See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/337067451

Antibacterial silver (Ag) containing titanium oxynitride (TiOxNy) coatings for

inhibiting surgical site infections (SSI)

Article · November 2019

DOI: 10.1002/mds3.10052

CITATION READS

1 69

4 authors, including:

Prabaha Sikder Ahalapitiya Jayatissa

University of Pittsburgh University of Toledo
19 PUBLICATIONS   112 CITATIONS    153 PUBLICATIONS   2,046 CITATIONS   

SEE PROFILE SEE PROFILE

Sarit B Bhaduri
University of Toledo
193 PUBLICATIONS   4,109 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Antibacterial Coatings on Various Orthopedic Implants View project

Surface Modification View project

All content following this page was uploaded by Prabaha Sikder on 22 December 2019.

The user has requested enhancement of the downloaded file.

 | |
Received: 17 June 2019    Revised: 11 October 2019    Accepted: 15 October 2019

DOI: 10.1002/mds3.10052

RESEARCH ARTICLE

Antibacterial silver (Ag) containing titanium oxynitride (TiOxNy)

coatings for inhibiting surgical site infections (SSI)

Prabaha Sikder | Lufei Liu | Ahalapitiya H. Jayatissa | Sarit B Bhaduri

Department of Mechanical, Industrial and

Manufacturing Engineering, The University
of Toledo, Toledo, Ohio
Correspondence
Prabaha Sikder, Department of Mechanical,
Industrial and Manufacturing Engineering,
The University of Toledo, Toledo, OH.
Emails: james.sikder@gmail.com; Prabaha.
sikder@utoledo.edu
Funding information
Center for Disruptive Musculoskeletal
Innovations (CDMI); NSF
Abstract
The Center of Disease Control and Prevention states that about one in every thirty-
one patients gets infected with ‘hospital-acquired infections’. Bacterial colonization
on surgical instruments remains one of the most significant factors for causing them.
Such infections not only hinder a patient's recovery but can evolve into life-threat-
ening scenarios. Further, the moderate surface hardness of instruments might de-
grade their performance. Considering these issues, the central goal of this paper is
to develop antibacterial coatings with favourable hardness. The main objective was
to take advantage of the hardness property of titanium oxynitride (TiOxNy) while
combining silver's (Ag) antibacterial properties. As surgical instruments are mainly
fabricated using Ti6Al4V, we chose this material as our substrate. Radiofrequency
magnetron sputtering was employed to develop several multi-phasic coatings. Phase
compositions, morphology and hardness values were analysed by X-ray diffraction,
scanning electron microscopy and nanoindentation, respectively. The antibacterial
efficacy was evaluated against Escherichia coli using zone of inhibition, plate counting
assay and Live/Dead assay. The effect of repetitive sterilization of coated samples
was also examined. Results indicated the successful formation of antibacterial coat-
ings with favourable hardness. Ag-containing coatings exhibited pronounced anti-
bacterial effect by ‘contact killing’ method with no functionality change even after
five sterilizing cycles.

KEYWORDS

antibacterial, coatings, surgical site infections, titanium oxynitride

1 |  I NTRO D U C TI O N susceptibility rate to contamination in a hospital environment, these

Titanium (Ti) and its related alloys (Ti6Al4V) have been widely
used in the industry to fabricate surgical instruments. Reasons like
high specific strength, high corrosion and wear resistance, chemi-
cal stability and non-toxic nature contribute in making them the
material-of-choice (Geetha, Singh, Asokamani, and Gogia (2009);
Koju, Sikder, Ren, Zhou, & Bhaduri, 2017). However, pristine sur-
faces of instruments possess a high chance of bacterial colonization
(Dancer, Stewart, Coulombe, Gregori, & Virdi, 2012). Given the high
infected surgical apparatuses can cause surgical site infections (SSI).
These infections not only interrupt with the proper working of im-
plants or the patient's recovery but can evolve into lethal conditions
(Anthony et al., 2018). In a recent article published by the American
College of Surgeons and Surgical Infection Society, it was reported
that SSI account for 20% of all hospital-acquired infections (HAI).
With an estimated annual SSI incidence of 160,000 to 300,000 in
the United States alone, they result in a 2- to 11-fold increase in
mortality risks. Further, to treat SSI by either antibiotics or implant

Med Devices Sens. 2019;00:e10052. wileyonlinelibrary.com/journal/mds3 |

© 2019 Wiley Periodicals, Inc.     1 of 12
https://doi.org/10.1002/mds3.10052
 |
2 of 12       SIKDER et al.
removal via revision surgeries, the annual cost might go up to ap-
proximately $3.5 to $10 billion (Ban et al., 2017; Sikder, Grice, Lin,
Goel, & Bhaduri, 2018).
Furthermore, it has been reported that even post-sterilization,
contamination from surgical instruments have resulted in the in-
crease of deep SSI in orthopaedic and ophthalmic surgeries (Dancer
et al., 2012). Despite strict aseptic surgical protocols and periopera-
tive measures, it is indeed a challenge to ensure sterile maintenance
of surgical instruments. To treat endemic infections, systemic an-
tibiotic administration has been used conventionally over decades
(Prokuski, 2008). However, an increasing rate of antibiotic-resistant
bacterial mutation compelled researchers to devise alternative strat-
egies (Li & Webster, 2018). One such strategy is coating the instru-
ment surfaces and implants with antimicrobial agents. It has been
proven significantly effective to reduce the incidence of SSI (Ferraris
and Spriano, (2016); Zhao, Chu, Zhang, & Wu, 2009). Specifically,
antibacterial properties of metallic elements and their compounds
such as silver (Ag) (Sikder, Koju, et al., 2018), copper (Cu)/ copper
oxide (CuO) (Das, Nath, Phukon, & Dolui, 2013) and (Zn)/ zinc oxide
(ZnO) (Sirelkhatim et al., 2015) are well accepted in the literature.
Among them, Ag is outstanding because of the following reasons:
(a) it exhibits antibacterial activity against a broad-spectrum of mi-
crobes including antibiotic-resistant bacterial strains, (b) it is difficult
to mutate Ag-resistant bacterial strains, (c) it is stable in a physio-
logical medium, and (d) it has no toxicity against human cells up to a
critical concentration (Abdelghany et al., 2018; Sikder, Grice, et al.,
2018; Sikder, Koju, et al., 2018; Sim, Barnard, Blaskovich, & Ziora,
2018). Ag has been exploited as an antibacterial agent on orthopae-
dic and dental implants in several notable cases (Croes et al., 2018;
Kelly et al., 2009; Sikder, Bhaduri, Ong, & Guda, 2019; Sikder, Grice,
& Bhaduri, 2019). The antibacterial working of Ag is complex, and
various mechanisms have been proposed in the literature over the
course of time (Jung et al., (2008)). Thus, considering Ag's versatile
antibacterial activity, we choose this noble metal as the antibacterial
agent in the present study.
A second aspect of this paper is to address the surface hardness
improvement of surgical instruments as they perform a variety of
machining processes within the body. A low value of surface hard-
ness might cause galling on the surfaces, blunting the instruments
edges, and even reduce the working life of the instruments. To over-
come this, starting from the 1980s and further on, the focus has
been on fabricating titanium nitride (TiN) coatings (Hove, Sierevelt,
Royen, & Nolte, 2015). For the last two decades, there has been a
significant rise in applying titanium oxynitride (TiOxNy) coatings on
biomedical devices (dental, orthopaedic or cardiovascular implants)
(Beshchasna et al., 2019; Karjalainen, Ylitalo, & Airaksinen, 2006;
Subramanian, Muraleedharan, Ananthakumar, & Jayachandran,
2011). The coating conditions of TiOxNy, as opposed to the well-
known TiN, are less stringent since the presence of oxygen is toler-
ated. Hardness value, however, is not compromised. Hence, from an
industrial perspective, TiOxNy is preferable. For instance, Koerner et
al. developed TiOxNy coatings with different resistivities on stainless
steel (Koerner, Butterworth, Mayer, Dasbach, & Busscher, 2002).
Banakh et al. optimized the O/N ratio so that the TiOxNy coatings
are best suitable for dental implantology (Banakh et al., 2014).
Recently, the in vitro dissolution rate of TiOxNy coatings was also
explored (Pichugin et al., 2016). It is worth mentioning that in some
instances, TiOxNy coatings outperformed TiN coatings. For instance,
in a comparative study by Subramanian et al., the authors showed
that TiON possesses higher corrosion resistance properties over TiN
(Subramanian et al., 2011). However, we believe the primary interest
in transition metal oxynitrides is because of the combining proper-
ties of nitrides and oxides just like TiOxNy combines the excellent
mechanical properties of TiN and the favourable biocompatibility of
titanium dioxide (TiO2) (Piscanec et al., 2004).
Given the successful applications of TiOxNy coatings on medi-
cal devices and the well-known antibacterial activity of Ag, the pri-
mary goal of this paper is to fabricate and characterize antibacterial
TiOxNy-Ag coatings on Ti6Al4V prototypes. Such coatings are de-
veloped with the intent of reducing or inhibiting bacterial contami-
nation for surgical instruments. The idea is to take advantage of the
favourable mechanical and antibacterial properties of TiOxNy and
Ag, respectively. To the best of our knowledge, this the first report
of combing Ag with TiOxNy taking advantage of the coating's indus-
trial importance. We note that there are several studies that report
the development of Ag-containing coatings by employing physical
vapour deposition (PVD) (Chen et al., 2006; Kelly et al., 2009), chem-
ical vapour deposition (CVD) (Xiao et al., 2007), micro-arc deposition
(Zhang, Gao, & Han, 2016) or anodization (Kelly et al., 2010). Among
these methods, magnetron sputtering is suitable for both laboratory,
and industrial use and the developed coatings exhibit good adhesion
strength to the substrate (Chawla, Jayaganthan, & Chandra, 2008).
Additionally, reactive sputtering is a well-established technique
where various kinds of gasses (like N2, O2) are introduced to react
with the sputtered phase. However, none of the above references
took the advantage of the TiOxNy-Ag coatings.
In the present study, TiOxNy coatings with varying Ag contents
were deposited by radiofrequency (RF) magnetron sputtering on
Ti6Al4V substrates. We will refer the substrates as Ti from now on.
The phase compositions and morphological studies of the coatings
were performed by X-ray diffraction (XRD) and scanning electron
microscopy (SEM), respectively. Nanoindentation was employed to
measure the hardness of the coated films. The antibacterial prop-
erties of coatings were evaluated by zone of inhibition (ZOI), plate
counting assay and Live/Dead cell assays against Escherichia coli
(E. coli). Coated instruments were subjected to several autoclave cy-
cles to simulate repeated sterilizing cycles and the change in morphol-
ogy and antibacterial functionality of the coatings were examined.
2 | E X PE R I M E NTA L
2.1 | Material preparation
Ti specimens with dimensions 10 mm × 10 mm × 1 mm were sec-
tioned from a bigger block. Both sides of the specimens were
SIKDER et al. |
      3 of 12

polished using silicon carbide (SiC) abrasive papers grid 240, 400 and 2.4 | Evaluation of antibacterial properties
600 in a sequential manner. Afterwards, they were cleaned ultra-
sonically with 70% ethanol and deionized (DI) water for 6 min each, 2.4.1 | ZOI method
dried and stored at room temperature.
The antibacterial properties of coatings were initially evaluated by
traditional ZOI method with an objective to determine the diffusion
2.2 | Coating preparation of Ag+ ions. E. coli (Strain: W3110, ATCC 39936) bacterial suspension
was diluted by super broth (SB) and adjusted to an optical density
The deposition chamber of the RF magnetron sputtering set-up was (OD) of 0.06 at 600  nm. Then, 100  µl diluted bacteria suspension
−6
pumped down to a base pressure of 1 × 10 millitorr by sequentially was added into the SB culture plates and distributed uniformly using
using a mechanical and molecular pump. Argon (Ar, sputtering me- circular beads. TiON-0Ag, TiON-3Ag, TiON-5Ag specimens and as-
dium) and nitrogen (N2, reactive gas) were fed into the chamber by polished Ti were placed into individual SB agar plates in a way so that
different mass flow controllers at the rate of 100 ccm, resulting in a the coated sides were in direct contact with the agar surface. The
total working pressure of 35 millitorrs. RF was set at 13.56 MHz, and plates were then put in an incubator overnight at 37°C. The effec-
the sputtering power was controlled at 250 W. Strips of Ag (≥99.99% tiveness of the antibacterial functionality of the coatings was evalu-
purity) plates were arranged on a Ø 6 inches Ti target while main- ated by the formation of ZOI.
taining a target–substrate distance of 2 inches. Further, to vary the
Ag content in coatings, Ag strips representing different surface
areas were chosen. 5 and 9 cm2 Ag plates were fixed on the Ti tar- 2.4.2 | Plate counting method
get. The specimens were referred to as TiON-3Ag and TiON-5Ag,
respectively. Coated specimens with no Ag (TiON-0Ag) served as In this assay, two different bacterial strains were used—E. coli
the control. and Staphylococcus aureus (S. aureus, Strain: Seattle 1945, ATCC
25923). In order to analyse the bactericidal that is bacteria-killing
property of coated Ti samples, the plate counting method was em-
2.3 | Coating characterization ployed wherein the colony-forming units (CFUs) were accounted.
Specimens were placed in a 24-well plate and bacterial suspensions
The phase compositions of the coated samples were analysed (107 cfus/ml) were pipetted onto their surface. The specimens were
using XRD (Ultima III, Rigaku) with parallel beam mode over the kept in an incubator at 37°C for 24 hr without any agitation. At
2θ range of 15–70°. Surface morphologies of coatings were ana- the end of the time period, the bacterial suspensions were seri-
lysed using SEM (S4800, Hitachi) equipped with an energy disper- ally diluted and plated out. After 24 hr, CFUs were counted and
sive X-ray spectroscopy (EDS, INCA, Oxford). Elemental analyses compared.
were performed at 20 kV with a 15-mm working distance. Both
secondary and back-scattered electron detectors were used to
image. Hardness test for the coatings was carried out by nanoin-
denter (Hysitron Ti-950 TriboIndenter; Bruker) equipped with a
standard transducer and three-sided pyramidal Berkovich stand-
ard probe (included angle: 142.3°, radius of curvature: ~150 nm),
which is suitable for coatings thicker than 100 nm. The load was
set at 3,000 µN and with a holding time of 10 s. Data were cal-
culated considering 20% lower part to 95% upper part of the un-
loading curves using the TriboScan software. The various coated
Ti coupons were immersed in DI water for 7 days. At every 24-hr
time point, the DI water containing the specimens were collected
and diluted for analysis with inductive coupled plasma-mass spec-
troscopy (ICP-MS, Xseries 2; Thermo Scientific). The ICP-MS
samples were diluted 200 times, and pure nitric acid was added
to the solution to make it in 3.5% nitric acid solution. For quan-
titative analysis, standards and internal standards were prepared
by using the certified ICP-MS standards from Inorganic Ventures.
Atomic force microscopy (AFM, Nanoscope V, Veeco Instruments)
was performed in the tapping mode using a 1Ω Si probe tip over a F I G U R E 1   XRD patterns of magnetron-sputtered (i) TiON-0Ag,
5 µm × 5 µm area. (ii) TiON-3Ag and (iii) TiON-5Ag coatings on Ti substrates
 |
4 of 12       SIKDER et al.

(a)

(b) (c)

F I G U R E 2   (a) SEM micrographs and EDS results of TiON coatings with varying Ag content. Images are captured using the secondary
electron (SE) detector and the back-scattered electron (BSE) detector. (b) Hardness values of various specimens as obtained by
nanoindentation technique. (c) Inductive coupled plasma—mass spectroscopy (ICP-MS) analysis of the coatings. *means statistically
significant with respect to bare Ti6Al4V control (ρ < 0.05 were considered significant)

2.4.3 | Live/Dead cell assay
The bacterial anti-adhesive nature of coatings was evaluated by
the Live/Dead cell assay. E. coli suspensions were diluted and ad-
justed to an OD of 0.03 at 600  nm. All specimens were placed in
individual wells of a 24-well culture plate. 1 ml of E. coli suspension
was added to each sample well, and then, placed in an incubator for
24 hr at 37°C. After the specific time frame, the culture medium was
removed, and samples were washed by phosphate-buffered saline
(PBS) three times to remove any non-adherent bacteria. Bacterial vi-
ability kit L7012 (LIVE/DEAD BacLight, Invitrogen Molecular Probes)
was employed as the staining solution. For the purpose of bacterial
SIKDER et al. |
      5 of 12

(a)

(b)

F I G U R E 3   ZOI assay results of Ti, TiON-0Ag, TiON-3Ag and

TiON-5Ag coatings incubated for 24h on E. coli cultured agar
surface

staining, 3 µl of each dye—Syto9 and propidium iodide (PI)—was

added into 1 ml of PBS and vortexed. After proper mixing, 250 µl
of the staining solution was added to each sample and placed in the (c)
dark for 15 min at room temperature (Berney, Hammes, Bosshard,
Weilenmann, & Egli, 2007). Following the staining process, 1 ml of
10% formalin solution was added to fix the bacterial cells. Finally,
the samples were immersed in PBS to remove excess formalin, dried
in air at room temperature and observed under cell imaging multi-
mode reader (Cytation 5, BioTek).

2.5 | Evaluation of the impact of autoclaving cycles

on antibacterial properties

For evaluating the impact of repeated sterilization on coatings, we

used gravity cycle in the autoclaving process. Specimens were first
cleaned ultrasonically with 70% ethanol and DI water for 10 min each,
dried at room temperature and placed in glass beakers. The set-up was
then transferred into an autoclave where they were exposed to the
hydrothermal cycle which maintained 121°C and 100 kPa for 15 min.
The samples underwent either one or five repetitive cycles. After spe-
cific cycle(s), the specimens were investigated under SEM to evaluate
the impact of autoclaving on the coating morphology. The antibacte-
rial functionality was also examined by plate counting method (Section
2.4.2) and Live/Dead cell assay (Section 2.4.3) post-sterilization.
2.6 | Cytocompatibility tests
Model osteoblastic cell line–mouse calvarial pre-osteoblasts des-
ignated as MC3T3-E1 (ATCC CRL-2593) were used to check the
cytocompatibility of the coated specimens. Cells were cultured in
F I G U R E 4   (a) Photograph of the coated side of the specimen
in contact with the bacterial agar surface. Red arrows indicate
the presence of abundant bacterial colonies on uncoated Ti block
and TiON-0Ag surface in contrast to no bacterial colonies or
contamination on TiON-3Ag. (b) Number of adherent colonies on
specimen surface after ZOI test. (c) Number of colony-forming units
(CFUs) formed in case of various specimens. *means statistically
significant with respect to bare Ti control (ρ < 0.05 were considered
significant)
minimum essential medium alpha (MEM-α; Thermo Scientific), sup-
plemented with 10% foetal bovine serum (FBS; Thermo Scientific
HyClone) and 1% penicillin/streptomycin (0.2 g/ml), also known as
complete MEM-α, at 5% CO2 and 37°C.
Specimens were placed in 24-well plates and seeded with
approximately 4 x 10 4 cells and incubated at 37°C with 5% CO2
flow for 1, 4 or 7 days. Complete medium was replenished every
 |
6 of 12       SIKDER et al.
(a) (b)
(c)
third day. At specific time points, well plates were retrieved from
the incubator and the specimens were treated with thiazolyl blue
tetrazolium bromide (MTT; Sigma-Aldrich) and incubated for
4h. Subsequently, dimethyl sulfoxide (DMSO) was used to dis-
solve the formazan followed by a short incubation for 10 min.
Eventually, OD540 readings were recorded by spectrophotometer
and compared.
2.7 | Statistical analysis
All test results represented means ± standard deviation in triplicates.
One-way analysis of variance with the Tukey test was conducted
to determine the statistical difference between groups and ρ < .05
were considered significant.
3 |  R E S U LT S & D I S CU S S I O N
3.1 | Physical characterization
The XRD spectra of various specimens are presented in Figure 1.
The highest intensity peaks correspond to Ti, which is from the sub-
strate. Majority of the diffraction peaks from the coatings corre-
lates with the TiO 4N (PDF# 97-017-3420) phase. The well-defined
nature of diffraction peaks indicates the crystalline nature of the
coatings. No additional peaks are observed in the case of TiON-3Ag
apart from titanium dioxide. However, TiON-5Ag exhibits diffrac-
tion peaks related to elemental Ag (PDF# 98-000-0398). This result
is due to the low Ag content in the former which might be lesser
than the detection limit of the XRD machine. Importantly, the for-
mation of broadened Ag diffraction peaks indicates the presence of
nanocrystalline particles on the crystalline parent phase of the coat-
ings (Sikder, Koju, et al., 2018). The nanocrystalline or amorphous
F I G U R E 5   (a) Nanorough surface of
polished Ti and (b) nanosmooth surface
of TiON coated Ti. (c) Atomic force
microscopy (AFM) analysis of bare Ti
surfaces and TiON coated Ti. Of note are
the horizontal lines representing grooves
on the Ti surface as opposed to smooth
surface of the TiON coatings
nature of the particles might also be another reason which makes it
difficult for detection of Ag in case of TiON-3Ag.
The high-magnification SEM images of TiON-0Ag, TiON-3Ag and
TiON-5Ag are shown in Figure 2(a). The images on the left are cap-
tured using the second electron (SE) detector, while the ones on the
right are captured via back-scattered electron (BSE) detector from
the same scanning location of the coating. The micrographs display
the compact nature of coatings with well-defined grain boundaries
of the particles. The results are in accordance with the XRD peaks
which also indicate the crystalline nature of coatings. In the case of
Ag-sputtered specimens, nanoparticles with an average diameter of
approximately 30 nm can be seen distributed throughout the coating
surface. Interestingly, most of them are located on grain boundaries
of the coating's main phase. de los Arcos et al. (2002). observed the
same trend when TiN–Ag nanocomposite films were co-sputtered
employing three targets (de los Arcos et al., 2002). With an increase
in the area ratio of Ag in the sputtering target, the concentration
of nanoparticles also increases. This result can be verified by BSE
SEM images. The widely distributed nanoparticles appear brighter
than the parent phase of the coating composition due to the detec-
tion of back-scattered electrons. Finally, EDS analyses reveal that
the dispersed nanoparticles all over coatings are Ag. It also picks up
Ti, N and O elements confirming the formation of either TiO 4N or
TiO 4N-Ag composite coatings.
Figure 2(b) shows the nanoindentation results of various speci-
mens. As the coating thickness is less than a micron, nanoindenta-
tion method helps in determining the hardness of the coating itself
without the influence of substrate. The TiON or TiON-Ag coatings
in the present case exhibit hardness in the range of 7–7.5 GPa, an en-
hancement of 113% as compared to bare Ti. The presence of Ag and
its varying concentration does not have any influence on the nano-
hardness of the coatings. However, the value is much lesser than
pure TiN films or even TiOXNy with higher content of nitride in it. Vaz
et al. (2004) studied the hardness of TiOXNy coatings as a function
SIKDER et al. |
      7 of 12

(a)

(b)

F I G U R E 6   (a) Fluorescence microscopy images of adhered Live/Dead E. coli. From left to right: images are taken under the GFP filter,
Texas red filter, and the overlapped images. (b) Quantification data of adhered Live/Dead E. coli. *means statistically significant with respect
to bare Ti control (ρ < 0.05 were considered significant)
 |
8 of 12       SIKDER et al.
of oxygen content and reported a significant reduction in hardness
with an increase of the oxygen content (Vaz et al., 2004). This re-
sult is in accordance with the present study where a higher oxygen
content in the coatings is the prime reason for a low hardness value.
Figure 2(c) shows the ICP-MS results of the coatings. This test
+
was done to analyse the extent of Ag ion release from the coatings.
The analysis shows very less amount of Ag+ ion release from both
the coatings—TiON-3Ag and TiON-5Ag. The maximum amount in
either case was less than 0.1 ppm. Previous studies have reported
+
that at least 0.1 ppm of Ag ions is required to witness antibacte-
rial effect, especially when Ag+ ions are responsible for the effect
(Sikder, Bhaduri, et al., 2019; Sikder, Grice, et al., 2019, 2018; Sikder,
Koju, et al., 2018).
3.2 | Antibacterial properties
The ZOI results for all the specimens are shown in Figure 3. No ZOI
was formed around Ti, TiON-0Ag coating, TiON-3Ag and TiON-5Ag
coated specimens. Similar results were observed by Kelly et al. and
Ahearn et al. where no ZOI was formed around the specimen coat-
ing which contained Ag (Ahearn, May, & Gabriel, 1995; Kelly et al.,
2009). Several studies have proven the fact that elementary Ag is
chemically stable with a low dissolution rate. However, the high spe-
cific area of Ag nanoparticles provides a possibility of Ag+ ion release
in an aqueous environment (Ferraris & Spriano, 2016).
Even the concentration of nanoparticles plays a significant role
in influencing the dissolution kinetics of Ag+ ions from the coatings
(Sikder, Grice, et al., 2019, 2018; Sikder, Koju, et al., 2018). However,
even though TiON-5Ag possess the highest concentration of Ag
nanoparticles distributed all over the coating (in this study), they
were unable to exhibit ZOI. Further, the trace amount of free Ag+
ions can only exist around the Ag nanoparticles and do not possess
the ability to be released to a longer distance due to its chemically
stable nature. Thus, even though there might be a release of Ag+
ions from the coatings, it is not enough to create a ZOI around itself.
However, the release was sufficient to not allow the attachment of
any bacterial colonies on the surface as shown in Figure 4(a-b). No
bacteria are seen on the surface of TiON-5Ag which confirms that
the Ag+ release was enough to inhibit any E. coli attachment on the
coating. In contrast, red arrows on bare Ti and TiON-0Ag show the
presence of abundant bacterial colonies indicating heavy contam-
ination (Arcos et al., 2002). Furthermore, these results are corrob-
orated by the results of the CFU assay. This assay helped to obtain
a better analysis of the antibacterial or specifically the bactericidal
effect of the coatings. As shown in Figure 4(c), TiON-5Ag showed
the highest reduction in bacterial colonies indicating the successful
contact-killing property of the coating. The reduction is more than
F I G U R E 7   (a) SEM images of TiON-0Ag, TiON-3Ag, and TiON-5Ag coatings post autoclave treatment. Pictures on the left indicate
results after one autoclaving cycle while images on the right display results after five autoclaving cycles. (b) Number of colony-forming
units (CFUs) formed in case of various specimens after 5 autoclaving cycles. (c) Live/Dead cell assay results of TiON-5Ag coating after five
autoclaving cycles. From left to right: images are taken under the GFP filter, Texas red filter, and the overlapped images
2Log kill. However, the reduction in CFUs in case of TiON-3Ag is not
statistically significant with respect to the control.
The results of Live/Dead cell assay as analysed with Cytation 5
are presented in Figure 5(a-b). In the fluorescence microscopy im-
ages, the green dots indicate live bacteria while the red dots indicate
dead bacteria. It should be noted that even though all the specimens
were incubated with the same bacterial concentration; however,
the amount of total adhered bacteria on polished Ti is quite lesser
than the TiON-coated ones. Seil and Webster (2012) reviewed the
effectiveness of nanotechnology on antimicrobial applications and
illustrated that surface roughness plays an essential role in bacterial
attachment (Seil & Webster, 2012). Since the Ti blocks were only
polished by abrasive paper, the cutting action of polish could have
created a nanorough surface with sharp nanosized edges on the sur-
face. Therefore, when E. coli tried to adhere to the nanorough sur-
face, a good number might have been destroyed by the sharp edges
or simply got washed away due to the lack of adhesion. Referring
to the SEM and AFM images of the coated and uncoated samples
(Figure 5(a-b)), the coatings created a considerably smoother surface
with spherical nanosized edges, hence, providing a favourable area
for bacteria adhesion.
More importantly, it is evident from the fluorescence microscopy
images that the concentration of dead bacteria (red dots) increases
with an increase in Ag concentration (Figure 6a). The quantification of
the microscopy images confirms the presence of significantly higher
number of dead bacteria on Ag-containing coatings (Figure 6b).
Thus, both TiON-3Ag and TiON-5Ag exhibit good antibacterial and
anti-adhesive properties. In the overlapped images, almost all the
attached bacteria on the TiON-3Ag and TiON-5Ag coatings show
yellow or red. These represent bacteria with either compromised cell
membranes or dead bacteria. Moreover, the bacteria attached on
TiON-5Ag coating are assumed to be more severely damaged, since
more PI stain penetrated the bacteria cell membranes and took the
place of Syto9, thus reducing the green fluorescence and showing
more red fluorescence (Stiefel, Schmidt-Emrich, Maniura-Weber, &
Ren, 2015).
Nucleic acid stain Syto9 generally labels all the bacteria both
with intact and compromised membranes and shows green fluores-
cence, whereas stain PI only labels the bacteria with compromised
membranes and shows red fluorescence. When both stains are pres-
ent, PI takes the place of Syto9 and reduces the green fluorescence.
Therefore, under an ideal condition, bacteria with red fluorescence
are treated as dead bacteria, and bacteria with green fluorescence
are treated as live bacteria. However, PI may not always take the
place of Syto9 entirely in dead bacteria due to the unfavourable
condition of the bacterial membrane or staining time, hence dead
bacteria may also show green fluorescence (Stiefel et al., 2015). Also,
such assay does not help in analysing the actual antibacterial effect
SIKDER et al. |
      9 of 12

(a)

(b)

(c)
 |
10 of 12       SIKDER et al.
F I G U R E 8   MTT assay results of various specimens with MC3T3
pre-osteoblast cells
at a cellular level that is how Ag have affected the morphology of
the bacteria (Sikder, Bhaduri, et al., 2019; Sikder, Grice, et al., 2019).
This assay determines the ‘contact killing' ability of the coatings.
Contact killing is an efficient way to prevent the biofilm formation or
any contamination on the surgical instruments as the antibacterial
surface does not allow the adhesion of bacteria. Even if it does, the
anti-adhesive antibacterial coating kills them once the bacteria come
in contact with the bactericidal agent, in this case, Ag nanoparticles
(Li, Lee, Sheng, Cohen, & Rubner, 2006). Overall, it is very important
that the antibacterial coatings suffice certain regulatory aspects in
the orthopaedic industry (Sikder & Bhaduri, 2019).
3.3 | Impact of autoclaving cycles on
antibacterial properties
Simulating the effect of sterilization on coatings and studying their
effect is an essential aspect of this study. Figure 7(a) shows SEM im-
ages of coatings post-autoclaving. The images on the left display the
effect after one autoclaving cycle while ones on the right show the
effect of five autoclaving cycles. No obvious morphological change
can be noticed in the case of the TiN-0Ag coatings. However, in the
case of Ag-containing TiON coatings, Ag nanoparticles appear more
crystallized and brighter after one autoclaving cycle. The effect is
more vivid after five cycles.
This result is due to the aggregation effect which increases the
particle size. In this case, the hydrothermal treatment increased the
spherical Ag nanoparticles considerably which made it look brighter
under the SEM (Sommerfeld, Schroeder, & Sabel, 1998). Memisoglu-
Bilensoy and Hincal made the same observations when they exposed
β-cyclodextrin nanoparticles to an autoclave treatment (Memisoglu-
Bilensoy & Hincal, 2006). In some cases, autoclaving might not
be favourable as it might alter the original nanoparticle charac-
teristics, especially those in a suspension (Memisoglu-Bilensoy &
Hincal, 2006; Sommerfeld et al., 1998). However, in the present
case, there were no chemical modifications. Gamma irradiation can
be an effective alternative in case of nanoparticles which are de-
signed for entrapping drugs and their release. The particle size is
not affected by radiation, though a change in zeta potential can be
recorded (Memisoglu-Bilensoy & Hincal, 2006). The EDS results of
all the autoclave-treated samples remain unaltered with no signifi-
cant change in composition. After five repetitive autoclave cycles,
the TiON-5Ag-coated specimens were again tested on grounds of
antibacterial functionality. The results are shown in Figure 7(b-c). As
seen in Figure 7b, no change in bactericidal effect of the coatings
was observed when compared to pre-sterilization results (as shown
in Figure 4c). The extent of Logkill or antibacterial functionality was
same. Furthermore, in corroboration with the CFU results, there
was very few or almost no live bacteria (green dots) on TiON-5Ag
specimens. Instead, good concentration of dead bacteria (red dots)
was present on the same surface. Thus, it can be concluded that the
coatings exhibit the same level of antibacterial efficacy even after
the exposure to repeated sterilization.
3.4 | Cytocompatibility
Preliminary cytocompatibility test is indeed important to evaluate
the biological responses of the biomaterials prior to in vivo assess-
ment (Ren, Sikder, Lin, & Bhaduri, 2018). Especially, when certain
concentrations of Ag have been proven cytotoxic in pre-osteoblast
cells (Sikder, Bhaduri, et al., 2019). Figure 8 shows the MTT assay
results of the various coatings. The cell viability measured by MTT
assay presents significant increase (p < .05) increase of all the coated
samples with respect to bare Ti control. This might be due to the
presence of TiO2 in the oxynitride coatings. This was one of the
primary reasons for choosing titanium oxynitride coating as it com-
bines of nitrides and oxides. The titanium nitride provide excellent
mechanical properties and the titanium oxide provide favourable
biocompatibility (Piscanec et al., 2004). Overall, the titanium oxyni-
tride coatings developed in the present study possess favourable
antibacterial functionality and are also cytocompatible in nature;
thus, highlighting their potential applications in the arena of surgical
instrument fabrication.
4 | CO N C LU S I O N S
In this work, TiOxNy coatings with varying Ag contents were suc-
cessfully developed on Ti by RF magnetron sputtering method. The
fabricated coatings exhibited dominant crystalline phases of TiO 4N
with elemental Ag only present in sputtered coatings with a higher
Ag area ratio. The microstructural studies revealed a dense and com-
pact arrangement of the parent phase and wide distribution of Ag
nanoparticles on top of the coatings. The enhancement in hardness
of 113% was recorded as compared to bare Ti. However, it was not
comparable to other hard coatings present in the literature because
of the presence of oxygen. ZOI and Live/Dead cell assay indicated
that both TiON-3Ag and TiON-5Ag coatings possess effective
SIKDER et al. |
      11 of 12

antibacterial properties. However, the method of antibacterial func- Dancer, S. J., Stewart, M., Coulombe, C., Gregori, A., & Virdi, M. (2012).
+ Surgical site infections linked to contaminated surgical instru-
tionality is mainly ‘contact killing’ and not Ag ionic diffusion in the
ments. Journal of Hospital Infection, 81(4), 231–238. https​ ://doi.
case of TiON-3Ag, whereas coatings with higher Ag concentration— org/10.1016/j.jhin.2012.04.023
TiON-5Ag promoted diffusion of Ag+ ions on the agar surface along Das, D., Nath, B. C., Phukon, P., & Dolui, S. K. (2013). Synthesis and eval-
with noticeable anti-adhesive effect. The repetitive autoclaving cy- uation of antioxidant and antibacterial behavior of CuO nanoparti-
cles did not affect the antibacterial properties of the coatings, but cles. Colloids and Surfaces B: Biointerfaces, 101, 430–433. https​://doi.
org/10.1016/j.colsu​r fb.2012.07.002
an aggregation effect significantly increased the size of the Ag na-
de los Arcos, T., Oelhafen, P., Aebi, U., Hefti, A., Düggelin, M., Mathys,
noparticles. Combined, these coatings hold the potential to be surgi- D., & Guggenheim, R. (2002). Preparation and characterization of
cal instrument coatings capable of inhibiting bacterial contamination TiN–Ag nanocomposite films. Vacuum, 67(3–4), 463–470. https​://
and thus decrease SSI chances. doi.org/10.1016/S0042-207X(02)00232-4
Ferraris, S., & Spriano, S. (2016). Antibacterial titanium surfaces for medi-
cal implants. Materials Science and Engineering: C, 61, 965–978.
AC K N OW L E D G E M E N T S Geetha, M., Singh, A. K., Asokamani, R., & Gogia, A. K. (2009). Ti based
The authors gratefully acknowledge the support received from the biomaterials, the ultimate choice for orthopaedic implants–A re-
Center for Disruptive Musculoskeletal Innovations (CDMI) at the view. Progress in Materials Science, 54(3), 397–425. https​ ://doi.
org/10.1016/j.pmats​ci.2008.06.004
University of Toledo (UT), an NSF-funded I/UCRC and Professor
Jung, W. K., Koo, H. C., Kim, K. W., Shin, S., Kim, S. H., & Park, Y. H.
Vijay K. Goel, the local site director at UT. (2008). Antibacterial activity and mechanism of action of the sil-
ver ion in Staphylococcus aureus and Escherichia coli. Applied and
REFERENCES Environmental Microbiology, 74(7), 2171–2178. https​://doi.org/10.1128/
AEM.02001-07
Abdelghany, T., Al-Rajhi, A. M., Al Abboud, M. A., Alawlaqi, M., Magdah,
Karjalainen, P. P., Ylitalo, A., & Airaksinen, J. K. (2006). Titanium and ni-
A. G., Helmy, E. A., & Mabrouk, A. S. (2018). Recent advances in
tride oxide-coated stents and paclitaxel-eluting stents for coronary
green synthesis of silver nanoparticles and their applications: About
revascularization in an unselected population. Journal of Invasive
future directions. A review. BioNanoScience, 8(1), 5–16.
Cardiology, 18(10), 462.
Ahearn, D., May, L., & Gabriel, M. (1995). Adherence of organisms to
Kelly, P., Li, H., Benson, P., Whitehead, K., Verran, J., Arnell, R. D., &
silver-coated surfaces. Journal of Industrial Microbiology, 15(4), 372–
Iordanova, I. (2010). Comparison of the tribological and antimicrobial
376. https​://doi.org/10.1007/BF015​69993​
properties of CrN/Ag, ZrN/Ag, TiN/Ag, and TiN/Cu nanocomposite
Anthony, C. A., Peterson, R. A., Sewell, D. K., Polgreen, L. A., Simmering,
coatings. Surface and Coatings Technology, 205(5), 1606–1610. https​
J. E., Callaghan, J. J., & Polgreen, P. M. (2018). The seasonal vari-
://doi.org/10.1016/j.surfc​oat.2010.07.029
ability of surgical site infections in knee and hip arthroplasty. The
Kelly, P., Li, H., Whitehead, K. A., Verran, J., Arnell, R. D., & Iordanova, I.
Journal of Arthroplasty, 33(2), 510–514.e1. https​://doi.org/10.1016/j.
(2009). A study of the antimicrobial and tribological properties of TiN/
arth.2017.10.043
Ag nanocomposite coatings. Surface and Coatings Technology, 204(6–
Ban, K. A., Minei, J. P., Laronga, C., Harbrecht, B. G., Jensen, E. H., Fry, D.
7), 1137–1140. https​://doi.org/10.1016/j.surfc​oat.2009.05.012
E., … Duane, T. M. (2017). American College of Surgeons and Surgical
Koerner, R., Butterworth, L., Mayer, I., Dasbach, R., & Busscher, H.
Infection Society: Surgical site infection guidelines, 2016 update.
(2002). Bacterial adhesion to titanium-oxy-nitride (TiNOX) coatings
Journal of the American College of Surgeons, 224(1), 59–74.
with different resistivities: A novel approach for the development of
Banakh, O., Moussa, M., Matthey, J., Pontearso, A., Cattani-Lorente, M.,
biomaterials. Biomaterials, 23(14), 2835–2840.
Sanjines, R., … Durual, S. (2014). Sputtered titanium oxynitride coat-
Koju, N., Sikder, P., Ren, Y., Zhou, H., & Bhaduri, S. B. (2017). Biomimetic
ings for endosseous applications: Physical and chemical evaluation
coating technology for orthopedic implants. Current Opinion in Chemical
and first bioactivity assays. Applied Surface Science, 317, 986–993.
Engineering, 15, 49–55. https​://doi.org/10.1016/j.coche.2016.11.005
Berney, M., Hammes, F., Bosshard, F., Weilenmann, H.-U., & Egli, T.
Li, B., & Webster, T. J. (2018) Bacteria antibiotic resistance: New chal-
(2007). Assessment and interpretation of bacterial viability by using
lenges and opportunities for implant-associated orthopedic infec-
the LIVE/DEAD BacLight Kit in combination with flow cytometry.
tions. Journal of Orthopaedic Research, 36(1), 22–32.
Applied and Environmental Microbiology, 73(10), 3283–3290. https​://
Li, Z., Lee, D., Sheng, X., Cohen, R. E., & Rubner, M. F. (2006). Two-level
doi.org/10.1128/AEM.02750-06
antibacterial coating with both release-killing and contact-killing
Beshchasna, N., Ho, A. Y. K., Saqib, M., Kraśkiewicz, H., Wasyluk, Ł.,
capabilities. Langmuir, 22(24), 9820–9823. https​://doi.org/10.1021/
Kuzmin, O., … Ficai, A. (2019). Surface evaluation of titanium oxyni-
la062​2166
tride coatings used for developing layered cardiovascular stents.
Memisoglu-Bilensoy, E., & Hincal, A. A. (2006). Sterile, injectable cyclo-
Materials Science and Engineering: C, 99, 405–416.
dextrin nanoparticles: Effects of gamma irradiation and autoclaving.
Chawla, V., Jayaganthan, R., & Chandra, R. (2008). Structural char-
International Journal of Pharmaceutics, 311(1–2), 203–208.
acterizations of magnetron sputtered nanocrystalline TiN thin
Pichugin, V. F., Pustovalova, A. A., Konishchev, M. E., Khlusov, I. A.,
films. Materials Characterization, 59(8), 1015–1020. https​ ://doi.
Ivanova, N., Zhilei, S., & Gutor, S. (2016). In-vitro dissolution and
org/10.1016/j.match​ar.2007.08.017
structural and electrokinetic characteristics of titanium-oxyni-
Chen, W., Liu, Y., Courtney, H., Bettenga, M., Agrawal, C., Bumgardner,
tride coatings formed via reactive magnetron sputtering, Journal
J., & Ong, J. (2006). In vitro anti-bacterial and biological properties
of Surface Investigation. X-ray, Synchrotron and Neutron Techniques,
of magnetron co-sputtered silver-containing hydroxyapatite coating.
10(2), 282–291. https​://doi.org/10.1134/S1027​45101​6020166
Biomaterials, 27(32), 5512–5517. https​ ://doi.org/10.1016/j.bioma​
Piscanec, S., Ciacchi, L. C., Vesselli, E., Comelli, G., Sbaizero, O., Meriani,
teria​ls.2006.07.003
S., & De Vita, A. (2004). Bioactivity of TiN-coated titanium implants.
Croes, M., Bakhshandeh, S., van Hengel, I., Lietaert, K., van Kessel, K.,
Acta Materialia, 52(5), 1237–1245. https​://doi.org/10.1016/j.actam​
Pouran, B., … Fluit, A. (2018). Antibacterial and immunogenic be-
at.2003.11.020
havior of silver coatings on additively manufactured porous tita-
Prokuski, L. (2008). Prophylactic antibiotics in orthopaedic surgery.
nium. Acta Biomaterialia, 81, 315–327. https​ ://doi.org/10.1016/j.
JAAOS-Journal of the American Academy of Orthopaedic Surgeons,
actbio.2018.09.051
12 of 12       |
16(5), 283–293. https​://doi.org/10.5435/00124​635-20080​
5000-00007​
Ren, Y., Sikder, P., Lin, B., & Bhaduri, S. B. (2018). Microwave assisted
coating of bioactive amorphous magnesium phosphate (AMP) on
polyetheretherketone (PEEK). Materials Science and Engineering: C,
85, 107–113.
Seil, J. T., & Webster, T. J. (2012). Antimicrobial applications of nanotech-
nology: Methods and literature. International Journal of Nanomedicine,
7, 2767.
Sikder, P., & Bhaduri, S. B. (2019). Regulatory aspects of medical de-
vices and biomaterials. In L. Yang, S. Bhaduri, & T. Webster (Eds.),
Biomaterials in Translational Medicine, (pp. 23–36). Cambridge, MA:
Academic Press.
Sikder, P., Bhaduri, S. B., Ong, J. L., & Guda, T. (2019). Silver (Ag) doped
magnesium phosphate microplatelets as next-generation antibacte-
rial orthopedic biomaterials. Journal of Biomedical Materials Research
Part B: Applied Biomaterials, https​://doi.org/10.1002/jbm.b.34450​
[Epub ahead of print]
Sikder, P., Grice, C., & Bhaduri, S. B. (2019). Processing-structure-
property correlations of crystalline antibacterial magnesium phos-
phate (newberyite) coatings and their in vitro effect. Surface and
Coatings Technology, 374, 276–290. https​://doi.org/10.1016/j.surfc​
oat.2019.06.007
Sikder, P., Grice, C. R., Lin, B., Goel, V. K., & Bhaduri, S. B. (2018).
Single-phase, antibacterial trimagnesium phosphate hydrate
coatings on polyetheretherketone (PEEK) implants by rapid
microwave irradiation technique. ACS Biomaterials Science & Engi-
neering, 4(8), 2767–2783. https​://doi.org/10.1021/acsbi​omate​rials.
8b00594
Sikder, P., Koju, N., Ren, Y., Goel, V. K., Phares, T., Lin, B., & Bhaduri, S.
B. (2018). Development of single-phase silver-doped antibacte-
rial CDHA coatings on Ti6Al4V with sustained release. Surface and
Coatings Technology, 342, 105–116. https​://doi.org/10.1016/j.surfc​
oat.2018.02.100
Sim, W., Barnard, R., Blaskovich, M., & Ziora, Z. (2018). Antimicrobial sil-
ver in medicinal and consumer applications: A patent review of the
past decade (2007–2017). Antibiotics, 7(4), 93.
Sirelkhatim, A., Mahmud, S., Seeni, A., Kaus, N. H. M., Ann, L. C., Bakhori,
S. K. M., … Mohamad, D. (2015). Review on zinc oxide nanoparticles:
Antibacterial activity and toxicity mechanism. Nano-Micro Letters,
7(3), 219–242.
View publication stats
SIKDER et al.
Sommerfeld, P., Schroeder, U., & Sabel, B. A. (1998). Sterilization of un-
loaded polybutylcyanoacrylate nanoparticles. International Journal
of Pharmaceutics, 164(1–2), 113–118. https​ ://doi.org/10.1016/
S0378-5173(97)00394-3
Stiefel, P., Schmidt-Emrich, S., Maniura-Weber, K., & Ren, Q. (2015).
Critical aspects of using bacterial cell viability assays with the fluo-
rophores SYTO9 and propidium iodide. BMC Microbiology, 15(1), 36.
https​://doi.org/10.1186/s12866-015-0376-x
Subramanian, B., Muraleedharan, C., Ananthakumar, R., & Jayachandran,
M. (2011). A comparative study of titanium nitride (TiN), titanium oxy
nitride (TiON) and titanium aluminum nitride (TiAlN), as surface coat-
ings for bio implants. Surface and Coatings Technology, 205(21–22),
5014–5020. https​://doi.org/10.1016/j.surfc​oat.2011.05.004
van Hove, R. P., Sierevelt, I. N., van Royen, B. J., & Nolte, P. A. (2015).
Titanium-nitride coating of orthopaedic implants: A review of the
literature. BioMed Research International, 2015, 1–9. https​ ://doi.
org/10.1155/2015/485975
Vaz, F., Cerqueira, P., Rebouta, L., Nascimento, S., Alves, E., Goudeau, P.,
… De Rijk, J. (2004). Structural, optical and mechanical properties of
coloured TiNxOy thin films. Thin Solid Films, 447, 449–454. https​://
doi.org/10.1016/S0040-6090(03)01123-4
Xiao, L., Yan, D., He, J., Zhu, L., Dong, Y., Zhang, J., & Li, X. (2007).
Nanostructured TiN coating prepared by reactive plasma spraying
in atmosphere. Applied Surface Science, 253(18), 7535–7539. https​://
doi.org/10.1016/j.apsusc.2007.03.062
Zhang, L., Gao, Q., & Han, Y. (2016). Zn and Ag co-doped anti-micro-
bial TiO2 coatings on Ti by micro-arc oxidation. Journal of Materials
Science & Technology, 32(9), 919–924. https​ ://doi.org/10.1016/j.
jmst.2016.01.008
Zhao, L., Chu, P. K., Zhang, Y., & Wu, Z. (2009). Antibacterial coatings
on titanium implants. Journal of Biomedical Materials Research Part B:
Applied Biomaterials, 91(1), 470–480.
How to cite this article: Sikder P, Liu L, Jayatissa AH, Bhaduri
SB. Antibacterial silver (Ag) containing titanium oxynitride
(TiOxNy) coatings for inhibiting surgical site infections (SSI).
Med Devices Sens. 2019;00:e10052. https​://doi.org/10.1002/
mds3.10052​