Choi et al.

International Journal of
International Journal of Implant Dentistry (2023) 9:24
https://doi.org/10.1186/s40729-023-00488-w Implant Dentistry

RESEARCH Open Access

Antibacterial activity and biocompatibility

of silver coating via aerosol deposition
on titanium and zirconia surfaces
Sunyoung Choi1,2, Ye‑Hyeon Jo3, Jung‑Suk Han1, Hyung‑In Yoon1, Jae‑Hyun Lee1 and In‑Sung Luke Yeo1*   

Abstract
Purpose The purpose of this in vitro study was to investigate the antibacterial effect and biocompatibility of silver
coatings via aerosol deposition on titanium and zirconia surfaces.
Methods The surfaces of titanium and zirconia specimens were polished and coated with silver via aerosol deposi‑
tion. After silver coating, the elemental composition, surface roughness and amount of silver released from the coated
surfaces were measured. The bacterial growth on the silver-coated surfaces was investigated via crystal violet assay
after incubation with Streptococcus gordonii for 24 h, Fusobacterium nucleatum for 72 h and Porphyromonas gingivalis
for 48 h. Human gingival fibroblasts and mouse preosteoblasts were also cultured on the silver-coated specimens
to examine the biocompatibility of the coating.
Results After silver coating via aerosol deposition, the surface roughness increased significantly, and the released sil‑
ver ranged from 0.067 to 0.110 ppm. The tested bacteria formed significantly less biofilm on the silver-coated titanium
surfaces than on the uncoated titanium surfaces. In contrast, biofilm formation on the silver-coated zirconia surfaces
was greater than that on the uncoated zirconia surfaces. Human gingival fibroblasts and mouse preosteoblasts prolif‑
erated on the silver-coated surfaces without significant differences from the uncoated surfaces.
Conclusions Silver coating via aerosol deposition provided an antibacterial effect against oral bacteria on titanium
surfaces, whereas it promoted more bacterial growth on zirconia surfaces. The proliferation of fibroblasts and osteo‑
blasts was not significantly affected by the silver coating on both titanium and zirconia surfaces.
Keywords Silver, Titanium, Zirconia, Aerosol deposition, Bacterial proliferation, Biocompatibility

Background implant materials and treatment procedures, the long-

The use of dental implants has become a widely accepted term survival rates of dental implants have been reported
and preferred treatment modality to rehabilitate patients to be higher [1, 4]. However, despite the high survival rate
with edentulous sites [1–3]. With the improvement of of dental implants, the risk of complications remains.
Peri-implantitis is one of the representative compli-
cations of dental implants. It is a host response to the
*Correspondence: microbial challenge occurring in tissues surrounding
In‑Sung Luke Yeo
[email protected] implants and characterized by the destructive inflamma-
1
Department of Prosthodontics, School of Dentistry and Dental tion in the connective tissue and the progressive loss of
Research Institute, Seoul National University, 101 Daehak‑Ro, Jongro‑Gu, supporting bone, which can lead to implant loss [5–8].
Seoul 03080, Korea
2
Department of Prosthodontics, One‑Stop Specialty Center, Seoul Since the normal flora lives in the oral cavity, the surfaces
National University Dental Hospital, Seoul, Korea of implant prostheses in the oral cavity inevitably become
3
Dental Research Institute, Seoul National University, Seoul, Korea habitats for bacteria to proliferate [9]. Furthermore,

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
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Choi et al. International Journal of Implant Dentistry (2023) 9:24 Page 2 of 8

bacterial proliferation on implant surfaces is closely silver via aerosol deposition would have antibacterial
related to peri-implantitis. Several specific periodon- effect and biocompatibility.
tal pathogens, such as Porphyromonas gingivalis, were
observed more frequently in peri-implantitis than in Methods
healthy tissue surrounding implants [10], and a history of Specimen preparation
chronic periodontitis and poor plaque control are known Titanium specimens were machined from grade 4 tita-
risk factors for peri-implantitis with strong evidence [7, nium bars (Seoul Titanium Co., LTD., Siheung, Korea)
11]. In an animal study, the progression of peri-implan- into disc shapes 20 mm in diameter and 0.5 mm in thick-
titis was induced by breaks of the mucosal seal around ness. Three percent yttria-stabilized tetragonal zirconia
the implants and submucosal bacterial biofilm formation polycrystalline discs that were 15 mm in diameter and
[12]. 2 mm in thickness were prepared through cold isostatic
To evade biofilm formation on the surfaces of dental pressing of the powder mixture (Tosoh, Tokyo, Japan)
implants, various surface treatments, such as topographi- at 200 MPa, followed by sintering at 1500 °C for 2 h.
cal modifications or loading of antibacterial agents on The titanium and zirconia discs were polished follow-
surfaces, have been introduced [13, 14]. In the surface ing a previously published method [25] until the surface
coating method, effective antibacterial agents and dura- roughness was lower than 0.01 μm. The polished discs
ble coating methods are required for reliable inhibition were coated with silver using the aerosol deposition
of biofilm formation on the implant surfaces. Silver is a method. First, the discs as substrates were sequentially
widely used inorganic agent with antibacterial effects in washed using acetone, ethanol and water and mounted
various fields, including medical implants [15, 16]. Dop- in the deposition chamber. Commercially available silver
ing silver directly on the surfaces without additional nanoparticles (ENB KOREA Co., LTD., Daejeon, Korea)
layers, growing a titanium oxide layer with silver, and mixed with alumina powder (Showa Denko, Tokyo,
depositing a coating with silver on the surfaces were pro- Japan) at a concentration of 1 wt% were preheated and
posed as techniques for silver application on titanium aerosolized by vibration with a carrier gas in the aerosol
implant surfaces [14]. Aerosol deposition is a method to chamber. The silver aerosol was accelerated by the pres-
coat surfaces with ceramic or ceramic mixtures at room sure difference between the two chambers and deposited
temperature without a high-temperature sintering pro- onto the substrates through a slit nozzle at room tem-
cess [17]. Durable coating via aerosol deposition on the perature. The thickness of the coating was confirmed by
titanium surface has also been reported [18]. scanning electron microscopy to be approximately 1 μm.
Titanium has long been established as the standard
material for dental implants with a proven record of suc- Surface characterization
cess [1, 4, 19]. Its physical properties and biocompatibil- The coated surfaces were observed using field emission
ity are suitable for use as an implant material. However, scanning electron microscopy (SEM, S-4700, Hitachi
with increasing esthetic demands on dental treatments, High-Tech Corporation, Tokyo, Japan), and elemen-
the gray color of titanium has become a major draw- tal analysis of the surfaces was performed using energy
back. Zirconia not only has proper physical properties dispersive X-ray spectroscopy. The arithmetic mean
and excellent biocompatibility, but also has outstanding heights of the surfaces of uncoated and coated speci-
esthetics; thus, its use as an implant material is rapidly mens were measured using a three-dimensional confo-
expanding [20, 21]. Furthermore, although still contro- cal laser microscope (LSM 800, Zeiss, Jena, Germany).
versial, some studies have shown that zirconia is more The amount of silver released from each coated disc
resistant to bacterial colonization than titanium and can was measured by incubating it in a tube containing
potentially be beneficial against bacterial contamination phosphate-buffered saline (PBS) at 37 °C. After 24 h of
[22–25]. However, zirconia implants are not completely incubation, the disc was removed from the tube, and
free from peri-implant mucositis or peri-implantitis [26]. the amount of silver in the solution was measured using
Both titanium and zirconia surfaces require extra treat- inductively coupled plasma–mass spectrometry (ICP-
ment to reduce the potential for bacterial colonization MS, NexION 350D, Perkin-Elmer, Waltham, MA, USA).
that can cause peri-implant inflammation.
The purpose of this in vitro study was to examine the Bacterial adhesion and proliferation on the surface
antibacterial effect of silver coating on titanium and zir- Based on previously published data [25], we determined
conia surfaces. Silver was coated on the surfaces via aer- the optimum growth conditions for each bacterium in
osol deposition method and the biocompatibility of the our experiments. Streptococcus gordonii ATCC 10558

(BHI broth; Bacto™ Brain Heart Infusion, BD, Franklin

coating was also investigated. The null hypothesis of this cells cultivated in sterilized brain heart infusion broth
study was that titanium and zirconia surfaces coated with
Choi et al. International Journal of Implant Dentistry (2023) 9:24 Page 3 of 8

Lakes, NJ, USA) were harvested through centrifugation, oxide and placed in 12-well plates. Human gingival
resuspended in fresh media and adjusted to a bacterial fibroblasts (HGFs; PCS-201-018, ATCC, Manassas, VA,
concentration of approximately 4.8 × ­104 colony forming USA) cultured in fibroblast basal medium (PCS-201-
units (CFU)/mL, corresponding to an optical density of 030, ATCC, Manassas, VA, USA) using growth kit-low
0.03 at 600 nm. The silver-coated and uncoated discs were serum (PCS-201-041, ATCC, Manassas, VA, USA) were
sterilized by ethylene oxide and incubated with 1 mL collected and seeded onto the specimens at a density of
of the bacterial suspension under aerobic conditions at ­105 cells/mL. After 24 h of incubation, the medium was
37 °C in 12-well plates, and bacterial growth was assessed replaced with another freshly prepared medium con-
after 24 h. Cultured Fusobacterium nucleatum ATCC taining MTT in each well, and the 12-well plates were
25586 cells in sterilized BHI broth under anaerobic con- incubated at 37 °C for 4 h. After removing the medium
ditions (80% N ­ 2, 10% ­H2, 10% ­CO2) were harvested and containing MTT in each well, an MTT solubilization
washed with fresh medium. Then, the cells were adjusted solution was added, and the absorbance at 570 nm was
to an optical density of 0.064 at 600 nm, corresponding to measured using a microplate reader (Epoch 2, BioTek).
a bacterial concentration of approximately 2 × ­108 CFU/ The background absorbance at 690 nm was also meas-
mL. The sterilized specimens were inoculated with 1 mL ured and subtracted from the 570 nm measurement
of the bacterial suspension and 3 mL of fresh media in values.
12-well plates and incubated in an anaerobic chamber at Mouse preosteoblast cells (MC3T3-E1, ATCC, Manas-
37 °C for 72 h. Porphyromonas gingivalis ATCC 33277 sas, VA, USA) cultured in α-minimal essential medium
cells cultivated in sterile BHI broth supplemented with containing 10% fetal bovine serum and 1% penicillin/
0.5 mg/mL hemin and 5 mg/mL vitamin K under anaero- streptomycin were also seeded on the sterilized speci-
bic conditions were harvested, washed and adjusted to a mens in the 12-well plates. After 4 and 7 days of cul-
concentration of 5.1 × ­107 CFU/mL; the suspension had ture, MTT assays were performed as described above.
an optical density of 0.01 at 600 nm. The sterilized speci- The negative controls were prepared by culturing the
mens were inoculated with 2 mL of the bacterial suspen- cells without specimens and the absorbance values were
sion in 12-well plates and then incubated in an anaerobic expressed as a percentage of the controls.
chamber at 37 °C for 48 h.
After incubation, each specimen was gently rinsed
using PBS to remove unadhered bacteria from the sur- Statistical analysis
face. Thereafter, the remaining adherent bacteria were The Shapiro–Wilk test was used to assess the normal-
stained with 1% crystal violet solution for 10 min, washed ity of variables and the Levene’s test was performed to
gently with PBS and destained using 400 μL of a destain- assess the equality of variances. Since the results showed
ing solution (a mixture of 80% ethanol and 20% acetone). non-normal distribution of the measurement values, the
The absorbance of each solution was then measured comparison of the biofilm formation and cell prolifera-
using a microplate spectrophotometer (Epoch 2, BioTek, tion between silver-coated and uncoated surfaces of each
Winooski, VT, USA) at 590 nm. The absorbance values material was carried out using the Mann–Whitney U
were calculated per unit area of each disc. test. Version 26.0 of the IBM SPSS Statistics (IBM Corp.,
Armonk, NY, USA) software was used for all statistical
Silver solution experiment analyses, and the level of significance was set at 0.05.
Silver solutions were prepared with commercially avail-
able silver nanoparticles (ENB KOREA Co., LTD.) in PBS
at concentrations of 0.00001, 0.0001, 0.01 and 0.1 wt%. Results
After sterilizing 5 mL of the prepared silver solutions at Surface characterization
121 °C for 15 min, S. gordonii, F. nucleatum and P. gin- Analysis of the elemental components of the coated
givalis were cultured in the solutions. The absorbance of surfaces using electron dispersive X-ray spectroscopy
each bacterial suspension was measured over time. confirmed the presence of silver on both titanium and
zirconia surfaces (Table 1). The arithmetic mean heights
Cell proliferation assay of the surfaces of the coated specimens are listed in
Human gingival fibroblasts and mouse preosteoblasts Table 2. The silver coating was found to significantly
were cultured on the silver-coated and uncoated discs, increase the surface roughness of both materials, and no
and the cellular proliferation was evaluated using a significant difference was observed when comparing the
3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazolium two materials. The amount of silver released from the sil-
bromide (MTT) kit (TOX-1, Sigma-Aldrich, St. Louis, ver-coated titanium was 0.067 ± 0.020 ppm and that from
MO, USA). The specimens were sterilized by ethylene the silver-coated zirconia was 0.110 ± 0.033 ppm.
Choi et al. International Journal of Implant Dentistry (2023) 9:24 Page 4 of 8

Table 1 Elemental analysis using electron X-ray dispersive concentrations above 0.01 wt% were shown to inhibit
spectroscopy bacterial growth.
Silver-coated titanium Silver-coated zirconia
Cell proliferation assay
Element wt% atomic% Element wt% atomic%
The absorbance measured in the MTT assay indicated
C 3.88 6.52 O 25.24 64.84 the proliferation of cells cultured on each specimen
O 45.33 57.17 Al 1.37 2.08 (Fig. 4). For both titanium and zirconia, there was no sig-
Al 46.12 34.48 Y 3.65 1.69 nificant difference in the cell proliferation between the
Ti 4.09 1.72 Zr 69.29 31.22 silver-coated and uncoated specimens. The silver coat-
Ag 0.58 0.11 Ag 0.45 0.17 ing via aerosol deposition using a 1 wt% silver mixture on
Total 100 100 Total 100 100 titanium and zirconia surfaces showed no cytotoxic effect
C carbon, O oxygen, Al aluminum, Ti titanium, Ag silver, Y yttrium, Zr zirconium
on the proliferation of the HGF and MC3T3-E1 cells.

Discussion
Since bacterial colonization on implant surfaces is closely
Table 2 Arithmetic mean heights of the silver-coated specimens related to implant survival rate, many studies have been
Smooth Silver-coated Smooth Silver-coated performed to suppress the growth of bacteria by anti-
titanium titanium zirconia zirconia bacterial application to the implant surfaces [14, 27]. In
Sa (μm) 0.000 ± 0.000 0.220 ± 0.022 0.001 ± 0.000 0.208 ± 0.018 this study, we investigated silver coating as an antibacte-
rial treatment for implant surfaces. Silver is one of the
Values are shown as the mean ± standard deviation
Sa arithmetic mean height of the surface
most commonly used antimicrobial agents with various
clinical applications. It has been shown to have a broad-
spectrum antibacterial effect, high efficacy even at low
Crystal violet assay concentrations, and stability enabling straightforward
The three bacteria exhibited the same tendencies in bio- application on surfaces using various techniques [14,
film formation. There was a significant decrease in the 28–30]. Although the underlying mechanisms remain
biofilm formation on the silver-coated titanium surface unclear, it has been widely accepted that the antibacte-
compared to the uncoated bare titanium surface and a rial properties of silver can be attributed to the silver ion,
significant increase on the silver-coated zirconia surface which contributes to oxidative stress, protein dysfunction
compared to the uncoated zirconia surface (Figs. 1 and 2). and membrane damage in bacteria [15, 29]. In addition,
according to recent research, silver nanoparticles them-
Silver solution experiment selves also have antibacterial effects by binding to the
Figure 3 shows the growth curves of S. gordonii, F. bacterial cell membrane [16].
nucleatum and P. gingivalis cultured in 0.00001, 0.0001, The amount of the biofilm formation on surfaces
0.01 and 0.1 wt% silver solutions. The bacterial growth was investigated by crystal violet staining assay. The
was unaffected by 0.0001 wt% silver solution, whereas absorbance values in crystal violet assay are directly

Fig. 1 Absorbance of the crystal violet solution on titanium surfaces incubated with A S. gordonii for 24 h, B F. nucleatum for 72 h and C P. gingivalis
for 48 h. The bars and intervals represent the mean absorbance values and standard deviations, respectively (n = 3; *p < 0.05)
Choi et al. International Journal of Implant Dentistry (2023) 9:24 Page 5 of 8

Fig. 2 Absorbance of the crystal violet solution on zirconia surfaces incubated with A S. gordonii for 24 h, B F. nucleatum for 72 h and C P. gingivalis
for 48 h. The bars and intervals represent the mean absorbance values and standard deviations, respectively (n = 3; *p < 0.05)

Fig. 3 Growth curves of A S. gordonii, B F. nucleatum and C P. gingivalis cultured in 0.00001%, 0.0001%, 0.01% or 0.1% silver solution. Ag silver

proportional to the amount of biofilm formation on indicated the low level of biofilm formation on the sur-
the surfaces. The results showed that the smooth tita- face, suggesting the low affinity of the surface for bacte-
nium surface before coating was a favorable substrate rial growth. After the silver coating, bacterial growth on
for bacterial colonization, whereas the biofilm forma- the surface was significantly increased. The bacterial col-
tion was significantly reduced after silver coating on the onization on surfaces is affected by several surface prop-
surface. For zirconia surfaces, the low absorbance values erties such as surface roughness, hydrophobicity, charge
Choi et al. International Journal of Implant Dentistry (2023) 9:24 Page 6 of 8

Fig. 4 Viability of human gingival fibroblasts (HGF) for 24 h and mouse preosteoblasts (MC3T3-E1) for 4 and 7 days in MTT assay for A titanium
and B zirconia specimens. The values are presented as the percentage of control. The bars and intervals represent the mean values and standard
deviations, respectively. MTT 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazolium bromide

and chemical composition [31–33]. Since the silver coat- Thus, the surface roughness and surface charge after
ing of the surfaces can alter the surface properties, the silver coating make the surfaces more favorable for bac-
biofilm formation of the coated surface might be affected terial growth. The bacterial colonization on the silver-
by the coating. coated surfaces would be affected by the bacteria-friendly
After silver nanoparticles mixed with alumina at a con- surface properties as well as antibacterial effect of coated
centration of 1 wt% were coated onto polished surfaces silver.
via aerosol deposition, the surface roughness was signifi- The amount of silver released from the coated speci-
cantly increased up to 0.250 μm. Surface roughness is a mens was approximately 0.1 ppm, and there was no sig-
critical factor affecting bacterial adhesion on the surface. nificant difference between the titanium and zirconia
In particular, a surface roughness of 0.2 μm is the thresh- substrates. To evaluate whether that amount of silver
old roughness that does not affect bacterial adhesion and was effective in inhibiting bacterial growth in dispersed
colonization even if the surface roughness is lower than state, the bacteria were cultured in silver solution at
it [34]. Therefore, an increase in surface roughness from the same concentration of released silver, 0.00001 wt%.
nearly 0 μm on a smooth surface to more than 0.2 μm Higher concentrations were also experimented to explore
could cause a significant increase in bacterial adhesion. the effective silver concentration for antibacterial activ-
Another surface property that could be altered after ity. The results show that the amount of silver released
silver coating is surface charge. Since bacteria are often from the coated surface is insufficient to inhibit bacteria
negatively charged by surface components, they colonize growth in solution, and concentrations of silver solu-
more readily on surfaces with positive to neutral charges tion above 0.01% can exhibit antibacterial effect. The
than on those that exhibit negative charges [33]. Zirco- difference in effective concentration between the coated
nia is a metal oxide that is covered with hydroxyl groups surface and the solution may indicate difference in the
under aqueous conditions, and thus, its net surface antibacterial mechanism of silver in the coated surface
charge is affected by the pH of the liquid where it is sub- and solution.
merged [35]. Since the pH of the BHI broth was 7.4 ± 0.2 The surfaces of dental implants are specifically engi-
and the isoelectric point of zirconia was reported to be neered for efficient osseointegration and mucosal sealing.
less than 7 [36], the net surface charge of the zirconia The incorporation of silver via aerosol deposition could
specimen was negative, which could repulse bacterial col- interrupt the cellular response to the implant surfaces.
onization on the surface. However, on the silver-coated However, the coating via aerosol deposition using a 1 wt%
zirconia surfaces, the silver nanoparticles released posi- silver mixture had no significant effect on the fibroblasts
tively charged silver ions, which neutralized the negative and osteoblasts, which implied its biocompatibility. In
surface charge of bare zirconia surfaces. The disruption this study, human gingival fibroblasts and mouse pre-
of the negative surface charge after silver coating can osteoblasts cultured in zirconia specimens showed lower
potentially interrupt the low affinity of smooth zirconia viability than controls. The results in MTT assay can be
surfaces for bacterial colonization, causing an increase in affected by the several experimental conditions such as
bacterial growth after silver coating on the surfaces. The seeding cell number, the concentration of MTT reagent
coated silver also affects the surface charge of the tita- and incubation time with MTT [37]. Thus, the absolute
nium surface in a same way. value should be analyzed with caution.
Choi et al. International Journal of Implant Dentistry (2023) 9:24 Page 7 of 8

Our study was limited to the colonization of a single Competing interests

The authors declare that they have no competing interests.
bacterium on the implant surfaces. As bacterial spe-
cies typically form complex structures in the oral envi-
ronment, the adhesion and proliferation of bacterial Received: 14 April 2023 Accepted: 22 August 2023
complexes on implant surfaces need to be further inves-
tigated. Another limitation was the differing dimensions
of the titanium and zirconia discs. Directly comparing
the bacterial adhesion properties between titanium and References
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