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Impact of engineered surface microtopography on biofilm formation of S

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The surface of an indwelling medical device can be colonized by human pathogens that can form biofilms and cause infections. In most cases, these biofilms are resistant to antimicrobial therapy and eventually necessitate removal or replacement of the device. An engineered surface microtopography based on the skin of sharks, Sharklet AF, has been designed on a poly(dimethyl siloxane) elastomer (PDMSe) to disrupt the formation of bacterial biofilms without the use of bactericidal agents. The Sharklet AF PDMSe was tested against smooth PDMSe for biofilm formation of Staphylococcus aureus over the course of 21 days. The smooth surface exhibited early-stage biofilm colonies at 7 days and mature biofilms at 14 days, while the topographical surface did not show evidence of early biofilm colonization until day 21. At 14 days, the mean value of percent area coverage of S. aureus on the smooth surface was 54% compared to 7% for the Sharklet AF surface (p<0.01). These results suggest that surface modification of indwelling medical devices and exposed sterile surfaces with the Sharklet AF engineered topography may be an effective solution in disrupting biofilm formation of S. aureus.
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Impact of engineered surface microtopography on biofilm formation
of Staphylococcus aureus
Kenneth K. Chung
Department of Materials Science and Engineering, University of Florida, Gainesville, Florida 32611
James F. Schumacher
J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, Florida
32611
Edith M. Sampson
Department of Otolaryngology, University of Florida, Gainesville, Florida 32611
Robert A. Burne
Department of Oral Biology, UF College of Dentistry, University of Florida, Gainesville, Florida 32611
Patrick J. Antonelli
Department of Otolaryngology, University of Florida, Gainesville, Florida 32611
Anthony B. Brennana
Department of Materials Science and Engineering, and J. Crayton Pruitt Family Department of Biomedical
Engineering, University of Florida, Gainesville, Florida 32611
Received 29 March 2007; accepted 30 May 2007; published 29 June 2007
The surface of an indwelling medical device can be colonized by human pathogens that can form
biofilms and cause infections. In most cases, these biofilms are resistant to antimicrobial therapy and
eventually necessitate removal or replacement of the device. An engineered surface
microtopography based on the skin of sharks, Sharklet AF™, has been designed on a polydimethyl
siloxaneelastomer PDMSeto disrupt the formation of bacterial biofilms without the use of
bactericidal agents. The Sharklet AF™ PDMSe was tested against smooth PDMSe for biofilm
formation of Staphylococcus aureus over the course of 21 days. The smooth surface exhibited
early-stage biofilm colonies at 7 days and mature biofilms at 14 days, while the topographical
surface did not show evidence of early biofilm colonization until day 21. At 14 days, the mean value
of percent area coverage of S. aureus on the smooth surface was 54% compared to 7% for the
Sharklet AF™ surface p0.01. These results suggest that surface modification of indwelling
medical devices and exposed sterile surfaces with the Sharklet AF™ engineered topography may be
an effective solution in disrupting biofilm formation of S. aureus.© 2007 American Vacuum
Society. DOI: 10.1116/1.2751405
I. INTRODUCTION
Bacterial biofilms are a major concern in the development
of biomaterials, ultrafiltration systems, and underwater ves-
sels. In the biomedical arena, bacterial colonization of sur-
faces compromises the effectiveness of implanted materials
and ultimately can result in persistent infections.1,2 Biomate-
rial surfaces for tissue constructs and implants are subject to
a competition between microbial adhesion and tissue integra-
tion, where an ideal surface would prevent the former while
promoting the latter.3However, the chemical, mechanical,
and physical properties of tissue construct materials are in-
herently meant to enhance all forms of biogrowth, making it
equally likely that such a surface would submit to bacterial
colonization.
Microorganisms colonize biomedical implants by devel-
oping biofilms, structured communities of microbial cells
embedded in an extracellular polymeric matrix that are ad-
herent to the implant and/or the host tissues.2,4 Biofilms ren-
der microorganisms resistant to host defenses and antibiotic
therapy.5,6 The clinical means of managing biofilms has been
prevention.
Preventing biofilm-associated infections has traditionally
been through use of prophylactic antibacterial agents,
whether delivered systemically or released directly from the
biomaterial.7Pharmacokinetics and toxicity of the antibacte-
rial agents incorporated in biomaterials have limited the ef-
fectiveness of such therapies.7–10 Antibiotics, antibodies, and
phagocytes can clear planktonic cells released by the biofilm,
but the sessile communities themselves are resistant to such
agents.11,12 Antibiotic therapy resulting in incomplete eradi-
cation of biofilm has been linked with the emergence of
antibiotic-resistant bacteria, which may compromise the ef-
fectiveness of these agents for even non-biofilm-mediated
infections.13–16
Another strategy for preventing the development of bio-
films has been to alter the biomaterial surface properties.
Surface modification techniques to tailor the surface energy
via surface chemistry and surface topography have been de-
veloped to study the effects of changes in these surface prop-
aElectronic mail: abrennan@mse.ufl.edu
89 89Biointerphases 22, June 2007 1934-8630/2007/22/89/6/$23.00 ©2007 American Vacuum Society
erties on biofilm formation.17,18 Bacterial adhesion has been
investigated on surface topographies that range from random
structures to ordered arrays. There appears to be a trend to-
ward increased bacterial coverage as the Ra-roughness values
increased on electropolished steel.19 Conversely, P. aerugi-
nosa was less likely to foul hydrophilic, electrically neutral,
smooth polymeric surfaces.20 Interestingly, bacterial adhe-
sion was reduced on stainless steel surface microtopogra-
phies that were generated by a one-directional polishing fin-
ish relative to smooth surfaces.21 The aforementioned studies
examined surfaces that were randomly roughened and did
not examine specific surface features. The effects of a non-
random topography consisting of etched grooves of varying
widths in silicon coupons with P. aeruginosa and P. fluore-
scens showed that rates of attachment were independent of
groove size and greatest on the downstream edges of
grooves.22 More recently, microbial retention on a defined
microtopography in the form of etched pits was determined
to be dependent on both the size of the surface defect and the
cell.23
The purpose of our study was to investigate the potential
for bacterial attachment and colonization on an engineered
topography with a well-defined structure. Our research is fo-
cused on the design and characterization of surface microto-
pographies that effectively control bioadhesion, with the goal
to produce a biomaterial with topography variants that can
effectively switch from biofilm forming to biofilm inhibition.
To achieve this, a surface energy model was created corre-
lating wettability with bioadhesion to characterize and de-
velop settlement-enhancing as well as antifouling
topographies.24 The application of this model led to the de-
sign concept of a biomimetic structure inspired by the skin of
fast-moving sharks Sharklet AF™, and an engineered
roughness index ERIwas developed as an extension of this
model to study and develop other unique engineered
topographies.25
In this article, a surface of uniform chemistry with an
engineered microtopography was investigated for the inhibi-
tion of bacterial biofilm formation. The most successful de-
sign to date is Sharklet AF™. This particular microtopogra-
phy is unique from the aforementioned ones in that it has
nonrandom, clearly defined surface features that are typically
tailored to the critical dimensions of the fouling organism.
Recent results on the Sharklet AF™ and other engineered
microtopographies designed at a 2
m feature width and
spacing have shown a strong correlation between the ERI
and the inhibition of settlement by the zoospores 共⬃5
min
diameterof the most common ship fouling alga, Ulva.25 In
addition, the Sharklet AF™ microtopography designed at a
20
m feature width and spacing has been demonstrated to
be a strong inhibitor of the settlement of barnacle cyprids of
B. amphitrite.26 This organism’s attachment disk measures
2530
m.27 For the present study, the Ulva-specific Shar-
klet AF™ surface was selected for the potential to inhibit
biofilm formation of Staphylococcus aureus based on the ap-
proximate match between the size of the bacteria and the
critical dimensions of this surface 2
m feature width and
spacing, 3
m feature height. It was hypothesized that the
dimensions of the topography would be slightly too large to
effectively reduce the attachment of the bacteria in the size
range of 1–2
m but could be effective at physically dis-
rupting the further colonization of additional bacteria and
subsequent formation of biofilm Fig. 1.S. aureus was se-
lected as the bacterial pathogen due to both its size and its
association with nosocomial infections in implanted devices,
such as cochlear implants, sutures, and heart valves.10,28
II. MATERIALS AND METHODS
A. Materials
Dow Corning® Silastic® T-2, a platinum-catalyzed poly-
dimethyl siloxaneelastomer PDMSe, was used for its low
modulus, low surface energy, and propensity for minimal
bioadhesion.29 Silastic® brand silicone elastomers are bio-
materials used in numerous medical devices including tub-
ing, catheters, and pacemaker leads. The elastomer was pre-
pared by mixing one part by weight of curing agent with ten
parts by weight of resin, then degassing under vacuum
2830 in. Hgfor 30 min. The mixture was cured at
22 °C for 24 h.
FIG. 2. Locations 关共AC兲兴 for scanning electron microscopy SEManaly-
sis for 8 mm circular PDMSe samples.
FIG. 1. Sharklet AF™ topography on polydimethyl si-
loxaneelastomer PDMSewith 2
m feature width
and spacing and 3
m feature height. ALight micro-
graph with top-down view. BScanning electron mi-
crograph with top down view. CScanning electron
micrograph taken at 35° tilt to show protruding
features.
90 Chung et al.: Impact of engineered surface microtopography 90
Biointerphases, Vol. 2, No. 2, June 2007
B. Sharklet AF™ design and fabrication of
topographical molds
The Sharklet AF™ design24,25 consists of 2
m wide rect-
angular ribs of varying lengths ranging from 4 to 16
m.
The ribs of varying lengths are combined into a periodic,
diamondlike array at a fixed spacing of 2
m between neigh-
boring features Fig. 1. The final, resultant Sharklet AF™
topography in PDMSe was created by replication of silicon
wafer molds. Silicon wafer molds were fabricated by first
transferring the Sharklet AF™ design to photoresist-coated
silicon wafers using photolithographic techniques as previ-
ously described.30 Next, the patterned silicon wafers were
deep reactive ion etched to a depth of 3
m before being
cleaned of residual photoresist with an O2plasma etch. The
etched silicon wafer surfaces were then methylated via va-
por depositionwith hexamethyldisilazane to prevent adhe-
sion. These wafers served as negative molds for topographi-
cal replication of the Sharklet AF™ topography at a feature
height of 3
m onto a PDMSe surface.
C. Sample preparation
Silicon molds were replicated into PDMSe to produce
0.4 mm thick films containing protruding topographical
features. Briefly, the PDMSe material components were pre-
pared and mixed as described,24,25 poured over the silicon
wafer molds, and the entire system was pressed between two
larger glass plates with the appropriate spacers to produce a
0.4 mm thick film. After curing for 24 h, the PDMSe film
was removed from the silicon wafer with protruding topo-
graphical features forming the Sharklet AF™ topography on
the PDMSe surface Fig. 1. PDMSe samples of smooth
replicated from an unmodified silicon waferand topo-
graphically modified were punched out with a circular die
8 mm in diameter. Five replicates each of smooth PDMSe
and Sharklet AF™ PDMSe disks were placed into 3 in. Petri
dishes one dish for each day examinedfor the growth assay
and gas sterilization.
D. S. aureus biofilm formation assay
Staphylococcus aureus ATCC 29213was subcultured in
tryptic soy broth TSBgrowth medium and grown at 37 °C
overnight in static conditions. Optical absorbance was mea-
sured, serial dilutions were performed, and growth curve and
linear optical density-colony-forming unit CFUregression
were plotted. Bacterial concentration was determined via
spectrophotometry by interpolating CFU per milliliter from
the linear optical density-CFU regression. Samples were
statically immersed in a 107CFU/ml bacterial suspension
for up to 21 days. Every day, dishes were put on a rocker for
1 min at 30 rpm and the medium was replaced to allow for
continued bacterial growth. Dishes were removed on days 0,
2, 7, 14, and 21. For each removed dish, areas surrounding
FIG.3. 关共Aand B兲兴 SEM images 2000of S. au-
reus on smooth and topographically modified PDMSe
surfaces after 7 day exposure. 关共Cand D兲兴 Processed
SEM images of smooth and Sharklet AF™ PDMSe sur-
faces using MACROMEDIA FIREWORKS®. Bacteria covered
areas were outlined and blackened.
FIG. 4. Grouping and numbering of bacteria colonies as
measured by IMAGEJ software. The analysis pictured
was conducted on the processed SEM images in Fig. 3.
ABacteria coverage on a smooth PDMSe surface was
detected as two colonies. BBacteria coverage on a
Sharklet AF™ PMDSe surface was detected as over 40
individual colonies of bacteria.
91 Chung et al.: Impact of engineered surface microtopography 91
Biointerphases, Vol. 2, No. 2, June 2007
the samples on the placement grid were rinsed with de-
ionized water using a Pipet-Aid® and aspirated to eliminate
nonadherent cells. This rinsing procedure was repeated for a
total of three times, and the dish was then put on an orbital
rocker for 1 min at 30 rpm. Each sample in the dish was then
treated with 20 ml of 10 mM cetyl-pyridinium chloride fixa-
tive and allowed to air dry overnight. Another dish exposed
only to sterile medium was incubated with the samples for
21 days and served as a negative control.
E. Characterization
Samples were dehydrated in a graded ethanol series of
25%, 50%, 75%, 95%, and 100% at 10 min intervals.
Samples were washed twice with hexamethyldisilazane with
a 5 min interval between washings, followed by drying using
a vacuum desiccator. Each sample was attached to a beveled
disk and sputter coated with Au/Pd and imaged with a JEOL
6400 scanning electron microscope SEM. SEM images at
2000from areas A, B, and C for each replicate were used
to quantify bacterial growth on each surface Fig. 2.
Biofilms were identified by the presence of microorgan-
isms and exopolymeric matrix. Biofilm growth was esti-
mated by measuring the percent area of coverage of bacteria.
To obtain this value, SEM images were first processed using
MACROMEDIA FIREWORKS® software to outline and blacken
the area containing bacteria and/or biofilm e.g., Fig. 3.
Processed SEM images were analyzed for percent cover-
age using IMAGEJ software.31 Areas of coverage were num-
bered and outlined, and the total summation of area covered
was measured e.g., Fig. 4. For each replicate, the results for
areas A, B, and C are combined to reflect the percentage
coverage of bacteria for that specific replicate. Results for
each replicate are reported as percent coverage of bacterial
colonies.
F. Statistical analysis
The mean value standard errorof percent area cover-
age of bacteria on both smooth and Sharklet AF™ PDMSe
surfaces at days 0, 2, 7, 14, and 21 was calculated. Statistical
differences were evaluated by a two-way analysis of variance
for the factors of “surface” smooth versus Sharklet AF™
and “day” 0, 2, 7, 14, and 21followed by Tukey’s test for
multiple comparisons. Statistical differences were considered
at the 95% confidence level.
III. RESULTS
On day 0, individual cells were seen on the surfaces of
smooth PDMSe Fig. 5A兲兴, while individual cells appeared
in the recesses between the protruding features for Sharklet
AF™ PDMSe surfaces Fig. 5B兲兴. On day 2, microcolonies
of bacteria began to form on the smooth surfaces Fig. 5C兲兴,
and the Sharklet AF™ surfaces continued to have isolated
cells accrete between features Fig. 5D兲兴. Growth of the
microcolonies increased on day 7 for the smooth surfaces
into early-stage biofilms Fig. 5E兲兴. The Sharklet AF™ sur-
faces day 7continued to exhibit small-sized clusters of bac-
teria, with no evidence of early-stage biofilm development;
the clusters were positioned similar to day 2 in the recesses
between protruding topographical features Fig. 5F兲兴.On
day 14, the smooth surfaces had the first evidence of mature
biofilms Fig. 5G兲兴, while the Sharklet AF™ surfaces had a
slight increase in the number of small clusters of cells com-
pared to day 7 but still no evidence of early biofilm devel-
opment or formation Fig. 5H兲兴. On day 21, a significant
portion of the smooth PDMSe surfaces was colonized by
biofilms Fig. 5I兲兴, and biofilms first appeared in isolated
areas on the Sharklet AF™ PDMSe surfaces Fig. 5J兲兴.Ar-
eas surrounding the large bacterial colonies on the topo-
graphical surfaces for day 21 were virtually devoid of adher-
ent bacteria. SEM images of the negative control samples
exposed to only TSB media showed no cells.
FIG. 5. Representative SEM images of S. aureus on PDMSe surfaces over
the course of 21 days areas of bacteria highlighted with color to enhance
contrast. On the left are smooth PDMSe surfaces and the right column
shows Sharklet AF™ PDMSe surfaces. Aand Bday 0, Cand Dday
2, Eand Fday 7, Gand Hday 14, and Iand Jday 21.
92 Chung et al.: Impact of engineered surface microtopography 92
Biointerphases, Vol. 2, No. 2, June 2007
Image and statistical analysis indicated that smooth
PDMSe samples had significant increases in bacterial cover-
age for pooled time points Tukey’s test, p0.05, with the
first evidence of biofilm on day 7 samples. The Sharklet
AF™ samples had significantly lower values of percent area
coverage for days 7, 14, and 21 Tukey’s test, p0.05, with
biofilm colonies not appearing until day 21. Even on day 21,
biofilm colonies covered only isolated areas on Sharklet
AF™ samples, with little to no evidence of biofilms or bac-
terial cells in other areas. The mean value standard error
of percent area coverage of bacteria on both smooth and
Sharklet AF™ PDMSe surfaces at days 0, 2, 7, 14, and 21
was calculated and is graphically displayed Fig. 6.
IV. DISCUSSION
Most in vitro studies involving S. aureus have examined
the adhesion behavior over the course of a few hours.16,19
However, for transcutaneous devices such as catheters, the
time frame for biofilm formation is typically 14 days.7Thus,
the focus of this study was to test the effects of an engineered
microtopography on bacterial colonization and biofilm for-
mation for a period of time that extended beyond 14 days.
The growth assay parameters included optimized conditions
for S. aureus colonization and spanned 21 days. To date, this
is the first in vitro study involving surface topography and S.
aureus over a time period approximating that of short-term
indwelling devices.
Material selection in this study was predicated upon the
popularity of silicone as the choice material for molded im-
plants, such as cochlear implants and long-term catheters,
despite being shown to have nearly a tenfold greater risk of
infection than other polymer materials.32 The static culture
provided for biofilm growth was chosen to represent the
most challenging environment for the material surface in the
presence of bacteria, as opposed to the low shear dynamics
of indwelling catheters. The results of this study strongly
suggest that the surface modification of existing silicone de-
vices with the Sharklet AF™ topography may prolong the
service life and improve the efficacy of these devices. It is
also encouraging to note that the topographical modification
of a surface used in this study involves no chemical changes
of the biomaterial surface and does not rely on the release of
any antibacterial agents.
Both qualitative e.g., development of extracellular ma-
trixand quantitative measures of biofilm formation revealed
inhibition of biofilm development on PDMSe with Sharklet
AF™ microtopography. The results confirm the hypothesis
that cells can fit in the recessed regions between the pro-
truded topographical features, but evidence of biofilm forma-
tion did not occur on the Sharklet AF™ features until day 21.
The raised features could potentially reduce the surface area
exposed to bacteria if the channels failed to fill with growth
media. However, finding bacteria strictly in the Sharklet
AF™ channels speaks against this possibility. Observations
of adhered bacteria would suggest that the protruded features
of the topographical surface provided a physical obstacle to
deter the expansion of small clusters of bacteria present in
the recesses into microcolonies. It was at day 21 when bac-
teria were observed to form small, multilayered colonies
within the recesses in order to extend over the protruding
features and connect to other isolated colonies. This phenom-
enon may be the explanation for the delay of early-stage
biofilm development to day 21 that was evident on the
smooth surface at day 7.
In the context of the “race for the surface” in
biomaterials,3our engineered surface approach suggests the
use of a hierarchy of surface topographies26 to control bio-
adhesion. Previous results detailing an engineered topogra-
phy capable of promoting cell growth33 can be integrated
into the Sharklet AF™ in this study to produce a hierarchical
topography for desirable competitive adhesion at the bioma-
terial surface. One can envision a surface that repels and
delays biofilm formation to the extent that host cells, vital to
the integration of the biomaterial with the physiological en-
vironment, can be established and proliferated on the de-
signed surface.
This study was performed using a highly simplified in
vitro model. A great deal of work is needed to determine if
FIG. 6. Mean value of percent area coverage of bacteria
on smooth and Sharklet AF™ PDMSe surfaces at vari-
ous time points. Bars represent± standard error, n=5.
93 Chung et al.: Impact of engineered surface microtopography 93
Biointerphases, Vol. 2, No. 2, June 2007
observations from this in vitro model are borne out in vivo.
Current research is evaluating the adhesion and biofilm for-
mation tendencies of other biofilm-forming bacteria on Shar-
klet AF™. Also, the application of the engineered roughness
index is being used to predict other engineered topographies
that may be effective at inhibiting biofilm formation. Consid-
erations for designing the optimal microtopography for an
implantable device will include the interactions with host
molecules and impact on fibrous capsule formation.
V. CONCLUSIONS
S. aureus was cultured on PDMSe surfaces for up to
21 days. Biofilms were established after 14 days on the
smooth PDMse surfaces whereas an engineered surface mi-
crotopography with nonrandom, clearly defined features elic-
ited a negative response. The Sharklet AF™ microtopogra-
phy disrupted S. aureus colonization and biofilm formation
without the use of bactericidal agents. Engineered surface
microtopographies present a promising means of blocking
biofilm development and reducing the rate of biomedical im-
plant infections.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the financial support
of the Office of Naval Research, Award No. N00014-02-1-
0325 to one of the authors A.B.B. Special thanks to Sean
Royston for his technical assistance in production and fabri-
cation of the engineered topography.
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... The adoption of biomimetics in various industries has increased over time, with innovations such as Michael Kelly's barbed wire inspired by the Osage orange bush [17] and micropatterns that mimic shark skin microstructured roughness which disrupt the formation of bacterial biofilms without the use of bactericidal agents [18,19] (Fig. 1). Line patterns observed on the periostracum of M. edulis (blue mussel) have a similar pitch and width as those used in an engineered structure recently published by Cordero-Guerrero (2023) [20] on an aluminum alloy; the former natural surface offers reduced algal spore attachment and germination [21] while the latter synthetic surface was proven to reduce E. coli attachment. ...
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... Prevention of bacterial infections from implants or medical devices is of utmost importance; therefore, proactive approaches are necessary. Adherence and proliferation of bacteria on the surfaces of medical materials can lead to the formation of biofilms and subsequent infections in the affected area [32]. Besides promoting the formation of new bone tissues, LL-37 also exhibits certain antimicrobial properties. ...
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Correspondence: Donraporn Daranarong (donraporn.d@cmu.ac.th) | Chayarop Supanchart (chayarop.supanchart@cmu.ac.th) ABSTRACT This study aims to determine the in vitro and in vivo biocompatibility and the differences in LL-37 releasing patterns of the PLC/ Col/LL-37 sustained releasing membrane and the rapid releasing Col/LL-37 membrane for use as a barrier membrane in guided bone regeneration. On Days 3 and 7, the PLC/Col/LL-37 membrane revealed generated osteoclasts, whereas the PLC/Col membrane indicated mature osteoclasts. PLC/Col/LL-37 was non-toxic to blood cells and inhibited human albumin adhesion to the surface. A microbial reduction was observed in the Col/LL-37 membrane, while both PLC/Col/LL-37 and Col/LL-37 may have caused early phases of skin sensitization. In animal testing, PLC/Col/LL37 and Col/LL37 exhibited the same levels of inflammation as the control group at all time points, while the different releasing patterns of LL-37 loaded on PLC/Col/LL-37 and Col/ LL-37 membranes had no effect on inflammation resolution in rat models.
... This section, briefly reviews the contributions previously made in existing literature on biofilm quantification using various imaging techniques through different physical or chemical features, as well as previous works on mathematical modelling of biofilm growth. In contemporary research, several methods of biofilm characterisation using statistical techniques has been reported on 2D SEM images [42][43][44][45][46][47][48] . Yang et al. 49 and Jackson et al. ...
Preprint
The present paper proposes a novel method of quantification of the variation in biofilm architecture, in correlation with the alteration of growth conditions that include, variations of substrate and conditioning layer. The polymeric biomaterial serving as substrates are widely used in implants and indwelling medical devices, while the plasma proteins serve as the conditioning layer. The present method uses descriptive statistics of FESEM images of biofilms obtained during a variety of growth conditions. We aim to explore here the texture and fractal analysis techniques, to identify the most discriminatory features which are capable of predicting the difference in biofilm growth conditions. We initially extract some statistical features of biofilm images on bare polymer surfaces, followed by those on the same substrates adsorbed with two different types of plasma proteins, viz. Bovine serum albumin (BSA) and Fibronectin (FN), for two different adsorption times. The present analysis has the potential to act as a futuristic technology for developing a computerized monitoring system in hospitals with automated image analysis and feature extraction, which may be used to predict the growth profile of an emerging biofilm on surgical implants or similar medical applications.
... Shark scales have a rational structure for infection prevention [19]. Plates made from silicone elastomers that mimic sharkskin scales have antimicrobial effects against various pathogens [20,21]. Furthermore, the technology has been commercialized under the brand name "Sharklet" and is now used in medical devices as well as smaller items, such as mouse pads and cell phone covers. ...
Article
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The increase in infections derived from biofilms from Staphylococcal spp. prompted us to develop novel strategies to inhibit biofilm development. Nanoscale protrusion structures (nanopillars) observed on the wings of dragonflies and cicadas have recently gained notable attention owing to their physical, antimicrobial, and bactericidal properties. Thus, they are not only expected to reduce the damage caused by chemical antimicrobial agents to human health and the environment, but also to serve as a potential countermeasure against the emergence of antimicrobial-resistant bacteria (ARB). In this study, we evaluated the anti-biofilm effects of cyclo-olefin polymer (COP) nanopillars by changing the wettability of surfaces ranging in height from 100 to 500 nm against Staphylococcus spp., such as Staphylococcus aureus NBRC 100910 (MSSA), Staphylococcus aureus JCM 8702 methicillin-resistant S. aureus (MRSA), and Staphylococcus epidermidis ATCC 35984. The results clearly show that the fabricated nanopillar structures exhibited particularly strong biofilm inhibition against MRSA, with inhibition rates ranging from 51.2% to 62.5%. For MSSA, anti-biofilm effects were observed only at nanopillar heights of 100–300 nm, with relatively low hydrophobicity, with inhibition rates ranging from 23.9% to 40.8%. Conversely, no significant anti-biofilm effect was observed for S. epidermidis in any of the nanopillar structures. These findings suggest that the anti-biofilm properties of nanopillars vary among bacteria of the same species. In other words, by adjusting the height of the nanopillars, selective anti-biofilm effects against specific bacterial strains can be achieved.
... Studies suggest cells generally adhere to surfaces with contact angles ranging from 40˚to 70˚ [12]. Chung et al. [13] investigated the effect of incorporating a rough pattern onto polydimethylsiloxane (PDMS) elastomer through photolithography, fabricating the Sharklet AF™ topography design. Over 21 days, Staphylococcus aureus cultures were grown on smooth and patterned surfaces. ...
Article
Full-text available
The rise of infections associated with indwelling medical devices is a growing concern, often complicated by biofilm formation leading to persistent infections. This study investigates a novel approach to prevent Candida albicans attachment on the surface by altering surface topography. The research focuses on two distinct surface topographies: symmetry (squares) and non-symmetry (lines), created through a direct laser photolithography process on a Cyclic olefin copolymer (COC) surface. The wettability of these patterned surfaces was then examined immediately after fabrication and plasma treatment to mimic the sterilization process of indwelling devices through UV plasma. The results reveal directional wettability in the line pattern and size-dependent wettability in both square and line patterns. Candida albicans were cultured on these surfaces to assess the efficacy of the topography in preventing biofilm formation. The study demonstrates that symmetry and non-symmetry pattern topography inhibit biofilm formation, providing a promising strategy for mitigating Candida-associated infections on medical devices. The research sheds light on the potential of surface modification techniques to enhance the biocompatibility of medical devices and reduce the risk of biofilm-related infections.
Article
Decellularization is the process of obtaining acellular tissues with low immunogenic cellular components from animals or plants while maximizing the retention of the native extracellular matrix structure, mechanical integrity and bioactivity. The decellularized tissue obtained through the tissue decellularization technique retains the structure and bioactive components of its native tissue; it not only exhibits comparatively strong mechanical properties, low immunogenicity and good biocompatibility but also stimulates in situ neovascularization at the implantation site and regulates the polarization process of recruited macrophages, thereby promoting the regeneration of damaged tissue. Consequently, many commercial products have been developed as promising therapeutic strategies for the treatment of different tissue defects and lesions, such as wounds, dura, bone and cartilage defects, nerve injuries, myocardial infarction, urethral strictures, corneal blindness and other orthopedic applications. Recently, there has been a growing interest in the decellularization of fish tissues because of the abundance of sources, less religious constraints and risks of zoonosis transmission between mammals. In this review, we provide a complete overview of the state-of-the-art decellularization of fish tissues, including the organs and methods used to prepare acellular tissues. We enumerated common decellularized fish tissues from various fish organs, such as skin, scale, bladder, cartilage, heart and brain, and elaborated their different processing methods and tissue engineering applications. Furthermore, we presented the perspectives of (i) the future development direction of fish tissue decellularization technology, (ii) expanding the sources of decellularized tissue and (iii) innovating decellularized tissue bio-inks for 3D bioprinting to unleash the great potential of decellularized tissue in tissue engineering and regenerative medicine applications.
Chapter
The discovery of antimicrobials has had a significant impact on the reduction of deaths related to infectious diseases. However, this advancement was no sooner followed by the emergence of resistance against these antimicrobials. Overuse and misuse of these agents have brought us to a pre-antibiotic world, with extremely limited agents targeting quickly evolving multidrug-resistant (MDR) bacterial pathogens. This scenario highlights the need to develop strategies to discover novel antibiotics that can overcome at least the presently known resistance mechanism. This review covers the newly emerging strategies that not only bypass the bacterial resistance mechanism but also potentiate available antibiotics against MDR bacterial pathogens. These nonantibiotic approaches include targeted antibacterial photodynamic therapy (APT) and phage therapy, bacterial protein degradation via BacPROTAC, genome editing utilizing clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas), reduction of bacterial pathogenesis by anti-virulence strategies such as targeting specialized secretion systems, regulating virulence gene expression and toxin production, and reducing drug-associated toxicity by utilizing biomimetic assemblies and surfaces, using immunotherapies and vaccines, and modulating the gut microbiome via fecal microbiota transplantation (FMT), prebiotics, probiotics, postbiotics, and hemofiltration devices. Additionally, this chapter explores the exciting advancements on the horizon, such as personalized medicine, nanotechnology-based therapies, and genome mining and artificial intelligence (AI)-driven approaches. In addition, this chapter discusses the potential of these innovations on the treatment landscape and the hope that they bring by overcoming antibiotic resistance associated with MDR bacterial pathogens.
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The interaction between bacteria and implanted medical device surfaces presents a significant challenge in healthcare. This interaction often leads to biofilm formation, resulting in prolonged bacterial exposure and operational complications. Consequently, the risk of developing multidrug-resistant infections increases, posing a serious threat to patient health and treatment efficacy. Effective prevention of biofilm development requires a comprehensive understanding of the physicochemical properties of biomaterials. Recent advancements in the study of natural antifouling mechanisms have provided valuable insights for developing materials resistant to bacterial colonization. These discoveries offer promising directions for creating more effective antifouling surfaces. However, the existing surface topographies of medical devices, originally designed for optimal tissue integration, may unintentionally facilitate microbial adhesion. This review highlights the crucial need to evaluate the biocompatibility of medical device surfaces, emphasizing the impact of their specific topographical features on bacterial adhesion and biofilm development. We emphasize that surface topography can either promote or inhibit bacterial colonization, depending on specific features such as roughness, pattern, and scale. Understanding these topography-dependent effects is crucial for designing surfaces that minimize bacterial adhesion while maintaining optimal functionality and biocompatibility for the intended medical application. Our analysis reveals significant findings regarding the complex relationship between bacteria and three-dimensional surface properties. This knowledge provides a foundation for further advancements in the development of efficient antifouling materials. By understanding the nuances of bacterial-surface interactions, researchers can design more effective strategies to prevent biofilm formation. Through an extensive examination of preclinical studies, this research not only elucidates the mechanisms of bacterial adhesion but also paves the way for innovative solutions.
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Musculoskeletal disorders are on the rise, and despite advances in alternative materials, treatment for orthopedic conditions still heavily relies on biometal-based implants and scaffolds due to their strength, durability, and biocompatibility in load-bearing applications. Bare metallic implants have been under scrutiny since their introduction, primarily due to their bioinert nature, which results in poor cell-material interaction. This challenge is further intensified by mechanical mismatches that accelerate failure, tribocorrosion-induced material degradation, and bacterial colonization, all contributing to long-term implant failure and posing a significant burden on patient populations. Recent efforts to improve orthopedic medical devices focus on surface engineering strategies that enhance the interaction between cells and materials, creating a biomimetic microenvironment and extending the service life of these implants. This review compiles various physical, chemical, and biological surface engineering approaches currently under research, providing insights into their potential and the challenges associated with their adoption from bench to bedside. Significant emphasis is placed on exploring the future of bioactive coatings, particularly the development of smart coatings like self-healing and drug-eluting coatings, the immunomodulatory effects of functional coatings and biomimetic surfaces to tackle secondary infections, representing the forefront of biomedical surface engineering. The article provides the reader with an overview of the engineering approaches to surface modification of metallic implants, covering both clinical and research perspectives and discussing limitations and future scope.
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The pathogenesis of vascular catheter infections has recently been extensively reviewed. This chapter summarizes existing understandings and presents details of new work published on vascular catheter infections since the recent reviews. Many factors have been shown to affect the risk of catheters becoming infected. These include the unique abilities of certain organisms, Staphylococcus epidermidis, Staphylococcus aureus, and Candida albicans, to cause catheter-related infections. Molecular typing studies increasingly are improving our understanding of the pathogenesis of S. epidermidis/CoNS catheter-related infection. Recent studies with isogenic S. epidermidis mutants increasingly suggest that production of a polysaccharide adhesin is crucial to the pathogenesis of foreign-body infection. This polysaccharide, first named PS/A, was initially described as a virulence factor in association with work examining the pathogenesis of endocarditis. Two additional findings of relevance to the pathogenesis of endocarditis and possibly vascular catheter infections are that binding to platelets facilitates endocarditis and S. aureus strains causing endocarditis are much more likely to be resistant to platelet microbicidal proteins. The pathogenesis of catheter-related thrombosis has been studied in greater depth in recent years. With peripheral catheters, ultrasonographic imaging has shown that early thrombus formation ( 24 h after insertion) occurs near the catheter tip. Recent in vitro studies have shown that surface manipulations of polyurethane can lead to differences in protein and platelet deposition with associated differences in bacterial adherence.
Chapter
The energetics of a polydimethylsiloxane (PDMS) elastomer biointerface were micro-engineered through topographical and chemical modification to elicit controlled cellular responses. The PDMS elastomer surfaces were engineered with micrometer scale pillars and ridges on the surface and variable mechanical properties intended to effect directed cell behavior. The topographical features were created by casting the elastomer against epoxy replicas of micropatterned silicon wafers. Using UV photolithography and a reactive ion etching process, highly controlled and repeatable surface microtextures were produced on these wafers. AFM, SEM and white light interference profilometry (WLIP) confirmed the high fidelity of the pattern transfer process from wafer to elastomer. Ridges and pillars 5 μm wide and 1.5 μm or 5 μm tall separated by valleys at 5 μm, 10 μm, or 20 μm widths were examined. Mechanical properties were modulated by addition of linear and branched nonfunctional trimethylsiloxy terminated silicone oils. The modulus of the siloxane elastomer decreased from 1.43 MPa for the unmodified formulation to as low as 0.81 MPa with additives. The oils had no significant effect on the surface energy of the siloxane elastomer as measured by goniometry. Two main biological systems were studied: spores of the green alga Enteromorpha and porcine vascular endothelial cells (PVECs). The density of Enteromorpha spores that settled increased as the valley width decreased. The surface properties of the elastomer were altered by Argon plasma, radio frequency glow discharge (RFGD) treatment, to increase the hydrophilicity for PVEC culture. The endothelial cells formed a confluent layer on the RFGD treated smooth siloxane surface that was interrupted when micro-topography was introduced.
Article
Bacterial adhesion on stainless steel may cause problems such as microbially induced corrosion or represent a chronic source of microbial contamination. The investigation focussed on how the extent and patterns of four bacterial species comprising three different phyla and a broad variety of physicochemical characteristics was influenced by the surface topography of AISI 304 stainless steel. Five types of surface finish corresponding to roughness values Ra between 0.03 and 0.89 m were produced. Adhesion of all four bacteria was minimal at Ra=0.16 m, whereas smoother and rougher surfaces gave rise to more adhesion. This surface exhibited parallel scratches of 0.7 m, in which a high proportion of bacteria of three of the strains aligned. Reduced overall adhesion was attributed to unfavorable interactions between this surface and bacteria oriented other than parallel to the scratches. Interaction energy calculations and considerations of micro-geometry confirmed this mechanism. Rougher surfaces exhibiting wider scratches allowed a higher fraction of bacteria to adhere in other orientations, whereas the orientation of cells adhered to the smoothest surface was completely random.
Article
Biofilm fouling is one of the major obstacles hindering the use of membranes in water processing systems. There are a series of events that take place during biofilm formation, one of the most interesting and important issues of biofouling is the initial attachment of microorganisms to the surface. Therefore, effects that surface properties have on biofilm fouling are important to attachment and were examined. Hydrophobicity, surface charge and roughness were measured for several polymeric surfaces of interest in water processing membrane systems. These surfaces were then subjected to conditioning layer formation and biofilm fouling, both of which were quantified. The results show that biofilm initiation by a strain of Pseudomonas aeruginosa increases as the surface becomes more rough and more hydrophobic, while fouling is minimal when surface charge is minimized and increases with increasing charge, whether positive or negative.
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Despite extensive studies on settlement of the cypris larva of Balanus amphitrite amphitrite Darwin (Crustacea: Cirripedia), the fine structure of the putative settlement receptors of this species has not been described. This study presents observations made with the scanning electron microscope of the fourth antennular segment and its associated setae.
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This article reviews the mechanisms of bacterial adhesion to biomaterial surfaces and the factors affecting the adhesion. The process of bacterial adhesion includes an initial physicochemical interaction phase (phase one) and a late molecular and cellular interaction phase (phase two), which is a complicated process affected by many factors, including the characteristics of the bacteria themselves, the target material surface, and the environmental factors, such as the presence of serum proteins or bactericidal substances. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res (Appl Biomater) 43: 338–348, 1998
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Integration – supporting multiple application classes with heterogeneous performance requirements – is an emerging trend in networks, file systems, and operating systems. We evaluate two architectural alternatives – partitioned and integrated – for designing next-generation file systems. Whereas a partitioned server employs a separate file system for each application class, an integrated file server multiplexes its resources among all application classes; we evaluate the performance of the two architectures with respect to sharing of disk bandwidth among the application classes. We show that although the problem of sharing disk bandwidth in integrated file systems is conceptually similar to that of sharing network link bandwidth in integrated services networks, the arguments that demonstrate the superiority of integrated services networks over separate networks are not applicable to file systems. Furthermore, we show that: an integrated server outperforms the partitioned server in a large operating region and has slightly worse performance in the remaining region; the capacity of an integrated server is larger than that of the partitioned server; and an integrated server outperforms the partitioned server by a factor of up to 6 in the presence of bursty workloads.
Article
Several reports indicate the emergence of subpopulations resistant to glycopeptides in some clinical isolates of Staphylococcus aureus . While the development of glycopeptide resistance in S. aureus is easily observed in vitro , the in vivo conditions promoting emergence of glycopeptide-resistant subpopulations are unknown. Using a rat model, subcutaneous implants were chronically infected with a methicillin-resistant strain of S. aureus , MRGR3, devoid of a significant (>10–7) glycopeptide-resistant subpopulation at 2 mg/L of either teicoplanin or vancomycin. After 3 weeks of infection in antibiotic-untreated animals, subpopulations emerged, growing on agar containing 10 mg/L of either glycopeptide. These subpopulations were detected in all tissue cage fluids containing >7 log cfu/mL at average frequencies of 4 × 10–5 and 2 × 10–5 on teicoplanin- and vancomycin-containing agar, respectively. While teicoplanin MICs increased two- to 16-fold, vancomycin MICs increased by less than two-fold. Population analysis and survival kinetic studies of three teicoplanin-selected subclones indicated that transfer from solid to liquid medium conditions decreased expression of teicoplanin resistance in the bacterial population. In Mueller–Hinton broth, >90% of cells remained fully resistant to antibiotic, but did not grow in the presence of teicoplanin for an initial period of at least 6 h. All three teicoplanin-resistant subclones expressed stable teicoplanin resistance with slight cross-resistance to vancomycin after a few transfers on teicoplanin-supplemented agar. These data suggest that some in vivo conditions may lead to selection of S. aureus subpopulations exhibiting decreased glycopeptide susceptibility and growing in the presence of otherwise inhibitory concentrations of these antimicrobial agents.