Journal of the mechanical behavior of biomedical materials 65 (2017) 872–880

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Journal of the Mechanical Behavior of Biomedical Materials

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Adhesion force of staphylococcus aureus on various biomaterial surfaces

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Fahad Alam, Kantesh Balani
Biomaterials Processing and Characterization Laboratory, Materials Science and Engineering, Indian Institute of Technology Kanpur, UP, India

A R T I C L E I N F O A BS T RAC T

Keywords: Staphylococcus comprises of more than half of all pathogens in orthopedic implant infections and they can
Bacterial adhesion cause major bone infection which can result in destruction of joint and bone. In the current study, adhesion
Stainless steel force of bacteria on the surface of various biomaterial surfaces is measured using atomic force microscope
Polymer (AFM). Staphylococcus aureus was immobilized on an AFM tipless cantilever as a force probe to measure the
Ceramic
adhesion force between bacteria and biomaterials (viz. ultra-high molecular weight poly ethylene (UHMWPE),
Atomic force microscope
stainless steel (SS), Ti–6Al–4V alloy, hydroxyapatite (HA)). At the contact time of 10 s, UHMWPE shows weak
adhesion force (~4 nN) whereas SS showed strong adhesion force (~15 nN) due to their surface energy and
surface roughness. Bacterial retention and viability experiment (3M™ petrifilm test, agar plate) dictates that
hydroxyapatite shows the lowest vaibility of bacteria, whereas lowest bacterial retention is observed on
UHMWPE surface. Similar results were obtained from live/dead staining test, where HA shows 65% viability,
whereas on UHMWPE, SS and Ti–6Al–4V, the bacterial viability is 78%, 94% and 97%, respectively. Lower
adhesion forces, constrained pull-off distance (of bacterial) and high antibacterial resistance of bioactive-HA
makes it a potential biomaterial for bone-replacement arthroplasty.

1. Introduction 1977), spinning–disc assay (García et al., 1997), centrifugation assay

It is known that Staphylococcus aureus (S. aureus) biofilm plays a
significant role in biomaterial associated infection and can cause the
failure of implants (Bisno, 2000; Lamagni et al., 2015; Zimmerli et al.,
2004). Establishment of infection starts from initial adhesion and leads
to biofilm formation (Bos et al., 1999; Drake et al., 1999; Gottenbos
et al., 2000). A biofilm consists of sessile communities of bacteria that
are protected by a self-produced, hydrated polysaccharide containing
matrix (Busscher and van der Mei, 2012; Nejadnik et al., 2008b). The
biofilm leads to a major physico-chemical hurdle for anti-microbial
therapies due to the reduced diffusion of antibiotics into the polymeric
matrix (Cochis et al., 2015; Dastgheyb et al., 2014; Wu et al., 2013).
Gradual changes in the microenvironment (e.g. oxygen, nutrients, pH)
within the biofilm matrix and the existence of persisted bacterial cells
further complicate the treatment of biofilm (Anwar and Costerton,
1992; Parra-Ruiz et al., 2012). Microbial cell development (Wang et al.,
2009), metabolic activity (Ribeiro et al., 2012), viability (Terada et al.,
2006) and biofilm formation (Nickzad and Déziel, 2014) on the surface
of biomaterials are strongly dependent on the bacterial adhesion (An
and Friedman, 1998). Therefore, it is critically important to study the
bacterial adhesion on different available biomaterials’ surfaces.
Till date there are different techniques in use for the measurement
of the adhesion strength: such as plate-and-wash assay (Klebe et al.,
(Reyes and García, 2003), step pressure technique (Salánki et al.,
2014), flow chamber (Nejadnik et al., 2008a), optical tweezers (Zhang
and Liu, 2008), atomic force microscopy (AFM) (Gilbert et al., 2007)
and scratch based technique i.e. nanoindentation (Lahiri et al., 2011).
Out of these techniques, most of them are non-quantitative and report
only approximation of the bacterial adhesion strength to a surface,
whereas AFM is one of the most effective and quantitative techniques
applicable in wide range of force quantification right from molecular
interaction of cell ligands to the cellular adhesion (Engel et al., 1999;
Müller and Dufrêne, 2011; Thewes et al., 2014; Toyoda et al., 2011).
In the current study, different class of widely used biomaterials {viz.
ultra-high molecular weight poly ethylene (UHMWPE), stainless steel
(SS), Ti–6Al–4V and hydroxyapatite (HA)} (Geetha et al., 2009;
Hryniewicz et al., 2009; Mucalo, 2015; Musib, 2011) are selected and
adhesion force of S. aureus was measured. Further, viability test of S.
aureus was carried out on these samples by using 3M™ petri film test
(Neut et al., 2011), tryptone soya broth (TSB) agar plates and live/dead
staining tests. AFM study (Younes et al., 2012) provides adhesion in a
very short duration (0, 5 and 10 s), 3M™ and TSB test elucidates the
bacterial viability whereas live/dead staining quantifies the number of
adhering bacteria along with the viability. The whole study will help to
select the implant material so that bacterial associated infection can be
reduced.

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Corresponding author.
E-mail address: kbalani@iitk.ac.in (K. Balani).

http://dx.doi.org/10.1016/j.jmbbm.2016.10.009
Received 28 August 2016; Received in revised form 16 October 2016; Accepted 18 October 2016
Available online 20 October 2016
1751-6161/ © 2016 Elsevier Ltd. All rights reserved.
F. Alam, K. Balani Journal of the mechanical behavior of biomedical materials 65 (2017) 872–880

2. Materials and methods

2.1. Materials

Medical grade UHMWPE powder (GUR™ 1020) with a density of

0.93 g/cm3, molecular weight 2.7×106 g/mol was supplied by Ticona
GmbH (WerkRuhrchemie) Germany with particle size of 10–300 μm.
The sheets were processed using compression molding (SCM-30 M/s
Santec Automation, Delhi) at a pressure of 7.5 MPa and 200 °C with a
dwell time of 1 h, followed by cooling of the die to room temperature in
air. Commercially available SS 316L and Ti–6Al–4V were cleaned by
ultra-sonication in the presence of ethanol. HA powders were pro-
cessed by wet chemical method (Mucalo, 2015) and the pellets were
prepared by conventional sintering (1100 °C for 3 h with a heating and
cooling rate of 5 °C/ min) in air. All the prepared samples were
autoclaved to sterilize them before testing.

2.2. Surface energy and surface roughness measurement

Contact angle (CA) of all samples with water were measured by

sessile drop method in static mode using contact angle goniometer
(Dataphysics Contact Angle System OCA). A single drop of water was
placed on the surface and the angle made between the drop and the
material on the either side was captured using the imaging software.
The tangent method was employed to measure the contact angle. Fig. 1. (a) Preparation of AFM probe; (i) add glue to the AFM cantilever by dipping into
Average CA (repeated nine times) for each sample is reported after PLL, (ii) attaching bacteria to the PLL coated AFM cantilever by dipping into the
performing measurements three times on three different coupons of bacterial suspension, (iii) Fluorescent imaging of the AFM probe after performing test
shows to confirm if the cell is still alive or has become dead, (b) typical AFM force-
the sample. The surface free energy of the samples was calculated by
distance curve, (i) Approaching AFM probe towards the sample, (ii) AFM probe is in
performing sessile drop (nine times with each liquid) using one polar contact with surface of the sample and (ii) retracting the AFM probe after providing a
(water) and three non-polar (chloroform, xylene, and toluene) liquids controlled dwell time (0–10 s).
and then the polar and dispersion components were calculated using
Owens–Wendt–Rabel–Kaelble geometric mean equations, whose de- Table 1
tail is provided in supplementary information (SI) equations 1–5 (Bos Water contact angle surface energy, polar and dispersion component of all the samples.
et al., 1999; Yuan and Lee, 2013).
Sample Water contact Surface Polar Dispersion
AFM was used to observe the microstructure of the selected
angle energy (mJ/ part part (mJ/m2)
biomaterials using “V” shaped AFM cantilever (Bruker, NP-10, (degrees)* m2)* (mJ/
Camarillo, CA, USA). Micrographs were taken at three randomly m2)
selected spots of each surface. The surface roughness was analyzed
by Nanoscope Analysis (Bruker, version 1.40) software. Three readings UHMWPE 81.9 ± 2.3 29.86 ± 0.50 3.06 26.80
SS 48.7 ± 1.9 32.43 ± 0.58 13.21 19.22
with a scan area of 50 μm× 50 μm on each sample were taken to obtain Ti–6Al–4V 68.8 ± 5.6 30.13 ± 0.39 8.56 21.56
an average roughness (Ra) of the sample. HA 94.9 ± 1.2 26.15 ± 0.68 1.03 25.12

*
2.3. Bacterial strain and culture condition Confidence level > 95% (p < 0.05).

S. aureus strain (ATCC25923) was grown in 10 ml tryptone soya
broth (TSB, Oxoid, Basingstoke, UK) overnight and transferred in fresh
200 ml TSB and incubated overnight at 37 °C. The bacteria were
harvested by centrifugation for 5 min at 5000 g (five thousand times
the acceleration due to gravity) at 10 °C, washed thrice with phosphate
buffered saline (PBS: 150 mM NaCl. 10 mM potassium phosphate; pH
7.0) and suspended in 10 ml of the same buffer. Number of bacteria
was counted, using a Bürker-Türk counting chamber, and their number
density was maintained at 106 bacteria/ml (for preparing AFM probe)
towards use in adhesion-force measurements.
2.4. Preparation of bacterial AFM probe for adhesion force
measurement
A micromanipulator (Narishige International, Tokyo, Japan) was
used to attach bacteria to a “V” shaped tipless AFM cantilever (Bruker,
NP-O10, Camarillo, CA, USA) under a microscope (Leica, DMIL,
Wetzlar, Germany) (Younes et al., 2012). On a glass slide, a very small
droplet (30 μl) of poly-L-lysine (PLL) solution was placed and the
pointed edge of the cantilever was dipped in the PLL droplet for 2 min.
After 2 min in PLL, the cantilever was air dried for 2 min, then a very
small droplet of bacterial suspension was placed on the glass slide and
the PLL coated cantilever was dipped for 2 min in the bacterial
suspension. To avoid any contamination and drying of the attached
bacteria the AFM probe was kept in a box with wet paper till it was
fitted in AFM. Fig. 1 explains the working protocol of the AFM setup
that was used to measure the adhesion forces. The description of
attachment of bacteria to the tipless AFM cantilever (to prepare AFM
probe) is described in Fig. 1a. The fluorescent image the of bacteria
attached to a cantilever with live/dead staining is shown (Fig. 1a. (iii))
after obtaining force-distance curve (Fig. 1b).
The prepared bacterial AFM probe was used immediately for
adhesion force measurement. All AFM measurements were carried
out at room temperature in sterile PBS (pH 6.0) using an optical lever
microscope (Nanoscope V, Digital Instruments, Woodbury, NY, USA)
with z-scan rates of 1.0 Hz under a maximal loading force of 5 nN. All
cantilevers were calibrated using AFM Tune-it Version 2 software to get
the spring constant of the cantilever. Force–Distance curves were
measured at different contact time (0, 5 and 10 s). All force curves were
analyzed using Nanoscope Analysis (Bruker, version 1.40) software.
For every test a freshly prepared probe was used, and on each sample,
force-distance curves were recorded thrice at three random spots and
averaged. Fig. 1b presents a typical interaction of the AFM tip with
attached bacteria and corresponding force-distance curve.

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F. Alam, K. Balani
Fig. 2. Surface properties of the samples: (a) digital image of the samples (b) AFM deflection images of the samples (c) water contact angle of the samples: (i) UHMWPE, (ii) SS (iii) Ti–
6Al–4V and (iv) HA. The total breadth and width of the AFM image is 50 μm×50 μm.
2.5. Is probe bacteria still alive or dead?
In order to verify whether or not a probe bacterium is alive after the
test, bacterial AFM probe was observed under a fluorescent micro-
scope. Bacterial AFM probe were stained with LIVE/DEAD Baclight
viability kit (i.e. fluorescent strain) 2μl of SYTO 9 (green-fluorescent
nucleic acid strain; 0.25 mM)/propidium iodide (red-fluorescent nu-
clear and chromosome counter strain; 1.5 mM) in 1:1 ratio in 1 ml of
PBS. AFM probe was incubated with this mixed strain for 15 min in
dark at room temperature and then observed under fluorescent
microscope.
2.6. Bacterial viability tests
To check the antibacterial property of various biomaterials, the
3M™ petrifilm (3M™ Microbiology, St. Paul, MN, USA) and TSB agar
plates tests were performed. 3M™ petrifilm is an aerobic count system,
consisting of two films, the bottom film contains standard nutrients, a
water gelling agent, and an indicator dye that facilitates counting of
colony forming units (CFUs). The top film encloses the sample within
the petrifilm system. Before putting the samples in the petrifilm, it was
hydrated with 1 ml of demineralized water for 1 h for the proper
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Journal of the mechanical behavior of biomedical materials 65 (2017) 872–880
gelation so that gel can be transported to top film. After the gel is
formed, the sample were inserted between the films of the petrifilm and
5 μl of 3×104 bacteria/ml suspension that was added on the surface of
samples and incubated for 48 h at 37 °C. Stand-alone 3M™ petrifilm
was considered as the control sample. After incubation, the red
colonies were counted. To check the bacterial viability through 3M™
petrifilm, the experiment was performed thrice and an average value of
CFUs is reported. In TSB agar plate tests, firstly the samples were
incubated at 37 °C with 106 bacteria/ml suspension for 4 h in 6 well
plates. After incubation, the sample coupons were transferred to the
test tubes and ultra-sonicated in the presence of fresh phosphate buffer
saline (PBS) thrice for 10 s at an interval of 30 s to detach the bacteria
attached to the sample surface. The resulting suspension contains the
detached bacteria (both live as well as dead). The number of detached
bacteria (both live as well as dead) were counted using counting
chamber and then suspension was diluted to three concentrations i.e.
10, 102 and 103 bacteria/ml. 100 μl aliquots from three concentrations
was plated on agar plate and incubated overnight and CFUs were
counted. Fresh bacterial suspension (not incubated with any sample)
with same dilutions were plated on the separate agar plate and
considered as the control. The TSB agar test was carried out three
times. All samples were incubated with 106 bacteria/ml suspension for
F. Alam, K. Balani Journal of the mechanical behavior of biomedical materials 65 (2017) 872–880

Fig. 3. Force–distance curves of all the samples; (a) UHMWPE, (b) SS, (c) Ti–6Al–4V and (d) HA at surface delay of 0 s, 5 s and 10 s indicated in the graphs. Bond rupture events in the
force-distance curve of surface delay of 10 s are indicated in circles. The lowest number of rupture event and smaller adhesion force was observed on UHMWPE whereas maximum
number of rupture event with highest adhesion force was observed on SS.

Table 2 3. Results and discussion

Summary of the force-distance curve (at 10 s surface delay) and the surface properties of
biomaterials.
3.1. Surface energy and surface roughness
*
Sample (Ra) in nm Max. adhesion force Pull-off distance
(nN)* (nm)* From the water contact angle (WCA), SS shows the lowest WCA of
48.7°, whereas UHMWPE, Ti–6Al–4V alloy and HA show WCA of
UHMWPE 70 ± 09.2 4.10 ± 0.65 131 ± 04.62
81.9°, 68.8° and 94.9°, respectively. Results of surface energy are
SS 219 ± 11.0 15.21 ± 1.41 425 ± 13.28
Ti–6Al–4V 289 ± 16.7 11.12 ± 1.07 242 ± 06.35
shown in Table 1. It is observed that among all the selected samples, SS
HA 229 ± 10.4 7.66 ± 0.67 221 ± 09.24 and Ti–6Al–4V have the higher surface energy of 32.43 mJ/m2 and
30.13 mN/m, respectively, whereas, UHMWPE and HA have the
*
Confidence level > 95.0% (p < 0.05). surface energy of 29.86 mJ/m2, 26.15 mJ/m2 respectively. The result
of all the CA show that UHMWPE and HA are hydrophobic in nature
4 h and live/ dead staining of bacterial grown on the surface of the and SS and Ti–6Al–4V are hydrophilic. Similarly, the adhesion force of
samples was done. Further the live and dead adhered bacteria was UHMWPE and HA is lower than the ones of SS and Ti–6Al–4V. Here it
counted and compared. is observed that higher the wettability, higher is the adhesion force.
Surface topography was analyzed by AFM in contact mode and it is
observed that UHMWPE was the smoothest among all and have the
lowest Ra (70 nm) whereas SS, Ti–6Al–4V and HA have Ra of 219, 289
and 229 nm, respectively. All surface properties measured are sum-
2.7. Statistical analysis marized and presented in Fig. 2., where column (a) represents the
digital image of samples, column (b) represents the deflection mode
The tests were performed for an average value of three independent image obtained from AFM for all the sample surfaces, and column (c) is
experiments (n=3) in triplicate (for contact angle, adhesion force and the WCA image of all the samples. So, wettability, surface energy and
pull-off distance), and for other experiments (surface roughness), an surface roughness play a strong role in dictating the adhesion of
average of three values is reported for each sample. For the statistical bacteria. Higher the surface roughness and surface energy, higher is the
quantification, the data was represented by the mean value along with adhesion force of bacteria (i.e. on SS and Ti–6Al–4V) and vice versa
the standard deviation. The statistical test was performed using a (i.e. on UHMWPE and HA).
Student's t-test with a confidence level > 95% (p < 0.05) in the
variation of means (surface roughness, contact angle and adhesion
force).

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16 16

14
(a) 14
(b)
12 12
Frequency

Frequency
10 10

8 8

6 6

4 4

2 2

0 0
-4 -3 -2 -1 0 -16 -14 -12 -10 -8 -6 -4 -2 0

Adhesion Force (nN) Adhesion Force (nN)

16 16

14 14
(c) (d)
12 12

Frequency
Frequency

10 10

8 8

6 6

4
4

2
2
0
0 -8 -7 -6 -5 -4 -3 -2 -1 0
-10 -8 -6 -4 -2 0

Adhesion Force (nN) Adhesion Force (nN)

Fig. 4. Histogram showing the distribution of adhesion forces at 10 s surface delay on the surface of samples: (a) UHMWPE, (b) SS, (c) Ti–6Al–4V and (d) HA.

Table 3 mentioned in SI, equations 6–11) of the force distance-curve of the

Number of colony forming units in various bacterial viability test, i.e. 3MTM petrifilm, bacterial adhesion force at a surface delay of 10 s is shown in the
TSB agar plates and adherent cell (live and dead) under florescent microscope. histogram (Fig. 4). In the histogram, the distribution of adhesion force
Sample Number Number Florescent microscopy live/dead test
ranges is plotted with the counts of ranges. It was observed that
of CFUs/ of CFUs/ (bacteria/mm2)* adhesion force curve on UHMWPE contains the highest count of −2 to
disc by disc by −2.5 nN whereas the SS, Ti–6Al–4V and HA contains highest count of
3M™ TSB agar −9 to −10 nN, −6 nN to −7 nN and −4 to −5 nN, respectively. The
petrifilm plate
study of deadhesion test for the quantification of adhesion force of
(5 μl from (100 μl
3×104/ from bacteria on the surface of biomaterials opens a new pathway to design
ml)* 1×103/ and optimize the biomaterial that are antibacterial in nature and less
ml)* prone to infection.
Live Dead Total

Control 120 ± 6 93 ± 3 – – –
3.3. Bacterial viability tests on bare biomaterial surfaces
UHMWPE 80 ± 4 63 ± 5 931 ± 14 (87.1%) 138 ± 27 1069 ± 41
SS 94 ± 5 77 ± 8 3538 ± 32 (93.5%) 246 ± 40 3784 ± 71
Ti–6Al–4V 90 ± 2 87 ± 6 2215 ± 24 (97.6%) 54 ± 16 2269 ± 40 CFUs grown on the surface of the sample in the 3M™ petrifilm are
HA 67 ± 9 37 ± 2 1631 ± 50 (65.4%) 861 ± 40 2492 ± 89 reported in the Table 3 and it is observed that all the samples, except
HA, show similar CFUs counts as that of control (120 ± 9).
*
Confidence level > 99.0% (p < 0.01).
Hydroxyapatite shows lower bacterial count and the number of CFUs
are 67 ± 13. Further, CFUs grown on TSB agar shows almost similar
3.2. Bacterial adhesion forces on biomaterial surfaces trend of bacterial viability as that of the 3M™ petrifilm test. Again, in
the case of hydroxyapatite, the number of CFUs is very less (37 ± 3)
The results of adhesion force of S. aureus on all the selected when compared to that of control (93 ± 4). Fluorescent microscopy
biomaterials are shown in Fig. 3. and summarized in the Table 2. images (Fig. 5) show the adherent bacterial cells as well as the viability
Maximum adhesion force was observed on the surface of SS, whereas of the bacteria on the surface of biomaterial. UHMWPE shows the
on UHMWPE, S. aureus shows the lowest adhesion force. The results lowest number (1069/mm2) of adhered bacteria but SS shows the
of adhesion force curves of S. aureus on all biomaterial surfaces are maximum number (3784/mm2) of adhered bacteria (Table 3). Ti–6Al–
summarized in the Table 2. 4V sample shows the lowest number of killed bacteria, whereas HA
A single force-distance curve is made up of a number of short range showed the maximum number of killed bacteria.
and long range adhesive bonds. The statistical analysis (with details In the current in-vitro study, adhesion force of S. aureus

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F. Alam, K. Balani Journal of the mechanical behavior of biomedical materials 65 (2017) 872–880

Fig. 5. Fluorescent images of S. aureus grown on the surface of samples (a) UHMWPE (b) Stainless steel (c) Ti–6Al–4V and (d) HA after 4 h incubation in TSB media. Green color
showing the live bacteria and red color (in white circles) is showing the dead bacteria/ mm2. (For interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)

19.2

14.4

9.6

4.8
Adhesion force (nN)

0.0
7.7

6.6

5.5

4.4

3.3

2.2

1.1

0.0

50 100 150 200 250 300 350 400 450

Pull-off distance (nm)
Fig. 6. (a) Adhesion force along with the pull-off distance with the variation of time (0 s, 5 s & 10 s), (b) is summary of adhesion force, pull-off distance and the water contact angle for
all the samples (the length of arrows qualitatively indicates the trend of change).

(ATCC225923) was quantified on four different kinds of biomaterials
(UHMWPE, SS, Ti–6Al–4V and HA). It was observed that the adhesion
force of S. aureus on the surface of UHMWPE was lowest (2.4 nN to
4 nN) whereas SS shows the maximum adhesion force among all
selected bare biomaterials ( Fig. 6 and Table 1). As shown in the Fig. 3,
with increase in the contact time (0–10 s), the adhesion force increases
on all the samples, and this change with contact time is maximum in SS
(0.8–16 nN) and minimum in UHMWPE (2.4–4 nN).
Fluorescent imaging was performed to find out the number of
adherent bacterial cell (both live and dead), and the adherent cell count
was very high (3784 bacteria/mm2) in the case of SS. The overall
relation of surface roughness of various substrates on the adhesion

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F. Alam, K. Balani Journal of the mechanical behavior of biomedical materials 65 (2017) 872–880

Fig.7. (a) Relation between the surface roughness, bacterial adhesion force and number of adhered bacteria. (b) Schematic representation of effect of surface roughness on the number
of adherent bacteria as well as the adhesion force. Surface with higher roughness values (stainless steel) has large number of adherent bacteria attached with large adhesion force and
surface with low surface roughness (UHMWPE) has lesser number of adherent bacteria with low force of adhesion.

6
4
2
Adhesion Force (nN)

0
0 100 200 300 400 5000 100 200 300 400 500 0 100 200 300 400 500 0 100 200 300 400 500
-2
Distance (nm) Distance (nm) Distance (nm)
-4 Distance (nm)
-6
-8
-10
-12
-14
-16

Fig. 8. Schematic representation of the bacterial adhesion force measurement by AFM. Pull-off event of AFM bacterial probe from surface of (a) UHMWPE, (b) SS, (c) Ti–6Al–4V and
(d) HA, inset is the contact angle measured on the same surfaces with water. Force-distance curves of bacterial adhesion on all sample after surface e delay of 10 s showing the pull-off
distance along with bond rupture event (e) UHMWPE, (f) SS, (g) Ti–6Al–4V and (h) HA.

force and the number of adhered bacteria/mm2 is shown in the graph
(Fig. 7a). From the results of maximum adhesion force and number of
adhered bacteria/mm2, schematic (Fig. 7b) explains the role of surface
roughness on the adhesion of S. aureus on the biomaterial surface.
Images from the fluorescent microscopy (Fig. 5) further confirm
that UHMWPE has the lowest number of adherent bacterial cell (total
bacteria i.e. both live and dead) after 4 h incubation with TSB media. It
was observed that the rougher (Ra ~225 nm) HA and SS show higher
count of adherent bacterial cells (~2500–3700 bacteria/mm2) when
compared to that of the smoother (Ra ~70 nm) UHMWPE (~1070/
mm2). So, it can be inferred that the surface roughness is one of the
important factors in governing the adhesion forces of the bacteria on
the biomaterial surface (Mei et al., 2011).
Figure 8 schematically presents the relationship between the

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F. Alam, K. Balani
wettability, surface roughness and adhesion-forces. It was observed
that smoother surface of UHMWPE (Ra ~70 nm) may provide only
limited binding sites for ligands, hence, lesser bond-rupture events
(Fig. 8a) were obtained. Whereas, in the case of rougher SS (Ra
~219 nm), enhanced attachment of ligands (Fig. 8b) has elicited higher
number of bond rupture events (i.e. 14 events). A bond rupture event
occurs when a ligand is detached from the adhering surface. The
corresponding bond rupture events are shown with circles (Fig. 8e–h).
It was also observed that the pull-off distance during deadhesion of
bacteria is directly related to the surface free energy of the biomaterial.
In case of SS and Ti–6Al–4V, the high surface free energy (i.e. 32.43
and 30.13 mJ/m2, respectively) show higher pull-off distance (i.e. 425
and 242 nm, respectively). On the contrary, in case of UHMWPE and
HA, lower surface free energies (i.e. 29.86 and 25.15 mJ/m2, respec-
tively) resulted in the lower pull-off distance (i.e. 131 and 221 nm,
respectively).
This study of estimating adhesion force during initial interaction of
S. aureus on few commonly used biomaterials was performed using
AFM. But, it may be pointed out that the current observations may
need to be confirmed using bacterial stains other than gram positive S.
aureus (such as gram negative E. coli). The current set of samples
marginally differ in their surface roughness (Ra~70–290 nm, with
polymer showing Ra of ~70 nm, whereas other samples showing
similar Ra of ~220–290 nm). Thus, further study may be necessitated
to understand the role of roughness on the adhesion strength of
bacteria on biosurfaces. In the current study, adhesion force for very
short contact time is quantified, but the temporal study of adhesion
strength may help identification of planktonic, interaction or lethal
regimes associated with the bacterial growth. Herein, viability test of
bacterial cell were performed using 3M™ petrifilm, TSB agar plates and
also from fluorescence microscopy. Among all the selected biomaterial
samples, only HA shows lower number of CFUs on its surface, when
compared to that of TSB plate. From the fluorescent microscopy, the
number of live and dead cells was counted among the observed
adherent cells and it was found that HA surface had maximum number
of dead bacteria when compared to that of other selected biomaterial
samples. Thus, HA may serve as potential bioactive implant coating
that provides lower deadhesion strength of bacteria when compared to
that of metallic implants.
4. Conclusions
In the summary S. aureus showed maximum adhesion on SS,
however, sample with lower surface energy and lower surface rough-
ness (UHMWPE) showed less adhesion of bacteria. It was observed
that surface energy, roughness and wettability play a vital role in the
adhesion of S. aureus. UHMWPE has lower surface energy (29.86 mN/
m), a smooth surface (72 nm) with high contact angle (81.9) and hence,
less adhesion force (4.1 nN) is observed whereas SS and Ti–6Al–4V
showed higher adhesion forces (15.21 and 11.12 nN respectively). In
case of HA, the roughness is high but the wettability and surface energy
is low, hence, lower adhesion force was observed when compared to
that of SS and Ti–6Al–4V. Bacterial viability, confirmed by 3M™
petrifilm and TSB agar plate on all samples are near or similar to
control, except HA. Lower bacterial adhesion on HA was further
confirmed by fluorescent microscopy. This comprehensive study has
correlated the adhesion forces of bacterial cell with wettability and
surface energy of various biomaterial surfaces, and linked it to their
anti-bacterial properties. UHMWPE and HA elicit minimum bacterial
adhesion force, but the enhanced antibacterial property of HA makes it
a potential bioactive bone replacement material.
Acknowledgements
Fahad Alam thanks “NAMASTE India-EU Mobility Project” for
funding his stay in the Department of Biomedical Engineering,
879
Journal of the mechanical behavior of biomedical materials 65 (2017) 872–880
University Medical Center Groningen, Groningen, The Netherlands.
He also acknowledges all people (especially Henny van der Mei and
Henk J. Busscher) who helped him solving few scientific and technical
problems.
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