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Available online at www.sciencedirect.com

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journal homepage: www.intl.elsevierhealth.com/journals/dema

Microbiome of titanium and zirconia dental

implants abutments

Cássio do Nascimento a,∗ , Murillo Sucena Pita a ,

Emerson de Souza Santos b , Nadia Monesi b , Vinícius Pedrazzi a ,
Rubens Ferreira de Albuquerque Junior a,c , Ricardo Faria Ribeiro a
a Faculty of Dentistry of Ribeirão Preto, Department of Dental Materials and Prosthodontics, Molecular Diagnosis
Laboratory, Av. Café s/n◦ , Monte Alegre, Ribeirão Preto, SP 14040-904, Brazil
b Faculty of Pharmaceutical Sciences of Ribeirão Preto, Department of Clinical Toxicological and Bromatologic

Analysis, University of São Paulo, Av. Café s/n◦ , Monte Alegre, Ribeirão Preto, SP 14040-903, Brazil
c Faculty of Dentistry, McGill University, Strathcona Anatomy & Dent, 3640, University Street, Montreal,

QC H3A 2B2, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Objectives. This study employed culture-independent molecular techniques to extend the
Received 27 April 2015 characterization of the microbial diversity of biofilm associated with either titanium or zir-
Received in revised form 8 July 2015 conia implant-abutments, including not-yet-cultivated bacteria species, and to identify and
Accepted 29 October 2015 quantify species recovered from peri-implantar/periodontal sulci, supragingival biofilm and
Available online xxx the internal parts of implants. Probing depth, clinical attachment level, bleeding on pro-
bing, and marginal bone level were also evaluated over time and correlated with biofilm
Keywords: formation.
Bacteria Methods. Twenty healthy participants were analyzed. DNA-Checkerboard and 16S-rDNA-
Biofilm Pyrosequencing were used to quantify and determine species identity.
Clinical outcomes Results. 161 bacterial taxa representing 12 different phylotypes were found, of which 25%
Dental implants were non-cultivable. Species common to all sites belonged to genera Fusobacterium, Prevotella,
Molecular genetics Actinomyces, Porphyromonas, Veillonella and Streptococcus. While some species were subject-
specific and detected in most sites, other species were site-specific. Moderate to higher levels
of unclassified species were found colonizing titanium-related sites. Pathogenic and non-
pathogenic species were detected colonizing oral sites in both materials. Titanium-related
sites presented the highest total microbial count and higher counts of pathogenic species.
Conclusions. Our results revealed differences regarding microbial diversity and microorgan-
isms counts in oral biofilm associated with titanium or zirconia. The obtained data suggests
a possible relation between microbiological findings and clinical outcomes.
Significance. Next-generation methods of detection have provided new insights on com-
plex microbiota colonizing different sites of oral cavity. The present study demonstrates
relevant differences in the communities and microbial counts colonizing different tested
substrates with consequent significant differences in the clinical-outcomes, suggesting a
probably different mechanism for specific bacterial adhesion.
© 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

∗
Corresponding author at: Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo Av. do Café s/n CEP: 14040-904 Ribeirão
Preto-SP Brazil. Tel.: +55 16 3315 4095; fax: +55 16 3315 0547.
E-mail address: [email protected] (C. do Nascimento).
http://dx.doi.org/10.1016/j.dental.2015.10.014
0109-5641/© 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

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São Paulo (Ribeirão Preto, Brazil). Potential participants were

1. Introduction invited to participate in the study if they: (a) were at least 18
years old; (b) were indicative of a cemented-retained single-
In spite of the high success rates of long-term implant-
unit implant-supported restoration in the anterior maxilla; (c)
supported restorations, recent studies have reported the
were indicative of a cemented-retained single-unit implant-
presence of relevant microbial adhesion on the implant
supported restoration in the posterior maxilla/mandible; (d)
components [1,2]. Microbial colonization of dental implant
possessed the contra-lateral and antagonists teeth; (e) have
assemblies is a consequence of the exposure of the compo-
had no treatment or professional cleaning in the previous
nents to the oral cavity [3,4] and one of the most important
3 months. Additional exclusion criteria were pregnancy, lac-
causes of early and late implant failure is related to the
tation, periodontal or antibiotic therapy in the previous 3
inflammatory process of the surrounding host bone and soft
months, and any systemic condition which could influence
tissues that occurs in response to microbial contamination
the course of periodontal status. The study was approved
[5,6].
by the local ethics committee (CAAE 0066.0.138.000-10) and
Bi-directional bacterial microleakage through the implant-
all the experiments were undertaken with the informed
abutment interface has been reported in both in vitro studies
and written consent of each subject according to ethical
and clinical investigations [3,7]. Microbial colonization of the
principles.
peri-implant sulci and prostheses occurs in approximately
60% of the rehabilitated patients [8] and therefore it consti-
tutes one of the major concerns related to the long term
success of the oral rehabilitation with implant-supported
2.2. Experimental design
restorations [2,5,6]. Oral microbiota represents a potential risk
when associated to two-part dental implant systems. The
Twenty healthy individuals, 17 women and 3 men (mean-age
gaps and cavities inherent to implant-abutment assemblies
45.5 years), fulfilled the study’s criteria. The clinical parameter
may act as traps, harboring bacterial species that can lead to
probing depth was selected as a primary variable in deter-
inflammatory reactions in the peri-implant soft tissues with
mining the progression of peri-implant disease and also for
consequent bone resorption and implant failure [9]. Chrcanon-
determining the sample size (N). To compare two independent
ivc et al. [10] performed a meta-analysis of several studies
groups (Titanium and Zirconia) with repeated measures per-
reported in the literature on the dental implant failure rates.
formed on the three proposed times (baseline, 3 months, and 6
The meta-analysis enrolled a total of 91 studies from 1989 until
months), and considering a standard deviation of 1.26 among
2014. A total of 52 357 dental implants were inserted, with 2224
individuals and 0.89 in the intra-individual analysis (values
failures (4–36%). Periodontitis/peri-implantitis was shown to
estimated from the deviations observed in the applied liter-
be a prevalent risk factor in implant failures.
ature), the sample size provided a statistical power (power of
Structural and topographical variations due to the dif-
study) equal to 84% for the factor “group” and 96% for the factor
ferent materials and manufacturing processes employed to
“time” and “group × time” interaction, with a significance level
fabricate implant components may influence the microbial
of 5%, and magnitude of effect (effect size) of 0.79 for the fac-
adhesion and can lead to significant differences in the formed
tor “group”, and 1.12 for the “time” factor and “group x time”
microbiome [1,11–13]. Nevertheless, studies about the micro-
interaction. Participants were enrolled into 2 groups of 10 par-
bial adhesion on the different abutment materials and the
ticipants each, according to the investigated abutments and
impact of the complex oral biofilm on the clinical param-
manufacturers’ recommendations: the first group (8 women
eters of implant restorations are still not conclusive. More
and 2 men, mean-age 47 years) comprehended individuals
recently, pyrosequencing of PCR-amplified 16S rDNA has pro-
who received a two-part dental implant with a morse taper
vided a more comprehensive way to characterize the oral
connection (Ankylos C/X, Dentsply, USA) in the anterior area
microbiome. In this context, we employed both DNA Checker-
of maxilla and were rehabilitated using esthetic pre-machined
board hybridization and pyrosequencing of PCR-amplified 16S
zirconia abutments (Ankylos Cercon Balance, Dentsply, USA)
rDNA to evaluate the microbial diversity of the biofilm associ-
and the second group (9 women and 1 men, mean-age 48
ated either with titanium or zirconia implant-abutments. The
years) comprehended individuals who received a two-part
microbiological findings were correlated with a set of clinical
dental implant with a morse taper connection (Ankylos C/X,
outcomes (probing depth, clinical attachment level, bleeding
Dentsply, USA) in the posterior area of maxilla/mandible
on probing and marginal bone level) at 3 different time points
and were rehabilitated using pre-machined titanium abut-
up to 6 months after functional loading. The null hypothesis
ments (Ankylos Regular Abutment C/X, Dentsply, USA). In both
tested in this study is that there is no significant difference in
groups, implants were placed at the level of the alveolar bone
the microbial profile from titanium or zirconia abutments.
crest followed by the insertion of transmucosal healing abut-
ments. The healing abutments were different lengths so that
the occlusal surface ended 1.0 mm above the gingival marginal
2. Material and methods level. After 75 days (following manufacturer’ instructions),
all the participants were rehabilitated with a total ceramic
2.1. Participants (in the anterior area) or a metalloceramic (in the posterior
area) single unit cemented-retained restoration. All surgical
Participants were recruited among partially edentate individ- and prosthetic procedures were performed by the same clini-
uals refereed to the prosthodontics clinic of the University of cian.

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2.3. Follow-up and data collection fragillis, Capynocitophaga gingivalis, Campylobacter rectus,
Escherichia coli, Eikenella corrodens, Enterococcus faecalis, Fusobac-
Microbiological samples and clinical data collection were con- terium nucleatum, Fusobacterium periodonticum, Kleibsella
ducted at baseline–implant loading (T0 ), 3-month (T1 ) and Pneumoniae, Lactobacillus casei, Mycoplasma Salivarium, Neis-
6-month (T2 ) after the intervention. seria mucosa, Porphyromonas aeruginosa, Peptostreptococcus
Anaerobios, Porphyromonas endodontalis, Porphyromonas gingi-
2.3.1. Clinical parameters assessment valis, Prevotella intermedia, Prevotella melaninogenica, Parvimonas
At each evaluation time point a complete peri-implantar/ micra, Prevotella nigrencens, Pseudomonas putida, Staphylococ-
periodontal examination using an electronic periodontal cus aureus, Streptococcus constelatus, Streptococcus gordonii,
probe (FP32, Florida Probe, USA) was conducted to record clin- Streptococcus mitis, Solobacterium moreei, Streptococcus mutans,
ical parameters (probing depth, clinical attachment level and Streptococcus oralis, Streptococcus parasanguinis, Staphylococcus
bleeding on probing). Each site (mesial, medial and distal in pasteuri, Streptococcus salivarius, Streptococcus sanguinis, Strep-
buccal and palatal/lingual aspects) was probed twice to reduce tococcus sobrinus, Treponema denticola, Tanerella forsythia and
the potential error in probing angulation and the final mea- Veillonella parvula) and 5 Candida species frequently found
surement for probing depth and clinical attachment level was harboring the oral microbiota of healthy and diseased individ-
a mean of the 2 evaluations. uals were investigated (C. albicans, C. dublinienses, C. glabrata,
Radiographic examinations were conducted at each time C. krusei and C. tropicalis). Descriptive statistics was used to
point to assess the marginal bone level around dental analyze the frequency of positive signals. The normality of
implants. Intraoral radiographs were obtained using the same data was assessed using a normality probability plot and
film (Kodak E-Speed, Kodak, São José dos Campos, São Paulo, Kolmogorov-Smirnov goodness of fit test. The Friedman
Brazil) and X-ray apparatus (Timex 70E, Gnatus, Ribeirão Preto, two-way analysis of variance by rank test for original-level
SP, Brazil). A paralleling technique was performed by means data was used to compare quantitation of each species for
of an oral device which allowed setting the film on the same repeated measures design. When applicable, post hoc analysis
position for all the radiographs taken from each participant. was performed by Wilcoxon test corrected by Dunn method.
The radiographs were digitized using a computerized scan- Differences were considered significant when p < 0.05. The
ning and images were evaluated using the software Image J SPSS 17.0.0 statistical software (SPSS Inc., Chicago, IL, USA)
Tool (Version 3.00 for Windows, University of Texas Health Sci- was used for data analysis.
ences Center, USA). The vertical and horizontal marginal bone
loss was measured using the most coronal point of the implant 2.4.2. Pyrosequencing of PCR-amplified 16S rDNA
as the reference point and the lowest point of marginal bone DNA from clinical samples was extracted according to Smith
around the implant as the bone level. Bone loss was measured et al. [15] and further purified using the QiAmp DNA mini kits
on the mesial and distal sides of the implants. (Qiagen, CA, USA). For multiplex sequencing a total of 18 for-
ward primers, each one with a different 10-mer nucleotide
2.3.2. Microbiological sampling barcode, was employed (Table A.1). 16S rDNA Amplification
Supragingival biofilm samples from the selected prostheses/ was performed in 25 ␮l reactions using the Fast Start High
contra-lateral teeth were recovered with sterile microbrushes. Fidelity PCR System from Roche (USA) and 5–250 ng of DNA.
Subgingival biofilm samples from peri-implantar sulci of PCR cycles were as follows: initial denaturation step of 95 ◦ C,
implants and from periodontal sulci of their contra-lateral for 2 min; 35 cycles of 95 ◦ C, for 30 s, 55 ◦ C, for 30 s and 72 ◦ C, for
teeth were collected with sterile paper points. Each subgin- 60 s; final extension, at 72 ◦ C, for 4 min. Amplicons were puri-
gival sample was a pool of 6 paper points exposed for 30 s in fied using Agencourt AMPure XP kit (Beckman Coulter, USA)
the peri-implant or periodontal sulcus, 3 in the buccal and 3 in and the quality of the purified amplicons was checked using
the palatal/lingual aspects, respectively in mesial, medial and the 2100 Bioanalyzer (Agilent, USA). The amplified libraries
distal positions. Biofilm samples from the internal parts of the were quantified by fluorometry using QuantiFluor® dsDNA
implants and abutments surfaces and screws were collected System (Promega, USA) and equal amounts (1010 molecules)
with sterile microbrushes after isolation of the implant site. of 18 different samples were pooled together for multiplex
Microbiological samples were recorded as follows: Implant- sequencing. Since 36 samples were to be sequenced, we
related sites—(1) supragingival biofilm of prosthesis, (2) performed 2 independent rounds of amplification, sample
subgingival biofilm of peri-implant sulcus, (3) biofilm from the pooling and multiplex sequencing. emPCR was performed
internal parts of implant, and (4) biofilm from implant abut- using the GS Junior emPCR Kit-Lib-L (454/Roche). Sequenc-
ment surfaces; Dental-related sites—(1) supragingival biofilm ing was executed according to the manufacturer’s instructions
of tooth crown, (2) subgingival biofilm of periodontal sulcus. using the Genome Sequencing Junior System (454/Roche).
Samples were stored at −20 ◦ C prior to laboratorial processing.
2.4.3. Sequencing analysis and taxonomic assignment
2.4. Molecular detection of microorganisms Initially low quality sequences, short fragments and
sequences derived from polyclonal amplification gener-
2.4.1. DNA checkerboard hybridization analysis ated during the emPCR step were removed. Subsequently
DNA Checkerboard hybridization procedures were performed sequences were demultiplexed and the data was deposited
as described by do Nascimento et al. [14]. Thirty-eight bac- and analyzed in the Metagenomics RAST server (MG-RAST,
terial species, including putative periodontal pathogens version 3) [16]. Reads were clustered at 97% identity, and the
(Aggregatibacter actinomycetemcomitans a and b, Bacteroides longest sequence was picked as the cluster representative.

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Table 1 – Clinical parameters from periodontal or peri-implantar related sites of individuals over the time after implant
loading.
Titanium

Implant Contra-lateral Tooth

T0 T1 T2 T0 T1 T2
Participants 10 10 10 10 10 10
Age (years-old, mean ± SD) 48 ± 3.6 48 ± 3.6 48 ± 3.6 48 ± 3.6 48 ± 3.6 48 ± 3.6
Gender (male/female) 9/1 9/1 9/1 9/1 9/1 9/1
Sampling Sites 120 120 120 120 120 120
Probing Depth (mm, mean ± SD) 1.79 ± 1.55A 2.25 ± 0.83B 2.05 ± 0.87 A 2.00 ± 0.70A 1.70 ± 0.64A 1.81 ± 0.59A
Clinical Attchment Level (mm, mean ± SD) 0.13 ± 0.27a 0.41 ± 0.86c 0.27 ± 0.60a 0.24 ± 0.52a 0.10 ± 0.30a 0.15 ± 0.40a
Bleeding on Probing (%) 1.66 23.33* 6.66 10.00 16.66 23.33*

Zirconia

Implant Contra-lateral Tooth

T0 T1 T2 T0 T1 T2
Participants 10 10 10 10 10 10
Age (years-old, mean ± SD) 47 ± 4.2 47 ± 4.2 47 ± 4.2 47 ± 4.2 47 ± 4.2 47 ± 4.2
Gender (male/female) 8/2 8/2 8/2 8/2 8/2 8/2
Sampling Sites 120 120 120 120 120 120
Probing Depth (mm, mean ± SD) 3.00 ± 1.68B 2.05 ± 0.91A 2.12 ± 0.70A 1.84 ± 0.68A 1.53 ± 0.81A 1.64 ± 0.61A
Clinical Attchment Level (mm, mean ± SD) 0.18 ± 0.61a 0.12 ± 0.39a 0.16 ± 0.42a 0.18 ± 0.34a 0.03 ± 0.19b 0.05 ± 0.23b
Bleeding on Probing (%) 5.00 33.33* 16.66 11.66 31.66* 18.33

Student-t test detected no significant differences comparing ages between Titanium and Zirconia groups (p = 0.8657). Different letters in the
rows mean significant differences detected by Kruskal-Wallis followed by Dunn’s post-test; p < 0.05; A < B; c > b > a.
∗
Differences sought by Chi-square test for trend; p < 0.05.

A BLAT search for the cluster representative was then per- (p > 0.05). Lower percentages of bleeding sites were recorded
formed against the ITS, Greengenes, Silva LSU, M5RNA, RDP and no relation could be found between bleeding on probing
and Silva SSU databases. A minimum cutoff of 98% and max- and deep sulci.
imum e-Value Cutoff of 1 × 10−5 were applied, which were The marginal bone loss (mm, mean ± SD) for each type of
sufficient for species identification. A minimum alignment abutment is shown in Table 2. The changes in bone levels were
length cutoff of 200 bp was used for analysis. Phylogenetic sought using Two-way repeated ANOVA. Implants restored
tree and heatmaps of taxonomic composition were generated with titanium abutments showed the highest amount of total
using the gplots library with relative frequencies per sample. bone loss after 6 months of functional loading (1.25 ± 0.27)
when compared to implants restored with zirconia abutments
(0.92 ± 0.36; p < 0.001). For both components main bone loss
3. Results occurred during the first 3 months of loading.

3.1. Clinical parameters 3.2. Microbial genome counts from DNA checkerboard
analysis
All the implants achieved the required initial stability after
surgery in all participants. The periodontal/peri-implantar All the implant-related sites and teeth assessed by DNA
status consisted of 1440 data collected from 20 participants. checkerboard showed microbial colonization over time. All
The mean values of Probing Depth (mm, ±SD), Clinical Attach- the 43 evaluated target species were encountered in the
ment Level (mm, ±SD) and Bleeding on Probing (%) of all investigated sites during the study and presented signifi-
sampling sites are shown in Table 1. There were significant cant differences in relation to genome counts (Friedman test
differences in probing depth and attachment level amongst followed by Dunn’s post-test; p < 0.0001). Overall, the micro-
the investigated samples time (Friedman; p < 0.0001). Overall, bial colonization in the titanium-related sites (including the
titanium-related sites presented an increase of probing depth inner parts of the implants, abutment surface, peri-implantar
after 3 months of loading returning to probing depths similar sulcus and supragingival biofilm) was higher than coloniza-
to the recorded at baseline after 6 months (p < 0.05). Differently, tion in the zirconia-related sites (p < 0.001). T. denticola, T.
implants restored with zirconia showed a striking reduction forsythia, P. gingivalis, F. nucleatum, P. intermedia and A. acti-
of probing depth after 3 months (p < 0.05), maintaining values nomycetemcomitans were recovered in relevant counts and
after 6 months similar to those found for titanium implants. showed significant differences between materials and sites
Titanium and Zirconia groups did not differ after 3 months of over time (p < 0.001) (Table 3). Other species associated to peri-
function. Contra-lateral teeth showed similar values for both implantitis (such as E. corrodens, P. nigrescens and P. micra), as
groups in all periods of sampling. The clinical attachment lev- well as Streptococcus spp. and opportunistic Candida spp., were
els were similar for titanium and zirconia groups over time found in higher counts in the titanium-related sites (p < 0.001).

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Table 2 – Marginal bone level reduction (in mm, mean ± SD) around dental implants restored with titanium or zirconia
abutments after 3 and 6 months of loading.
Follow-up

0–3 months 3–6 months Total Bone Loss

Titanium Abutments 0.93 ± 0.36 0.32 ± 0.12 1.25 ± 0.27a
Zirconia Abutments 0.78 ± 0.17 0.14 ± 0.06 0.92 ± 0.36

0: Baseline-Measures taken at implant loading (60 days after surgery).

a
Differences detected by Two-Way ANOVA; p < 0.05.

When species were analyzed as a pool of microorganisms, of the sequence reads was 326 bases. According to MG-RAST,
without discriminating among target species, Friedman test 99,4% of the sequences comprised bacterial rDNA. A total of
with Dunn’s Multiple Comparisons test showed significant 12 phyla, 104 genera and 596 species were identified and dif-
differences in the total genome count between the tested ferences between implant-related sites and teeth bacterial
materials, sites and periods of sampling, with the highest val- communities were observed at all phylogenetic levels.
ues also recorded for titanium-related sites (p < 0.0001; Fig. 1). In both materials (titanium and zirconia), the most preva-
lent species were Gram-negative bacteria belonging mainly
3.3. Microbiological profile by 16S rDNA to the phylum Proteobacteira, which includes the Neisseriaceae
pyrosequencing and Campylobacteraceae families, followed by the phyla Firmi-
cutes and Actinobacteria and Bacteroidetes (Figs. 2 and 3). The
The mean number of sequence reads and base pairs per library majority of the microbiota associated to zirconia was related
were, respectively, 5683 and 1,162,153 bps. The average length to the Proteobacteira phylum. The genera Klebsiella, Enterococcus,

Table 3 – Mean (×105) and standard deviation (±SD) of genome counts and respective p-values assessed by DNA
Checkerboard in the tested groups.
T. Denticola T. forsythia P. gingivalis F. nucleatum P. intermedia A. actinomy-
cetemcomitans

Mean ±SD Mean ±SD Mean ±SD Mean ±SD Mean ±SD Mean ±SD
Titanium Implant 5.37 0.31 4.76 0.36 5.08 0.43 3.79 0.32 5.34 0.26 3.77 0.33
T0 Abutment 5.35 0.31 4.28 0.34 6.28 0.52 2.92 0.3 5.32 0.29 4.26 0.37
Peri-implantar 1.95 0.41 0* 0 0* 0 0* 0 0* 0 0* 0
Sulcus
Supragingival 4.74 0.39 3.89 0.37 5.06 0.47 4.76 0.37 4.98 0.33 4.3 0.41
Implant 5.96 0.43 5.58 0.41 5.02 0.49 5.20 0.37 4.91 0.36 3.62 0.43
T1 Abutment 5.65 0.41 5.25 0.40 5.65 0.51 4.62 0.33 5.65 0.27 3.11 0.39
Peri-implantar 2.74 0.35 0* 0 2.26 0.31 2.35 0.29 2.85 0.29 2.04 0.27
Sulcus
Supragingival 6.37 0.42 5.74 0.42 7.50 0.56 6.21 0.41 6.22 0.31 4.76 0.29
Implant 4.31 0.37 4.31 0.37 6.04 0.52 4.53 0.41 5.01 0.39 4.26 0.33
T2 Abutment 4.60 0.38 4.03 0.36 4.03 0.41 3.23 0.39 4.77 0.41 3.12 0.37
Peri-implantar 2.31 0.36 2.24 0.29 2.72 0.32 2.79 0.33 2.87 0.27 2.19 0.26
Sulcus
Supragingival 6.46 0.44 6.32 0.43 7.15 0.47 6.17 0.32 7.47 0.36 4.68 0.32
Implant 4.69 0.41 2.32 0.27 2.66 0.31 3.70 0.29 4.27 0.29 1.85 0.29

Zirconia T0 Abutment 3.28 0.32 2.43 0.24 1.86 0.22 0* 0 1.86 0.23 2.12 0.26
Peri-implantar 0* 0 0* 0 2.11 0.20 0* 0 0* 0 0* 0
Sulcus
Supragingival 2.89 0.31 2.19 0.26 0* 0 1.87 0.27 2.49 0.27 2.48 0.27
Implant 2.99 0.33 1.99 0.21 2.74 0.27 2.64 0.29 2.28 0.25 3.54 0.31
T1 Abutment 4.15 0.33 2.85 0.27 0 0 2.90 0.29 0* 0 3.71 0.28
Peri-implantar 2.85 0.30 2.28 0.23 2.07 0.21 2.43 0.30 0* 0 0* 0
Sulcus
Supragingival 3.55 0.31 2.63 0.26 0* 0 0* 0 3.09 0.27 2.49 0.24
Implant 2.26 0.29 1.91 0.22 2.45 0.22 2.14 0.23 2.33 0.21 0* 0
T2 Abutment 2.24 0.27 2.19 0.23 2.21 0.29 2.32 0.26 2.29 0.26 0* 0
Peri-implantar 0* 0 2.57 0.25 0* 0 1.98 0.21 0* 0 0* 0
Sulcus
Supragingival 3.71 0.31 2.25 0.27 2.38 0.24 2.13 0.21 2.10 0.23 2.09 0.23

p-value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

∗
No significant differences sought by Friedman Test followed by Dunn’s post-tests; p > 0.05.

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subgingival biofilm samples in both titanium/zirconia sites

and teeth, whereas the the Firmicutes phylum (including Strep-
tococcus spp.) were found in all investigated sites. Within the
phylum Proteobacteira, the genus Neisseria and Eikenella were
found both in zirconia and titanium groups.
Periodontal pathogenic species were also found coloniz-
ing subgingival samples and implant-related sites from both
titanium and zirconia groups. Porphyromonas gingivalis, T. denti-
cola and Tanerella forsythia were recorded at low prevalence (up
to 22%) in samples from the inner parts of the implants and
abutments in both titanium and zirconia groups at all investi-
Fig. 1 – Checkerboard Hybridization data. Median (with gated times and in the subgingival sulci of contra-lateral teeth
maximum and minimum values), upper and lower at T0. Aggregatibacter actinomycetemcomitans, a Gram-negative
quartiles of total genome counts (×105 cells) of the 43 species commonly associated to aggressive periodontitis and
evaluated species recovered from the oral biofilms formed frequently found in implant-related sites, was recovered from
in the periodontal/peri-implantar sulci and implant-related the subgingival biofilm in both implant and contra-lateral
sites in both Titanium and Zirconia groups (I: inner parts of teeth at T0 and 3 and 6 months after loading in the titanium
the implants; A: abutment surface; Sub: subgingival sulcus; group, whereas in the zirconia group it was only found only
Supra: supragingival biofilm; Different letters mean at T0. Fusobacterium nucleatum was more prevalent in sam-
significant differences detected by Friedman test followed ples from the titanium groups when compared to samples
by Dunn’s post-test; p < 0.0001). from the zirconia group. Moderate to high reads of unclas-
sified sequences (derived from Bacteria) were found in all
samples.
Serratia and Stenotrophomonas were found exclusively in the Fig. 4 illustrates the percentage of reads from implant-
zirconia-related samples, including supra and subgingival related samples categorized as unclassified sequences. The
samples from contra-lateral teeth. The phyla Actinobacteria highest percentages of unclassified sequences were found
and Bacteroidetes were mostly found associated to supra and in Titanium-related samples, with the higher values for the

Fig. 2 – Circular maximum likelihood phylogenetic tree at genus level. The external band shows genera colored by phylum.
The inner band shows significant mean differences between evaluated sites and times of sampling. The magnitude of the
difference between sites and periods are indicated by the height of the bars.

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Fig. 3 – Heatmap showing microbiabundances within the 454 sequencing dataset at phylum level (1: Peri-implant Zirconia
T0; 2: Implant Titanium T0; 3: Implant Zirconia T2; 4: Peri-implant Zirconia T2; 5: Implant Zirconia T1; 6: Peri-implant
Titanium T1; 7: Abutment Zirconia T2; 8: Implant Titanium T2; 9: Abutment Titainum T2; 10: Abutment Titanium T0; 11:
Peri-implant Titanium T0; 12: Implant Zirconia T0; 13: Abutment Zirconia T0; 14: Abutment Titanium T1; 15: Abutment
Zirconia T1; 16: Peri-implant Titanium T2; 17: Peri-implant Zirconia T1; 18: Peri-implant Titanium T1).

Fig. 4 – Percentage (%) of unclassified sequence reads at genus level.

peri-implant sulcus at T0 (9.3%) and the inner parts of the have suggested that roughness is a major factor favoring the
implants at T1 (9.3%), followed by abutment surfaces at T0 microbial adhesion on titanium surfaces [17,18]. On the other
(7.9%) and T1 (7.2%). The mean percentage of unclassified hand, zirconia has been described to have a potentially lower
sequences for all the Zirconia-related sites was 2.26%. In the susceptibility for bacterial adhesion [19,20] and some studies
control samples, the mean percentages for Titanium and Zir- have suggested that the free energy surface is more important
conia groups were, respectively, 4.4% and 2.1%. Unassigned in biofilm formation on zirconia surfaces [21,22].
sequences were also found in lower prevalence in zirconia The DNA Checkerboard analysis revealed moderate counts
group (below 0.3%). of all 43 investigated target species. Species related to the
initial formation of the oral biofilm (such as L. casei and Strep-
tococcus spp.), as well as putative pathogens associated with
4. Discussion periodontitis/peri-implantitis were found in samples recov-
ered from both implants and contra-lateral teeth, suggesting
In the current study, we used two DNA based methodologies the translocation of microbiota from the remaining teeth to
to characterize the oral biofilm formed on different implant- the implant sites. Bacterial species commonly associated to
abutment surfaces and their restorations since implant mentioned diseases, were found in relevant amounts in the
loading until 6 months of function. The null hypothesis tested peri-implantar sulcus, abutments and in the inner parts of the
in this investigation was rejected once our results revealed implants from both titanium and zirconia groups.
relevant differences between the microbial composition of The pyrosequencing data agrees with the DNA Checker-
the biofilm formed on the titanium and zirconia-related oral board results in the sense that it shows that certain species
sites, both with respect to the number of recovered microor- are more common in a given material, and also provides a
ganisms detected by DNA Checkerboard hybridization and broader insight of overall community differences and com-
in what concerns the phylogenetic profile determined by plexity, thereby expanding the knowledge about both putative
16S pyrosequencing. Our results agree with previous studies pathogenic and unclassified species in the oral microbiota.
[12,13] that have shown that the titanium concentrates more Throughout our follow up, we recorded a total of 161 bac-
biofilm mass and higher amounts of microorganisms. Studies terial taxa representing 12 different bacterial phyla in 6 oral

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sites across all the 20 participants, of which 25% of the implants restored with titanium abutments was higher than
identified taxa were non-cultivable. Available data in the liter- those found for zirconia, which is indicative of a possible
ature indicate that there are more than 700 bacterial species relation between probing depth and high index of bacterial
or phylotypes colonizing the oral cavity, of which over 50% adhesion.
have not yet been cultivated [23]. The characterization of Our results reveal that pathogenic microorganisms are
the oral biofilm related to the different materials is impor- present in the implant-related sites since the initial load-
tant because it is necessary to identify both the cultivable ing and persist over time. Since bi-directional microbial
and not-yet-cultivated bacterial flora in a given environ- microleakage into and from implant-abutment interface have
ment before we can ascribe association to health or disease been associated to late implant failure [5,6] it is important
status. to follow procedures that avoid or minimize this coloniza-
Interestingly, higher incidences of unclassified species, tion. In this sense, our results are relevant and contribute
which may be related to non-cultivable or undescribed taxon, to the characterization of differences in the microbial adhe-
were found in samples of the Titanium group. These species sion on the implant-related oral sites using different materials
may act as a potential risk factor in the etiology of peri- and also to provide an insight about the impact of micro-
implantitis and may be a rationale for the clinical outcomes bial colonization on the clinical outcomes. These results
recorded for Titanium group, in which we have observed a should be interpreted with caution and should not be gen-
relevant bone loss during the first 3 months. eralized.
Although pathogenic species were found associated to The results reported in this clinical trial provide a more
both materials, no signals of clinical inflammation were complete insight on the oral microbiome that develops in
detected. Nevertheless, the clinical parameters assessed over implant-supported restorations with either titanium or zirco-
the time seem to reflect the microbiological findings. Partici- nia. We not only confirmed previous studies describing the
pants treated with titanium abutments presented an increase species associated with peri-implantitis but we also extended
of probing depth after 3 months of loading, while participants these findings to a large number of additional species that
treated with zirconia abutments showed a significant reduc- should be investigated in future studies. The characterization
tion at the same period. Conversely, the microbial genome of major changes in the oral microbiome composition provides
counts in the subgingival samples from titanium-related sulci the basis for further understanding of host-microbe interac-
were shown to increase 3 months after loading, which may tions in health and disease.
be suggestive of a positive relation. However, our findings
do not allow us to determine whether the higher counts of
Conflict of interest
microorganisms in titanium sites caused the increase in pro-
bing depth or were a consequence of this condition. We should
None of the authors does report any conflict of interest related
consider that increase of probing depth in titanium group
to the study or products involved.
may be related to the differences in the depth of transmu-
cosal pathway in the posterior region. In fact, the deep sulci
may facilitate microbial adhesion and colonization leading Acknowledgements
to inflammatory reactions. In addition, the microflora pro-
file, quantity of biofilm formation and hygiene quality after This work was supported by a grant from Fundação de
brushing may vary depending on the oral cavity region. Prasad Amparo à Pesquisa do Estado de São Paulo - FAPESP (Processes
et al. [24] showed, prior to and immediately after toothbrush- 2010/10442-2 and 2010/12830-0). The authors thank Prof. Dr.
ing, that anterior teeth harbored lower mean amounts of Sérgio Akira Uyemura and Prof. Dr. Valdir Antonio Muglia
biofilm than posterior teeth, irrespective of gender, subject, for their helpful technical and clinical support. The authors
or arch. The main rationale for that was the deep sulci and declare that they have no conflict of interest.
difficulty of hygiene. In contrast, previous investigations of
our group, found for both biofilm formation and microbial
count no significant differences between anterior and poste- Appendix A.
rior area for titanium or zirconia substrates [12,13]. Likewise,
in the present study, the marginal bone resorption around See Table A.1.

Table A.1 – Oligonucleotides used for the amplification of 16 rDNA. Both forward and reverse primers contained
454/Roche sequencing adaptors (italic), while forward primers contained barcode sequences (underline).
Oligonuleotide primer Sequence
Forward with barcode 1 CCATCTCATCCCTGCGTGTCTCCGACTCAGACGAGTGCGTGGAGGCAGCAGTRRGGAAT
Forward with barcode 2 CCATCTCATCCCTGCGTGTCTCCGACTCAGACGCTCGACAGGAGGCAGCAGTRRGGAAT
Forward with barcode 3 CCATCTCATCCCTGCGTGTCTCCGACTCAGAGACGCACTCGGAGGCAGCAGTRRGGAAT
Forward with barcode 4 CCATCTCATCCCTGCGTGTCTCCGACTCAGAGCACTGTAGGGAGGCAGCAGTRRGGAAT
Forward with barcode 5 CCATCTCATCCCTGCGTGTCTCCGACTCAGATCAGACACGGGAGGCAGCAGTRRGGAAT
Forward with barcode 6 CCATCTCATCCCTGCGTGTCTCCGACTCAGATATCGCGAGGGAGGCAGCAGTRRGGAAT
Forward with barcode 7 CCATCTCATCCCTGCGTGTCTCCGACTCAGCGTGTCTCTAGGAGGCAGCAGTRRGGAAT

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Table A.1 – (Continued)

Oligonuleotide primer Sequence
Forward with barcode 8 CCATCTCATCCCTGCGTGTCTCCGACTCAGCTCGCGTGTCGGAGGCAGCAGTRRGGAAT
Forward with barcode 9 CCATCTCATCCCTGCGTGTCTCCGACTCAGTAGTATCAGCGGAGGCAGCAGTRRGGAAT
Forward with barcode 10 CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTCTATGCGGGAGGCAGCAGTRRGGAAT
Forward with barcode 11 CCATCTCATCCCTGCGTGTCTCCGACTCAGTGATACGTCTGGAGGCAGCAGTRRGGAAT
Forward with barcode 12 CCATCTCATCCCTGCGTGTCTCCGACTCAGTACTGAGCTAGGAGGCAGCAGTRRGGAAT
Forward with barcode 13 CCATCTCATCCCTGCGTGTCTCCGACTCAGCATAGTAGTGGGAGGCAGCAGTRRGGAAT
Forward with barcode 14 CCATCTCATCCCTGCGTGTCTCCGACTCAGCGAGAGATACGGAGGCAGCAGTRRGGAAT
Forward with barcode 15 CCATCTCATCCCTGCGTGTCTCCGACTCAGATACGACGTAGGAGGCAGCAGTRRGGAAT
Forward with barcode 16 CCATCTCATCCCTGCGTGTCTCCGACTCAGTCACGTACTAGGAGGCAGCAGTRRGGAAT
Forward with barcode 17 CCATCTCATCCCTGCGTGTCTCCGACTCAGCGTCTAGTACGGAGGCAGCAGTRRGGAAT
Forward with barcode 18 CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTACGTAGCGGAGGCAGCAGTRRGGAAT
Reverse CCTATCCCCTGTGTGCCTTGGCAGTCTCAGCTACCRGGGTATCTAATCC

references titanium and zirconia abutment surfaces. Clin Oral Implants

Res 2014;25:337–43.
[14] do Nascimento C, de Albuquerque Jr RF, Monesi N,
[1] Yamane K, Ayukawa Y, Takeshita T, Furuhashi A, Yamashita Candido-Silva JA. Alternative method for direct DNA probe
Y, Koyano K. Bacterial adhesion affinities of various implant labeling and detection using the checkerboard hybridization
abutment materials. Clin Oral Implants Res 2013;24: format. J Clin Microbiol 2010;48:3039–40.
1310–5. [15] Smith GL, Socransky SS, Smith CM. Rapid method for the
[2] Dabdoub SM, Tsigarida AA, Kumar PS. Patient-specific purification of DNA from subgingival microorganisms. Oral
analysis of periodontal and peri-implant microbiomes. J Microbiol Immunol 1989;4:47–51.
Dent Res 2013;92:168–75. [16] Meyer F, Paarmann D, D’Souza M, Olson R, Glass EM, Kubal
[3] Quirynen M, Vogels R, Pauwels M, Haffajee AD, Socransky M, Paczian T, Rodriguez A, Stevens R, Wilke A, Wilkening J,
SS, Uzel NG, van Steenberghe D. Initial subgingival Edwards RA. The metagenomics RAST server—a public
colonization of ‘pristine’ pockets. J Dent Res 2015;84: resource for the automatic phylogenetic and functional
340–4. analysis of metagenomes. BMC Bioinformatics 2008;9:
[4] Subramani K, Jung RE, Molenberg A, Hammerle CH. Biofilm 386.
on dental implants: a review of the literature. Int J Oral [17] Quirynen M, De Soete M, van Steenberghe D. Intra-oral
Maxillofac Implants 2009;24:616–26. plaque formation on artificial surfaces. In: Lang NP, Karring
[5] Palma-Carrió C, Maestre-Ferrín L, Peñarrocha-Oltra D, T, Lindhe J, editors. Proceedings of the 3rd european
Peñarrocha-Diago MA, Peñarrocha-Diago M. Risk factors workshop on periodontology. Berlin: Quintessence Books;
associated with early failure of dental implants. A literature 1999. p. 102–29.
review. Med Oral Patol Oral Cir Bucal 2011;16:514–7. [18] Quirynen M, De Soete M, van Steenberghe D. Infectious risks
[6] Sakka S, Baroudi K, Nassani MZ. Factors associated with for oral implants: a review of the literature. Clin Oral
early and late failure of dental implants. J Invest Clin Dent Implants Res 2002;13:1–19.
2012;3:258–61. [19] Scarano A, Piattelli M, Caputi S, Favero GA, Piattelli A.
[7] Cosyn J, Van Aelst L, Collaert B, Persson GR, De Bruyn H. The Bacterial adhesion on commercially pure titanium and
peri-implant sulcus compared with internal implant and zirconium oxide disks: an in vivo human study. J Clin
suprastructure components: a microbiological analysis. Clin Periodontol 2004;75:292–6.
Implant Dent Relat Res 2011;13:286–95. [20] Hisbergues M, Vendeville S, Vendeville P. Zirconia:
[8] Lindhe J, Meyle J. Peri-implant diseases: Consensus Report of established facts and perspectives for a biomaterial in
the Sixth European Workshop on Periodontology. J Clin dental implantology. J Biomed Mat Res Part B Appl Biomater
Periodontol 2008;35:282–5. 2009;88:519–29.
[9] Jansen VK, Conrads G, Richter EJ. Microbial leakage and [21] Bürgers R, Hahnel S, Reichert TE, Rosentritt M, Behr M,
marginal fit of the implant-abutment interface. Int J Oral Gerhard T, Handel G, Gosau M. Adhesion of Candida
Maxillofac Implants 1997;12:527–40. albicans to various dental implant surfaces and the
[10] Chrcanovic BR, Albrektsson T, Wennerberg A. Dental influence of salivary pellicle proteins. Acta Biomater 2010;6:
implants inserted in male versus female patients: a 2307–13.
systematic review and meta-analysis. J Oral Rehabil [22] Zhao B, van der Mei HC, Subbiahdoss G, de Vries J,
2015;42:709–22. Rustema-Abbing M, Kuijer R, Busscher HJ, Ren Y. Soft tissue
[11] Bussche HJ, Rinastiti M, Siswomihardjo W, van der Mei HC. integration versus early biofilm formation on different
Biofilm formation on dental restorative and implant dental implant materials. Dent Mater 2014;30:716–27.
materials. J Dent Res 2010;89:657–65. [23] Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining
[12] do Nascimento C, Pita MS, Pedrazzi V, de Albuquerque Junior the normal bacterial flora of the oral cavity. J Clin Microbiol
RF, Ribeiro RF. In vivo evaluation of Candida spp. adhesion 2005;43:5721–32.
on titanium or zirconia abutment surfaces. Arch Oral Biol [24] Prasad KV, Sreenivasan PK, Patil S, Chhabra KG, Javali SB,
2013;58:853–61. DeVizio W. Removal of dental plaque from different regions
[13] do Nascimento C, Pita MS, Fernandes FH, Pedrazzi V, de of the mouth after a 1-minute episode of mechanical
Albuquerque Junior RF, Ribeiro RF. Bacterial adhesion on the oralhygiene. Am J Dent 2011;24:60–4.

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