J Clin Periodontol 2017; 44: 403–409 doi: 10.1111/jcpe.

12699

Chemotherapeutic Sebastien Dostie1,2, Lubna T.

Alkadi1,3, Gethin Owen1, Jiarui Bi1,
Ya Shen1, Markus Haapasalo1 and

decontamination of dental Hannu S. Larjava1

1
Faculty of Dentistry, Department of Oral
Biological and Medical Sciences, University

implants colonized by mature of British Columbia, Vancouver, BC,

Canada; 2Canadian Armed Forces,
Petawawa, ON, Canada; 3Department of

multispecies oral biofilm Dentistry, College of Dentistry, King

Abdulaziz Medical City, King Saud bin
Abdulaziz University for Health Sciences,
Riyadh, Saudi Arabia

Dostie S, Alkadi LT, Owen G, Bi J, Shen Y, Haapasalo M, Larjava HS.

Chemotherapeutic decontamination of dental implants colonized by mature
multispecies oral biofilm. J Clin Periodontol 2017; 44: 403–409. doi: 10.1111/jcpe.
12699.

Abstract
Aim: No studies have tested disinfectants on mature multispecies oral biofilms on
titanium substrata. The aim of this study was to investigate the efficacy of com-
monly used antimicrobial agents in decontamination of multispecies mature oral
biofilm on sandblasted, large-grit, acid-etched (SLA) titanium implants.
Methods: SLA titanium disks were inoculated with dental plaque and cultured
anaerobically for 21 days. The disks were rinsed with 0.9% NaCl, exposed for
2 min. to tetracycline paste, 1% Chlorhexidine gel (CHX), 35% phosphoric acid
gel (Etch) or a novel chemical formula (0.3% cetrimide, 0.1% CHX and 0.5%
EDTA) and then rinsed again with 0.9% NaCl. Bacteria were quantified from
scanning electron micrographs of the implant surfaces. Living bacteria were quan-
tified with confocal laser scanning microscopy (CLSM).
Results and Conclusions: Rinsing the surfaces with 0.9% NaCl removed the major-
ity of the biofilm. However, bacteria persisted in all specimens and none of the dis-
Key words: biofilm; chlorhexidine;
infectants was superior to the double saline rinse group. CLSM analysis showed decontamination; implant; phosphoric acid;
that CHX and Etch groups had a statistically significant reduction of viable bacte- tetracycline
ria, although small. Overall the results show that many disinfection agents used in
the clinic are ineffective in biofilm removal and leave live bacteria on the surface. Accepted for publication 19 January 2017

Despite the high success rates of Mombelli et al. 2012, Marrone et al.
osseointegrated dental implants, they 2013, Derks et al. 2016). Peri-
are susceptible to peri-implantitis, implantitis is defined as mucosal
which may eventually lead to inflammation and loss of supporting
implant loss (Mir-Mari et al. 2012, bone. It is infectious in nature and
the oral microflora is the primary
Conflict of interest and source of source of pathogens. Removal of
funding statement bacterial biofilm is routinely per-
The authors have stated explicitly formed as part of the treatment of
that there are no conflicts of interest peri-implantitis. Due to the screw-
in connection with this article. shaped design and the microstructure
This study was kindly supported by a of the implant surface, mechanical
grant from Alpha Omega Foundation debridement alone is incapable of
of Canada. removing all of the biofilm. Clini-
cians are often using adjunctive
© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
chemotherapeutic agents, such as
chlorhexidine (CHX), phosphoric
acid gel and tetracycline paste, in
order to decrease the microorgan-
isms to a level compatible with peri-
odontal health (Strooker et al. 1998,
Wiltfang et al. 2012). However, there
are limited in vitro studies support-
ing the use of one agent over the
others (Zablotsky et al. 1992, Den-
nison et al. 1994, Gosau et al. 2010,
Ntrouka et al. 2011, B€ urgers et al.
2012). Moreover, these studies used
single-species immature oral biofilms.
It is known that distinctive
403

404 Dostie et al.
subgingival microbiota become
established 3–12 weeks after
supragingival plaque formation
(Listgarten 1976, Listgarten 1994)
and that the maturity of the bio-
film increases its resistance to anti-
microbial agents (Shen et al. 2011,
Stojicic et al. 2013). An in vitro
biofilm model described by Shen
et al. (2009) could potentially be
used to assess implant surface
decontamination. To date, no stud-
ies have tested disinfectants on
mature multispecies biofilms using
titanium substrata.
The aim of this study was to use
the mature biofilm model on Strau-
mannâ implant rough SLA surface
and determine the susceptibility to
antimicrobial agents commonly used
in clinical practice, namely CHX
(Dennison et al. 1994, de Waal et al.
2015), tetracycline paste (Zablotsky
et al. 1992) and 35% phosphoric
acid (Wiltfang et al. 2010). A new
CHX decontamination agent for-
mula was also tested.
Materials and Methods
Culture of mature biofilms on SLA disks
The experiment was repeated in a
triplicate format. Each experiment
consisted of 18 sterile SLA disks
(5 mm diameter by 1 mm thickness;
Straumannâ, Basel, Switzerland),
which served as the biofilm sub-
strate. All disks were coated with
bovine dermal type I collagen
(10 lg/ml collagen in 0.012 M HCl
in water; Cohesion, Palo Alto, CA,
USA) overnight at 4°C in the wells
of a 24-well tissue culture plate con-
taining 2 ml of the selected solution.
The disks were then rinsed with 2 ml
of sterile phosphate-buffered saline
(Sigma-Aldrich, Saint Louis, MO,
USA) and placed in a 24-well tissue
culture plate containing 2 ml of dis-
persed subgingival dental plaque in
brain heart infusion broth (Becton-
Dickinson, Sparks, MD, USA) with
a minimum bacterial cell concentra-
tion of 3.2 9 107 CFU/ml. The pla-
que was collected from subgingival
sites of three healthy volunteers (one
donor per experiment). The disks
were incubated under anaerobic con-
ditions (AnaeroGen; Oxoid, Basing-
stoke, Hampshire, UK) at 37°C for
21 days and the medium was chan-
ged every 7 days (Shen et al. 2011).
1600051x, 2017, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcpe.12699 by Guatemala - OARE Access, Wiley Online Library on [20/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
with 0.9% NaCl to form a thick paste
Chemical treatments of the contaminated
SLA disks
(Tetracycline group) and a novel
chemical formula consisted of a mix
The schematic presentation of the of 0.3% cetrimide, 0.1% CHX and
methodology used is presented in 0.5% EDTA (pH 7.0) in 3% methyl-
Fig. 1. Three disks per experiment cellulose gel (C.C.E. Group). Subse-
were not rinsed or treated with chemi- quently, each agent was removed
cal agent and served as controls. To with six increments of 1 ml 0.9%
start the decontamination experi- Sodium Chloride Irrigation (0.9%
ment, the remaining disks were first NaCl; Braun, Irvine, CA, USA) as
rinsed with six increments of 1 ml described previously. To control the
0.9% NaCl (total 6 ml; Rinse group) mechanical effect of rinsing, three
to remove the established biofilm samples per experiment were rinsed
applied with a manual Fisherbrand with 12 increments of 1 ml 0.9%
Finnpipette II (100 ll to 1000 ll; NaCl (Double Rinse group) or dis-
Fisherbrand, Pittsburgh, PA, USA) tilled sterile water without any of
to produce a constant flow (1 ml/s) them being subjected to chemical
similar to a rinse in a clinical setting agents.
using a Monoject syringe Covidien Each disk was then transferred
Canada Saint-Laurent (Covidien into a well containing 1 ml of 0.1M
Canada, Saint-Laurent (Quebec)). PIPES buffer (pH 7.4) (Sigma-
The approximate fluid pressure at the Aldrich, Saint Louis, MO) for 2 min.
implant surface was about 2500 Pa. Proteins were then fixed with 2.5%
Each triplicate disk group was then glutaraldehyde in 0.1 M PIPES (pH
subjected to 2 min. treatment with a 7.4) for 30 min. Each disk was then
different chemical agent routinely rinsed again in 0.1 M PIPES (pH 7.4)
used by clinicians to disinfect the sur- for 5 min. and further fixed in 1 ml
face during surgical treatment for of 1% osmium tetroxide in 0.1 M
peri-implantitis. This time point was PIPES buffer (pH 6.8) for 60 min.
chosen to reflect practical application After rinsing, the specimens were
of these agents during surgery. The dehydrated in a successive increasing
agents applied were: 1% CHX in concentration of ethanol (EtOH;
methylcellulose gel (CHX group), Electron Microscopy Sciences, Hat-
35% phosphoric acid gel (Ultra-Etch, field, PA) for 5 min. each at 50%,
Ultradent, South Jordan, UT, USA) 60%, 70%, 80%, 90% and 3 times
(Etch group), tetracycline paste made 5 min. at 100%. Following dehydra-
by mixing 250 mg tetracycline tablet tion, the disks were placed in 1 ml of
Fig. 1. Schematic view of the experimental methodology. SLA implant disks with mul-
tilayer and multi-species 3-week-old biofilm were rinsed with 0.9% NaCl to remove
loose biofilm (“rinse” specimens) and then treated with chemical agents for 2 min
(“Test”). The chemical agent was then rinsed off the disk with 0.9% NaCl and control
disks received identical additional rinse (“double rinse”). All the specimens (N = 9 in
each group in triplicate separate experiments) including undisturbed control biofilm
samples were then processed and analyzed by SEM or CLSM.
© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

100% hexamethydilazane (HMDS;
Sigma-Aldrich, Saint Louis, MO)
three times for 5 min. to dry the sam-
ples. Each disk was then attached to
an aluminum stub using carbon con-
ducting double-sided adhesive, and
the contour of each disk/stub was
painted with colloidal silver to
improve the electrical conductivity.
Samples were sputter with 8 nm of
iridium using a Leica EM MED020
Coating System (Leica Microsystems,
Wetzlar, Germany).
Each sample was examined using
scanning electron microscopy (SEM)
(Helios NanoLab 650 Focussed Ion
Beam SEM, Oregon, USA). Images
of the center of each disk were taken
at a voltage of 1 kV with fields of
view that correspond roughly to a
magnification of 1500 and 5000
times respectively. Using ImageJ
1.47v software (National Institutes
of Health, Bethesda, MD, USA), a
bacterial cell count was performed
for each 50009 magnification image.
Determination of live/dead bacteria
To determine the viability of the
bacteria after treatment, each SLA
disk sample was rinsed or treated as
above but was observed under con-
focal laser scanning microscopy
(CLSM; Fig. 1) after staining the
samples for live/dead bacteria using
LIVE/DEAD BacLight Bacterial
viability kit L-7012 (Molecular
Probes, Eugene, OR, USA). Each
disk was scanned in three randomly
selected areas of the biofilm. Fluo-
rescence from stained cell was
viewed by using a CLSM (Nikon
Eclipse C1; Nikon Canada, Missis-
sauga, ON, Canada). Simultaneous
dual-channel imaging was used to
display green and red fluorescence
(Stojicic et al. 2013).
The surface areas occupied by live
or dead bacteria were calculated with
Imaris 7.3.0v software (BITPLANE,
South Windsor, CT, USA). The vol-
ume ratio of green fluorescence (live
bacteria) to green-and-red fluores-
cence (live and dead bacteria) was cal-
culated in three randomly selected
fields, indicating the percentage of live
bacteria in the remaining biofilms.
Statistical analysis
Distribution analyses of the results
were completed with the
© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Decontamination of dental implants
Kolmogorov-Smirnov Goodness-of-
Fit Test and the results of all the
SEM experiments were analyzed
using one-way analysis of variance
(ANOVA) test with Tukey post hoc
analysis at a significance level of
p < 0.05. The results of the CLSM
experiments were analyzed using
Kruskal-Wallis Test with Tamhane
post hoc analysis at a significance
level of p < 0.05. All statistical anal-
yses were performed by SPSS ver-
sion 16.0 software (SPSS Inc.,
Chicago, IL, USA). The sample size
was calculated based on differences
between “rinse” and “double rinse”
samples. With 0.05 significance level,
the sample size in each treatment
group should be at least six disks.
Nine disks were used for all the
treatment groups.
Results
Formation of mature biofilms on SLA
disks
After three weeks, the biofilm thick-
ness reached about 25–30 lm and
showed the presence of a multitude
of rod and coccoid shape micro-
organisms (Fig. 2A). In areas of arti-
ficial separation, numerous coccoid
organisms were present in the SLA
surface lacunae. Dividing cells and
clusters of bacteria that are closely
packed and interlaced by a well-
developed inter-microbial matrix
were observed in this biofilm model.
Coccoid cells surrounding filamen-
tous organisms suggesting of corn-
cob formations were also seen. These
characteristics are typical of natural
structural network of mature bio-
films. Using the estimates of bacte-
rial loads based on dental plaque
(1 mg contains approximately 108
bacteria), after three weeks of culti-
vation each disks contained about
6 9 107 bacteria.
Effect of chemical treatments on bacterial
counts
Most of the mature biofilm was
easily removed, leaving relatively
small proportion of bacteria on the
SLA titanium surface after the first
set of saline rinses (Fig. 2B). Accord-
ing to our estimates, the loads of
bacteria reduced from about 6 9 107
to 1 9 103 per disk. The remaining
bacteria were mainly located in the
1600051x, 2017, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcpe.12699 by Guatemala - OARE Access, Wiley Online Library on [20/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
405
lacunae of the SLA surface. Coccoid
bacteria dominated with occasional
rod-shaped bacteria. The mean bac-
terial count  SEM per mm2 was
7.4 9 105  0.9 9 105 for the Rinse
group (Figs 2B and 3A), 4.6 9
105  0.6 9 105 for the Double
Rinse group (Figs 2C and 3A),
4.9 9 105  0.6 9 105 for CHX
group (Figs 2D and 3A), 5.5 9
105  0.5 9 105 for the Etch group
(Figs 2E and 3A), and
4.9 9 105  0.7 9 105 for the Tetra-
cycline group (Figs 2F and 3A). The
Kolmogorov-Smirnov distribution
test showed the results of this study
were normally distributed (p =
0.200). Therefore a One-way ANOVA
with post hoc Tuckey test was used
for the statistical analysis. When
comparing the groups, only the Dou-
ble Rinse group removed significantly
more bacteria than the Rinse group
(p = 0.047). However, there were no
significant differences between the
Double Rinse group and the three
chemical agents groups that
received same rinsing effect as the
Double Rinse control group
(Fig. 3A). In addition, distilled
water had a comparable effect to
saline solution (data not shown),
again suggesting that the mechani-
cal effect of rinsing was responsible
for removal of the bacteria and not
the chemical effect.
When comparing the percentage
of bacteria remaining on the disks
using the Rinse group as control
(100% remaining), the Double Rinse
group removed 37.6% more bacteria,
CHX group removed 33.2% more
bacteria, Etch group removed 26.1%
more bacteria and Tetracycline group
removed 33.9% more bacteria,
respectively. The differences were sta-
tistically significant for the Double
Rinse group (p = 0.009), the CHX
group (p = 0.028) and the Tetracy-
cline group (p = 0.027). Only the Etch
group did not remove significantly
more bacteria than the Rinse group
using this method (p = 0.158). Once
again, there were no significant differ-
ences between the Double Rinse
group and disinfectant groups.
Effect of chemical treatments on bacterial
viability
A commercial viability test was used
to find out whether the remaining
bacteria in the SLA lacunae were
 1600051x, 2017, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcpe.12699 by Guatemala - OARE Access, Wiley Online Library on [20/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
406 Dostie et al.

Fig. 2. Scanning electron microscopy images (50009) of the 3-week-old biofilms on SLA titanium disks after treatment with disin-
fecting agents. (A) undisturbed control biofilm; (B) SLA disk after biofilm has been rinsed with 6 9 1 ml of 0.9% NaCl (rinse
group); (C) SLA disk after biofilm has been rinsed with 12 9 1 ml of 0.9% NaCl (double rinse group); (D) SLA disk after biofilm
has been rinsed with 6 9 1 ml of 0.9% NaCl followed by 2 min treatment with 1% chlorhexidine gel and then further rinsed with
6 9 1 ml of 0.9% NaCl (CHX group); (E) SLA disk after biofilm has been rinsed with 6 9 1 ml of 0.9% NaCl followed by 2 min
treatment with 35% phosphoric acid gel and then further rinsed with 6 9 1 ml of 0.9% NaCl (Etch group); (F) SLA disk after bio-
film has been rinsed with 6 9 1 ml of 0.9% NaCl followed by 2 min treatment with tetracycline paste and then further rinsed with
6 9 1 ml of 0.9% NaCl (Tetra group).

either dead or live. Randomly Kolmogorov-Smirnov distribution (p < 0.001). Therefore, Kruskal-
selected areas were used to assess the test showed the results of this study Wallis Test with Tamhane post hoc
percentage of live and dead cells. The were not normally distributed analysis was used for the statistical
© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
 1600051x, 2017, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcpe.12699 by Guatemala - OARE Access, Wiley Online Library on [20/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Decontamination of dental implants 407

Fig. 3. Quantification of bacteria on SLA titanium disks per mm2 area after treatment with disinfecting agents. The treatment
groups have been explained in the Figs 1 and 2 legends. (A) the effect of saline rinses and chemical treatment on bacterial counts
on the SLA disks. Only the double rinse group showed significantly lower bacteria counts compared to the baseline rinse group. (B)
the effect of the C.C.E chemical formula to the bacterial counts on the SLA disks. (C) scanning electron microscopy (SEM) images
of the biofilm contaminated SLA titanium disks after treatment with rinsing with 12 9 1 ml of 0.9% NaCl (Double rinse group);
(D) SEM images of the biofilm contaminated disks after biofilm has been rinsed with 6 9 1 ml of 0.9% NaCl followed by 2 min
treatment with a novel chemical agent (C.C.E; 0.3% cetrimide, 0.1% CHX and 0.5% EDTA in 3% methylcellulose gel) and then
further rinsed with 6 9 1 ml of 0.9% NaCl. The results show mean  SEM. (*p < 0.05).

analysis. The Control, Rinse and
Double Rinse disks showed insignifi-
cant percentage of dead cells with
ratio of live to total cells of 99.6%,
99.6% and 99.4% respectively
(Fig. 4). The CHX and Etch groups
showed, however, a small but signifi-
cant reduction of viable bacteria
within the biofilms compared with the
Control, Rinse and Double Rinse
disks. The proportion of killed cells in
the CHX and Etch was 11.8%
(p = 0.023) and 6.9% (p = 0.017),
respectively. The Tetracycline group
had a proportion of dead cells of
3.9%, which was not significantly
different from all other groups.
Testing of novel chemical treatments on
bacterial counts
Due to ineffectiveness of all the chem-
icals tested, a novel CHX formulation
© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
that contained cetrimide (another
antiseptic that contains quaternary
ammonium salts) and EDTA
(C.C.E.) was experimented. The Kol-
mogorov-Smirnov distribution test
showed the results of this study were
normally distributed (p = 0.002).
Therefore a One-way ANOVA with post
hoc Tukey test was used for the statis-
tical analysis. There was no signifi-
cant difference between the double
rinse and C.C.E. groups (Fig. 3B).
Discussion
The present study showed that multi-
species and mature live biofilms can
be grown on SLA titanium surface.
The thickness of the biofilms on SLA
observed in the present study (25–
30 lm) is narrower than what other
studies reported. Using the same
biofilm growing protocol on
hydroxyapatite collagen coated disks,
Shen et al. (2009) showed that the
thickness of the biofilms reached
155 lm after three weeks of anaero-
bic incubation. This is most likely
caused by the different substrata used
(Rupf et al. 2011, S anchez et al.
2014).
The choice of the chemotherapeu-
tic agents used in this study was
mainly based on their availability in
the general dental practice. These
agents have been reported in the
treatment of peri-implant diseases
(Leonhardt et al. 2003, Charalam-
pakis et al. 2011, de Waal et al. 2013,
2015). However, none of them were
tested on mature multispecies SLA
titanium surfaces. The results pre-
sented in the current study illustrate
that the agents used are not more
effective than the sterile sodium chlo-
ride rinse in removing bacteria from

408 Dostie et al.
Fig. 4. Effect of disinfectants on bacterial cell death. The groups have been explained
in the Fig. 1 legend. The ratios of live to total cells (%) were determined using LIVE/
DEAD Bacterial viability kit. The chlorhexidine (CHX) and 35% phosphoric acid
(Etch) groups showed modest but significant reduction in live bacteria (*p < 0.05).
The results show mean  SEM.
the surface of the SLA titanium disks.
This finding supports previous case
series and clinical trials that showed
no difference in the clinical parame-
ters between disinfectant solutions
and sterile saline rinse after the treat-
ment of peri-implantitis cases
(Zablotsky et al. 1992, Strooker et al.
1998, Romeo et al. 2005, Giardino
et al. 2007, Schwarz et al. 2011, de
Waal et al. 2013). It is quite interest-
ing to notice that the double rinse
group is the only group to have signif-
icantly more bacteria removed from
the SLA disks when compared to the
single rinse group. It is thought that
the mechanical effect of the rinsing
was the most important factor in bio-
film removal. It can also be specu-
lated that the thickness of the
disinfectant gels or pastes applied on
the SLA titanium disks acted as a
barrier, possibly preventing the
mechanical removal effect of the
rinse. An orthopaedic study con-
ducted by Bundy et al. (2001) used jet
impingement technique on titanium
joint surfaces, and showed that bacte-
rial detachment is caused mainly by
pressure and that bacterial adhesion
to metals is more affected by flow
rate. The same group found that
under the influence of jet impinge-
ment forces, bacterial cells attached
to metals detach by rupturing of cell
membranes (Richards et al. 1995).
The results showing that most bac-
teria were still alive were somewhat
disappointing. The best disinfection
in the current study was provided by
1% CHX gel with killing about 12%.
On the other hand, in a study by
Gosau et al. (2010) the least efficient
disinfectant agent was Plax with a
ratio dead to total ranging from 16%
to 39%, while the most efficient was
hydrogen peroxide ranging from 43%
to 92% after chemical agents were
applied for 1 min. on 12-h biofilms
on machined pure titanium substrata.
The same study reported results rang-
ing from 41% to 94% for 0.2%
CHX. The difference between the two
studies can be explained by the matu-
rity of the biofilm. This increase in
resistance is marked for the three-
week-old biofilms and remains similar
for even older biofilms (Shen et al.
2011, Stojicic et al. 2013). Interest-
ingly, this “three-week mark” coin-
cide with the formation of the
distinctive subgingival microbiota
characterized by Gram-negative and
anaerobic bacteria, which becomes
established 3–12 weeks after
supragingival plaque formation, as
described by Listgarten (1994). It can
also be hypothesized that the wetta-
bility capacity of the different chemi-
cal agents and their viscosity (Chen &
Bonaccurso 2014) may also play a
crucial role in the decontamination/
disinfection of any moderately rough
titanium surface. In addition, better
results in our disinfection protocol
might have been achieved with longer
chemical agents exposure.
Cetrimide was shown to weaken
the biofilm’s cohesive forces, disrupt-
ing the inter-microbial matrix respon-
sible for biofilms mechanical stability
(Sim~ oes et al. 2005). Furthermore,
when mixed with CHX, they have a
synergistic activity and have been
shown to kill E. faecalis, associated
with persistent root canal infection,
faster and at smaller concentration
than CHX alone (Portenier et al.
© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
1600051x, 2017, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcpe.12699 by Guatemala - OARE Access, Wiley Online Library on [20/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
2006, Arias-Moliz et al. 2010). The
combination of CHX, cetrimide and
EDTA was thought to provide an
effective chemotherapeutic agent to
reach the deepest lacunae of the SLA
titanium surface to weaken the bio-
film and facilitate its removal, while
killing the remaining bacteria. The
results of the present study, however,
clearly showed the limitations of the
chemical decontamination and disin-
fection of SLA titanium, even with
the enhanced formulation. The
remaining bacteria on the lacunae of
the rough implant surface after chem-
ical decontamination clearly presents
a significant obstacle for regenerative
therapies aiming at re-osseointegra-
tion.
One limitation of this study was
the difficulty to quantify the total
amount of biofilms remaining on the
titanium surfaces. Attempts were
made using ImageJ software to quan-
tify the percentage of the surface
occupy by the remaining biofilms
with shade of gray or shapes analysis.
However, it was not possible to differ-
entiate between the remaining bio-
films and the SLA surfaces. Another
limitation of this study is that only
SLA titanium disks were used as sub-
strata; similar study could evaluate
the effect of saline rinse on different
implant surfaces. Finally, it could be
argued that this in vitro model is not
representative of the in vivo model.
Indeed, the oral cavity provides a
very complex environment, which has
an important impact on the biofilm.
Nevertheless, it is the authors believe
that this mature multispecies biofilm,
grown in anaerobic condition, pro-
vides an excellent model to study
implant decontamination when com-
pared to planktonic and immature
biofilm models presented in the litera-
ture.
Future studies can use the
described study model, mature multi-
species biofilm, to investigate the
effectiveness of other chemicals and
techniques such as abrasive air, laser,
photodynamic therapy and plasma
jet that have also been used for
implant disinfection.
Acknowledgements
In addition, the authors would like
to thank Straumann AG, Basel,
Switzerland for their contribution in
providing the titanium disks used in

preparing the samples for this study
and Heraeus Kulzer LLC, South
Bend, IN, USA for the in-kind
donation of impression material.
References
Arias-Moliz, M. T., Ferrer-Luque, C. M.,
Gonzalez-Rodrıguez, M. P., Valderrama, M. J.
& Baca, P. (2010) Eradication of Enterococcus
faecalis biofilms by cetrimide and chlorhexi-
dine. Journal of Endodontics 36, 87–90.
Bundy, K. J., Harris, L. G., Rahn, B. A. &
Richards, R. G. (2001) Measurement of fibrob-
last and bacterial detachment from biomaterials
using jet impingement. Cell Biology Interna-
tional 25, 289–307.
B€
urgers, R., Witecy, C., Hahnel, S. & Gosau, M.
(2012) The effect of various topical peri-
implantitis antiseptics on Staphylococcus epider-
midis, Candida albicans, and Streptococcus san-
guinis. Archives of Oral Biology 57, 940–947.
Charalampakis, G., Rabe, P., Leonhardt, A. &
Dahlen, G. (2011) A follow-up study of peri-
implantitis cases after treatment. Journal of
Clinical Periodontology 38, 864–871.
Chen, L. & Bonaccurso, E. (2014) Effects of sur-
face wettability and liquid viscosity on the
dynamic wetting of individual drops. Physical
Review E 90, 022401.
Dennison, D. K., Huerzeler, M. B., Quinones, C.
& Caffesse, R. G. (1994) Contaminated
implant surfaces: an in vitro comparison of
implant surface coating and treatment modali-
ties for decontamination. Journal of Periodon-
tology 65, 942–948.
Derks, J., Schaller, D., H akansson, J.,
Wennstr€ om, J. L., Tomasi, C. & Berglundh, T.
(2016) Effectiveness of implant therapy ana-
lyzed in a Swedish population: prevalence of
peri-implantitis. Journal of Dental Research 95,
43–49.
Giardino, L., Ambu, E., Savoldi, E., Rimondini,
R., Cassanelli, C. & Debbia, E. A. (2007) Com-
parative evaluation of antimicrobial efficacy of
sodium hypochlorite, MTAD, and Tetraclean
against Enterococcus faecalis biofilm. Journal of
Endodontics 33, 852–855.
Gosau, M., Hahnel, S., Schwarz, F., Gerlach, T.,
Reichert, T. E. & B€ urgers, R. (2010) Effect of
six different peri-implantitis disinfection meth-
ods on in vivo human oral biofilm. Clinical
Oral Implants Research 21, 866–872.
Leonhardt, A., Dahlen, G. & Renvert, S. (2003)
Five-year clinical, microbiological, and radio-
logical outcome following treatment of peri-
implantitis in man. Journal of Periodontology
74, 1415–1422.
Listgarten, M. A. (1976) Structure of the micro-
bial flora associated with periodontal health
and disease in man: a light and electron micro-
scopic study. Journal of Periodontology 47, 1–
18.
Clinical Relevance
Scientific rationale for the study:
Decontamination of the rough
implant surface is a critical part of
treatment of peri-implantitis. Cur-
rently, there is no proven effective
way to decontaminate the implant
surface. The authors developed
© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Decontamination of dental implants
Listgarten, M. A. (1994) The structure of dental
plaque. Periodontology 2000 5, 52–65.
Marrone, A., Lasserre, J., Bercy, P. & Brecx, M.
C. (2013) Prevalence and risk factors for peri-
implant disease in Belgian adults. Clinical Oral
Implants Research 24, 934–940.
Mir-Mari, J., Mir-Orfila, P., Figueiredo, R., Val-
maseda-Castell on, E. & Gay-Escoda, C. (2012)
Prevalence of peri-implant diseases. A cross-
sectional study based on a private practice
environment. Journal of Clinical Periodontology
39, 490–494.
Mombelli, A., Muller, N. & Cionca, N. (2012)
The epidemiology of peri-implantitis. Clinical
Oral Implants Research 23, 67–76.
Ntrouka, V., Hoogenkamp, M., Zaura, E. & van
der Weijden, F. (2011) The effect of chemother-
apeutic agents on titanium-adherent biofilms.
Clinical Oral Implants Research 22, 1227–1234.
Portenier, I., Waltimo, T., Ørstavik, D. & Haa-
pasalo, M. (2006) Killing of Enterococcus fae-
calis by MTAD and chlorhexidine digluconate
with or without cetrimide in the presence or
absence of dentine powder or BSA. Journal of
Endodontics 32, 138–141.
Richards, R. G., ap Gwynn, I., Bundy, K. J. &
Rahn, B. A. (1995) Microjet impingement fol-
lowed by scanning electron microscopy as a
qualitative technique to compare cellular adhe-
sion to various biomaterials. Cell Biology Inter-
national 19, 1015–1024.
Romeo, E., Ghisolfi, M., Murgolo, N., Chia-
pasco, M., Lops, D. & Vogel, G. (2005) Ther-
apy of peri-implantitis with resective surgery: a
3-year clinical trial on rough screw-shaped oral
implants. Part I: Clinical outcome. Clinical
Oral Implants Research 16, 9–18.
Rupf, S., Idlibi, A. N., Marrawi, F. A., Hannig,
M., Schubert, A., von Mueller, L., Spitzer, W.,
Holtmann, H., Lehmann, A., Rueppell, A. &
Schindler, A. (2011) Removing biofilms from
microstructured titanium ex vivo: a novel
approach using atmospheric plasma technol-
ogy. PLoS ONE 6, e25893.
S
anchez, M. C., Llama-Palacios, A., Fern andez,
E., Figuero, E., Marın, M. J., Leon, R., Blanc,
V., Herrera, D. & Sanz, M. (2014) An in vitro
biofilm model associated to dental implants:
structural and quantitative analysis of in vitro
biofilm formation on different dental implant
surfaces. Dental Materials 30, 1161–1171.
Schwarz, F., Sahm, N., Iglhaut, G. & Becker, J.
(2011) Impact of the method of surface
debridement and decontamination on the clini-
cal outcome following combined surgical ther-
apy of peri-implantitis: a randomized
controlled clinical study. Journal of Clinical
Periodontology 38, 276–284.
Shen, Y., Qian, W., Chung, C., Olsen, I. & Haa-
pasalo, M. (2009) Evaluation of the effect of
two chlorhexidine preparations on biofilm bac-
teria in vitro: a three-dimensional quantitative
analysis. Journal of Endodontics 35, 981–985.
Shen, Y., Stojicic, S. & Haapasalo, M. (2011)
Antimicrobial efficacy of chlorhexidine against
mature multispecies oral biofilm
model on titanium implants and
tested the decontamination efficacy
of tetracycline paste, 1% chlorhexi-
dine gel, 35% phosphoric acid and
saline rinsing.
Principal findings: Rinsing with sal-
ine was effective in removing
1600051x, 2017, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcpe.12699 by Guatemala - OARE Access, Wiley Online Library on [20/07/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
409
bacteria in biofilms at different stages of devel-
opment. Journal of Endodontics 37, 657–661.
Sim~ oes, M., Pereira, M. O. & Vieira, M. J. (2005)
Effect of mechanical stress on biofilms chal-
lenged by different chemicals. Water Research
39, 5142–5152.
Stojicic, S., Shen, Y. & Haapasalo, M. (2013)
Effect of the source of biofilm bacteria, level of
biofilm maturation, and type of disinfecting
agent on the susceptibility of biofilm bacteria
to antibacterial agents. Journal of Endodontics
39, 473–477.
Strooker, H., Rohn, S. & Van Winkelhoff, A. J.
(1998) Clinical and microbiologic effects of
chemical versus mechanical cleansing in profes-
sional supportive implant therapy. The Interna-
tional Journal of Oral & Maxillofacial Implants
13, 845–850.
de Waal, Y. C., Raghoebar, G. M., Huddleston
Slater, J. J., Meijer, H. J., Winkel, E. G. & van
Winkelhoff, A. J. (2013) Implant decontamina-
tion during surgical peri-implantitis treatment:
a randomized, double-blind, placebo-controlled
trial. Journal of Clinical Periodontology 40,
186–195.
de Waal, Y. C., Raghoebar, G. M., Meijer, H. J.,
Winkel, E. G. & van Winkelhoff, A. J. (2015)
Implant decontamination with 2% chlorhexi-
dine during surgical peri-implantitis treatment:
a randomized, double-blind, controlled trial.
Clinical Oral Implants Research 26, 1015–1023.
Wiltfang, J., Zernial, O., Behrens, E., Schlegel,
A., Warnke, P. H. & Becker, S. T. (2010)
Regerative treatment of peri-implantitis bone
defects with a combination of autologous bone
and a demineralized xenogenic bone graft: a
series of 36 defects. Clinical Implant Dentistry
and Related Research 14, 421–427.
Wiltfang, J., Zernial, O., Behrens, E., Schlegel,
A., Warnke, P. H. & Becker, S. T. (2012)
Regenerative treatment of peri-implantitis bone
defects with a combination of autologous bone
and a demineralized xenogenic bone graft: a
series of 36 defects. Clinical Implant Dentistry
and Related Research 14, 421–427.
Zablotsky, M. H., Diedrich, D. L. & Meffert, R.
M. (1992) Detoxification of endotoxin-contami-
nated titanium and hydroxyapatite-coated sur-
faces utilizing various chemotherapeutic and
mechanical modalities. Implant Dentistry 1,
154–158.
Address:
Hannu S. Larjava
Faculty of Dentistry
The University of British Columbia
2199 Wesbrook Mall
Vancouver, BC
Canada V6T 1Z3
E-mail: larjava@dentistry.ubc.ca
majority of the biofilm. None of
the chemicals tested were superior
to saline rinsing.
Practical implications: Better
decontamination techniques are
needed to remove the bacteria from
rough implant surfaces during peri-
implantitis treatment.