MICROBIAL RECOLONIZATION OF THE INTERNAL
SURFACES OF THE IMPLANT-ABUTMENT JUNCTION AFTER DISINFECTION WITH IODINE
SOLUTION: A PILOT STUDY
Ferrari RB*, De Lorenzo JL**, Ferrari DS***, Shibli JA****, Sendyk WR*****
ABSTRACT: Infiltration of organic
fluids and microorganisms at the abutment/implant interface may result in bacterial infection of the peri-implant tissues. Internal
colonization of periodontal pathogens may be originated by bacteria trapped
during installation or by penetration of abutment/implant leakage. However,
there is few data on microbial recolonization in this interface. The aim of
this pilot study was to detect periodontal pathogens in the internal area of
dental implants after disinfection with iodine solution. Eight implants selected for this
preliminary evaluation. Before bacterial plaque sample
collection, the prosthetic elements (crown, abutment-screw, and abutment) of
the selected implants and the internal area of the implant were rinsed in a
mixture of 0.02% iodine-alcohol. At this time plaque samples
were taken after disinfection (baseline), and 30 and 90 days after-therapy.
Microbiological evaluation for Actinobacillus
actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Treponema denticola was performed by culture media and polymerase
chain reaction (PCR). After disinfection none of the target periodontal
pathogens could be detected. However, F. nucleatum,
T. denticola and P. intermedia,
P. gingivalis, P. intermedia,
and C. rectus were detected at 30 and 90 days
post-therapy, respectively. In conclusion, the disinfection of the abutment/implant
interface did not prevent bacterial contamination, and this leakage serves as a
reservoir of periodontal pathogens.
KEYWORDS: Dental
implants. Periodontal pathogens. Peri-implantitis. Microbiology; Leakage. Microgap.
RESUMO: A
infiltração de fluidos e microrganismos na interface implante/conector
protético pode resultar em infecção dos tecidos periimplantares. A colonização
interna dos conectores protéticos pode ocorrer durante a instalação dos
implantes ou pela microfenda presente na interface implante/conector protético.
Entretanto, existem poucos dados sobre a recolonização microbiana nesta
interface. Logo, o objetivo deste estudo piloto foi avaliar a detecção de
patógenos periodontais na área interna dos implantes após desinfecção com
solução de iodo. Oito implantes de 4 pacientes foram selecionados para esta
avaliação preliminar. Antes da coleta microbiológica, os componentes protéticos
(coroa, conector e parafuso) dos implantes selecionados e da sua porção interna
foram irrigados com uma mistura de 0,02% de álcool e iodo. Neste momento,
amostras microbiológicas foram obtidas logo após a desinfecção (inicial), 30 e
90 dias após terapia. A avaliação microbiológica para Actinobacillus
actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Fusobacterium
nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Tannerella
forsythia and Treponema denticola foi
realizada por meio de cultura e reação em cadeia da polimerase (PCR). Após
desinfecção, nenhum dos microrganismos pode ser detectado. F.
nucleatum, T. denticola, P. intermedia, P. gingivalis, P. intermedia, e C.
rectus foram detectados aos 30 e 90 dias após terapia. Conclui-se que a
desinfecção da interface do conector/implante não evitou a contaminação bacteriana,
e esta interface pode funcionar como reservatório de patógenos periodontais.
PALAVRAS-CHAVE: Implantes dentais. Patógenos periodontais. Peri-implantite. Microbiologia. Infiltração.
Micro-fendas.
INTRODUCTION
The
use of dental implants in oral rehabilitation has gained importance in daily
clinical practice. Despite the many advances, however, dental
implant failures have been reported1,2.
These failures can be attributed not only to bacterial contamination of the
peri-implant area but also may be attributed to dental implant design3-6.
Dental implants of a two-stage surgery dictate the insertion of the dental
implant body at or below the alveolar crestal bone. Once the prosthetic
abutment is positioned, a microgap at the dental implant interface is formed
that lies close to or just below the bony crest.
The microbial infiltration
and colonization at the abutment/implant interface can cause bad breath and
inflammation of peri-implant tissues6-9.
Earlier studies have investigated the microbial colonization inside the dental
implants10-12. Later in vitro and
in vivo studies demonstrated the
penetration of periodontal pathogens along implant components. Bacterial
species such as Actinobacillus
actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis andPrevotella intermedia were detected in
the internal area of the implant9,12,13.
In
addition, previous investigations utilizing two-piece implants presented showed
that peri-implant soft tissue develops a zone of inflammatory cells in connective
tissue below peri-implant epithelium11,12.
Microbial leakage at the abutment/implant interface is the most probable source
of the contamination in that area.7-12 This
contamination is assumed to originate from bacteria trapped during implant
installation or from penetration of the fixture-abutment interface, where the
microgap is related to bacterial leakage10.
Disinfection
with povidine-iodine, as well as solutions with iodine solutions, has been used
as an antiseptic in dentistry due to its microbial effect against Gram-negative
and Gram-positive bacteria, fungi, virus and protozoans13-17. In
addition, the solutions that contain iodine can act on periodontal pathogens
such as A. actinomycetemcomitans, P. gingivalis, P. intermedia and F. nucleatum14,17. Several studies have shown the bactericidal
effect as an adjunct to periodontal treatment in gingivitis, maintenance
subjects, refractory and chronic periodontitis, subjects with chronic
neutropenia associated with periodontal diseases, and furcation lesions17-22.
So
far, studies that evaluate recolonization of this area after disinfection are
scarce. The aim of this pilot study was to detect, in situ, the presence of periodontal pathogens inside of the dental
implant area that receive the abutment screw after disinfection with 0.02%
alcohol-iodine solution.
MATERIALS AND METHODS
Subject selection
Four
subjects, females, between 27 to 71 years of age (mean age 51.05+11.92
years) were enrolled in this pilot study. All subjects had at least two single
implant-supported crowns over two-stage dental implants, in function for at
least 6 months. These implants have no clinical and radiographical signs of
peri-implantitis such as bleeding on probing, suppuration, and bone loss >3mm.
Subjects
were excluded if they had taken antibiotics or anti-inflammatory drugs within 3
months prior to the clinical examination, had received periodontal or
peri-implant therapy within 3 months, had periodontal diseases, had a chronic
medical disease or condition, and if they were smokers.
The
study protocol was explained to each subject and signed informed consent was
obtained. The study was approved by the local Committee on
Research Involving Human Subjects.
Abutment/implant interface disinfection and
microbiological evaluation
The
prosthetic restorations were removed from the subjects, and the prosthetic
components (bridge/crown, abutments and abutment screws) as well as the
internal area of the implant were disinfected with a cotton swab soaked in a
0.02% alcohol-iodine solution and then rinsed with saline solution. After drying and
isolation with cotton rolls, microbiological plaque samples (baseline) were
taken from the internal surface of the abutment/implant interface using 2
sterile paper points left in position during 30s. One paper point was placed
in a microtube containing 3.5 ml of VMGA III (Viability-Medium Göteborg
Anaerobically) transport medium23 while the other was preserved in a
microtube containing sterilized Milli-Q water. All samples were collected
by the same operator and coded by an assistant for blind identification. The
microbiologic procedures were initiated within 24 hours.
After
microbiological collection, the abutments and prosthetic restorations were
repositioned. Torque of 20 N/mm was applied to the abutment screw, and the
prosthetic restoration was repositioned, with the proper torque (10 N/mm)
applied over the screw of the crown. Standardized intra-oral periapical
radiographs were taken to verify the adaptation of
the abutment and prosthetic restoration over the dental implant.
Following
that, the dental implants were randomly assigned to 2 group observations: 30
and 90 days after therapy. The remaining microbiological evaluations were
performed after 30 days and 90 days after the first collection, as performed in
the baseline. Additional therapy or disinfection was not employed until the
last microbiological plaque sample.
Microbiological evaluation
The
samples were centrifuged for 60s and serially diluted 10-fold in peptonated
water to between 10-1 and 10-6 for quantitative
evaluation of CFU/ml and to obtain isolated colonies for qualitative
identification. Aliquots of 0.1 ml of the dilutions were plated onto supplemented blood agar (SBA) and Tryptic
Soy-Serum-Bacitracin-Vancomycin agar (TSBV) in a standard manner. SBA plates
were incubated in anaerobic jars containing a mixed gas atmosphere (90%N2,
10%CO2) at 37oC for 10 to 15 days. TSBV agar plates were
incubated in a 5 to 10% CO2 atmosphere for 5 days at 37oC.
The bacterial species were identified from anaerobic cultures based on
gram-stain, aerotolerance, colony morphology esculin hydrolysis,24,25 nitrate reduction, indole production,
[alpha]-glucosidase and N-benzoyl-DL-arginine-2-naphthylamide (BANA)
hydrolysis,26 oxidase and catalase activities. Total viable count
(TVC) and cultivable microbiota, including Porphyromonas
gingivalis, Prevotella intermedia, Fusobacterium
nucleatum, Eikenella corrodens, Tannerella forsythia, and Campylobacter rectus. Actinobacillus actinomycetemcomitans
detection was performed based on colony morphology and positive catalase tests27.
In
addition to selective culture media, the polymerase chain reaction (PCR)
amplification of the conserved region of 16S ribossomal DNA was also tested for
periodontal pathogens including A. actinomycetemcomitans (forward primer
5’-GCTAATACCGCGTAGAGTCGG-3’ and reverse 5’-ATTTCACACCTCACTTAAAGGT-3’), C.
rectus (forward primer 5’-TTTCGGAGCGTAAACTCCTTTTC-3, and reverse
5’-TTTCTGCAAGCAGACACTCTT-3’), E.
corrodens (forward primer 5’-CTAATACCGCATACGTCCTAAG-3’ and reverse
5’-CTACTAAGCAATCAAGTTGCCC-3’), P.
intermedia (forward primer 5’-TTTGTTGGGGAGTAAAGCGGG-3’ and reverse
5’-TCAACATCTCTGTATCCTGCGT-3’), P.
gingivalis (forward primer 5’-AGGCAGCTTGCCATACTGCG-3’ and reverse
5’-ACTGTTAGCAACTACCGATGT-3’), T.
forsythia (forward primer 5’-GCGTATGTAACCTGCCCGCA-3’ and reverse
5’-TGCTTCAGTGTC AGTTATACCT-3’), and Treponema
denticola (forward primer 5’-TAATACCGAATGTGCTCATTTACAT-3’ and reverse
5’-TCAAAGAAGCATTCCCTCTTCTTCTTA-3’). All these PCR primers were obtained commercially
(Gibco BRL, São Paulo, SP, Brazil). Between 30 to 100ng of genomic DNA was
added to the PCR mixture which contained 1µmol/L of the primers, 2.5U of Taq
polymerase in 1x buffer and 0.2mmol/L of dCTP, dGTP, dATP, and dTTP in a total
volume of 50µL. Amplification was performed for 30 cycles of 30 seconds at 95oC,
30 seconds at 55oC and 30 seconds at 72oC in thermocycler
(Pekin Elmer, Gene Ampl PCR System, Norwalk, CT). Positive and negative
controls were included with each set. The negative control includes all the PCR
reagents except for the sample DNA. The positive control contained all the PCR
reagents together with positive controls for the target periodontal pathogens.
Twenty µL of each PCR reaction mixture was electroforesed in 1.0% agarose gel
in TBE buffer, and the amplification products were visualized under 302nm
ultraviolet light, on ethidium bromide-stained gels.
RESULTS
Microbiological
data were available for analysis from 8 sites/implants in 4 subjects (2 sites
per subject), in a total of 16 microbiological samples (8 microbial samples at
baseline; 4 microbial samples at 30 and 90 days). At baseline (after
disinfection with 0.02% iodate-alcohol), none of the microbiological samples
were able to detect the target periodontal pathogens. Therefore, at 30 days, P. intermedia, F. nucleatum, and T.
denticola were detected in 25%, 25% and 50% of the sites, respectively.
P. gingivalis and C. rectus were not detected at baseline. However, at 90 days, these
2 periodontal pathogens were detected in 100% and 25% of the sites,
respectively. A. actinomycetemcomitans,
E. corrodens, and T. forsytia were not detected for any of
the dental implants in this study.
DISCUSSION
In
our study, we verified the infiltration of bacteria in the abutment/implant
interfaces in dental implants of a two-stage surgery after disinfection with
iodine solution. Because of a potent antiseptic and present low cost, the
iodine solution showed very good results as an adjunct in periodontal treatment14,21,22. It was demonstrated that proliferation of
bacteria in the abutment/implant interface and inside the implant where the
prosthesis screw-type causes a fetid odor and tasting issues,28
and is a major dissatisfaction factor.
The
relevance of microbial penetration is clinically limited in view of the good
long-term results of two-stage surgery implant systems, since the development
of peri-implant disease is not related only to the presence of microorganisms,
but also to the quantitative prevalence of the periodontal pathogens.
It
is very important to identify the presence of the microorganisms directly
involved with periodontal and peri-implant diseases
in the microgap between the implant and the abutment screw, an anaerobic environment
that fosters the colonization and proliferation of microorganisms that could,
by means of fluid diffusion, reach the peri-implant
tissues and compromise the implant’s long-term success. It is also important to
achieve a manner to prevent this proliferation, since it is very difficult to
avoid the penetration of fluids and bacteria in the dental
implant and the area between prosthetic screws.
Only
5 of 8 target pathogens evaluated in our study were detected: P. gingivalis,
P. intermedia, F. nucleatum, T. denticola, and C. rectus. However, the occurrence of contamination
by P. gingivalis and C. rectus was verified only in the samples
collected from groups on the 90th day.
P.
gingivalis and P. Intermedia are frequently associated with the induction and
progression of peri-implantitis, as well as with periodontal diseases23.
F. nucleatum
and C. recuts which were also identified on some dental implant have also
been associated with peri-implant diseases, according to previous studies23,29-31.
Complementary, other investigations have associated the increase in
peri-implant bone loss with the detection of these microorganisms23,31-35.
The
absence of A. Actinomycetemcomitans
and T. denticola is not in accordance
with the previous studies that evaluated subgingival samples in peri-implant
defects32,34,35. However, we could
speculate that the leakage between abutment/implant may harbor the same
microbiota that occurs in subgingival environment. The difference between the
results of this study and the aforementioned studies is possibly related to
study design, microbial sample collection (curettes or paper points), use of
clorhexidine and antibiotics, and different microbiologic methods (culture
media, PCR and DNA probes).
There
are few studies, particularly in vivo, oriented
to shedding light on the issue of contamination of the internal components of
the dental implants. Our results confirm
the previous studies regarding the species identified inside the implant.9
In addition, the aforementioned authors have also
demonstrated that colonization occurs not only during dental implant placement
but also results from infiltration after prosthetic abutment placement. Our
results agree with these features, since after disinfection of the area inside
the implant as well as the abutment and abutment screws, there were no
periodontal pathogens at baseline.
The
results presented in our research could be matched with the work performed by
Groenendijk et al.36, who achieved some
results similar to the ones we achieved on the 30th day of
observation. Nonetheless, the aforementioned researchers have not extended the
observation period and did not identify the species found in the culture media so that we could compare the results with the
results achieved in our study.
Furthermore,
disinfection with 0.02% iodine solution demonstrated that it reduced or
eliminated the target periodontal pathogens at least that were evaluated in
this study at baseline. However, these data should be considered with caution
due the sample size utilized in this pilot study and also due to absence of a
control group (dental implant without disinfection). In addition, the
possibility that reduction of bacterial density in a fluid that filled the area
inside the implants can also caused by physical removal of the microorganisms
prior to the second sample collection by a stream of iodate-alcohol while
staining and suctioning, as it is well known that bacteria are quite loosely
attached to the infragingival surfaces or abutment/implant interface30.
Finally,
the presentation and analysis of the results achieved through our study may
represent a significant and valuable contribution to knowledge about the
occurrence of infiltrations in the external-hexagon abutment/implant interface
and about bacterial biofilm formation in the implant’s inner space, which may
increase the risk of peri-implantitis. Another aspect
that became clear is the need for new studies aimed at finding better resources
to minimize the infiltration and/or the microbial colonization of periodontal
pathogens in this area.
CONCLUSIONS
ACKNOWLEDGEMENTS
Dr.
Shibli was supported by grant# 301527/2006-7 from National Council of Research
(CNPq) Brazil.
DISCLOSURE
The
authors claim to have no financial interest in any company or any products
mentioned in this study.
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