of current methods and development of new fabrication techniques must be complemented with mathematical modeling and statistical approaches. It is important to recognize that the mere presence of nanoscale topographical features may yield an antibiofouling surface but not always a bactericidal surface.76,77 Though recent biomimetic approaches have yielded bactericidal surfaces exhibiting sharp nanopillars that can lyse the bacterial cells, not all such surfaces are cytocompatible. For use in biomedical implants, further optimization of the nanopillars is required such that the surface promotes the attachment and growth of mammalian cells. Perhaps softer polymeric pillars that rupture the small bacteria but remain conducive to much larger mammalian cells could offer a plausible solution. Further work is warranted to engineer such surfaces through development of novel micro- and nano-scale techniques.
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