UNIVERSIDADE DE SÃO PAULO Faculdade de Odontologia de Ribeirão Preto

Departamento de Materiais Dentários e Prótese Laboratório de Diagnóstico Odontológico Molecular

Trabalho de Conclusão de Curso de Graduação apresentado à Comissão Assessora para

Avaliação dos Trabalhos de Conclusão de Curso da FORP-USP / Ano 2018

Nome da aluna: Flávia Corrêa Raffaini

Nome do Orientador: Cássio do Nascimento

Modalidade: Pesquisa Científica

Título do Trabalho: Caracterização do biofilme microbiano inicial formado sobre

conectores protéticos de titânio ou zircônia em restaurações unitárias de implante

Agência de Fomento: Bolsa de Iniciação Científica PIBIC Edital 2016/2017 (Projeto

2016-2855)

Formato de Apresentação do TCC: Artigo Científico publicado na língua inglesa

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BiofoulingThe Journal of Bioadhesion and Biofilm Research

ISSN: 0892-7014 (Print) 1029-2454 (Online) Journal homepage: http://www.tandfonline.com/loi/gbif20

Genome analysis and clinical implications of thebacterial communities in early biofilm formationon dental implants restored with titanium orzirconia abutments

Flávia Correa Raffaini, Alice Ramos Freitas, Thalisson Saymo Oliveira Silva,Tarsis Cavagioni, Jessica Felix Oliveira, Rubens Ferreira Albuquerque Junior,Vinícius Pedrazzi, Ricardo Faria Ribeiro & Cássio do Nascimento

To cite this article: Flávia Correa Raffaini, Alice Ramos Freitas, Thalisson Saymo Oliveira Silva,Tarsis Cavagioni, Jessica Felix Oliveira, Rubens Ferreira Albuquerque Junior, Vinícius Pedrazzi,Ricardo Faria Ribeiro & Cássio do Nascimento (2018) Genome analysis and clinical implicationsof the bacterial communities in early biofilm formation on dental implants restored with titanium orzirconia abutments, Biofouling, 34:2, 173-182, DOI: 10.1080/08927014.2017.1417396

To link to this article: https://doi.org/10.1080/08927014.2017.1417396

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BIOFOULING, 2018VOL. 34, NO. 2, 173182https://doi.org/10.1080/08927014.2017.1417396

Genome analysis and clinical implications of the bacterial communities in early biofilm formation on dental implants restored with titanium or zirconia abutments

Flávia Correa Raffaini, Alice Ramos Freitas, Thalisson Saymo Oliveira Silva, Tarsis Cavagioni, Jessica Felix Oliveira, Rubens Ferreira Albuquerque Junior, Vinícius Pedrazzi, Ricardo Faria Ribeiro and Cássio do Nascimento

Faculty of Dentistry of Ribeirão Preto, Department of Dental Materials and Prosthodontics, University of São Paulo, Ribeirão Preto, Brazil

ABSTRACTThis cross-sectional study aimed to identify and quantify up to 42 target species colonizing the early biofilm of dental implants restored with titanium or zirconia abutments. A total of 720 samples from 20 healthy individuals were investigated. Biofilm samples were collected from the peri-implant sulci, inner parts of implants, abutment surfaces and prosthetic crowns over a functioning period of 30 days. Checkerboard DNA–DNA hybridization was used for microbial detection and quantitation. Clinical characteristics (probing depth, bleeding on probing, clinical attachment level and marginal bone loss) were also investigated during the monitoring period. Genome counts were low at the implant loading time point for both the abutment materials, and increased over time. Both the titanium and the zirconia groups presented similar microbial counts and diversity over time, and the microbiota was very similar to that colonizing the remaining teeth. Clinical findings were consistent with a healthy condition with no significant difference regarding marginal bone loss between the two materials.

Introduction

Implant-retained dental restorations have become a relia-ble treatment for the replacement of one or several miss-ing teeth (Muddugangadhar et al. 2015; Howe 2017). The reason for this success is research in surface treatments, implant designs, materials, and techniques, which have increased the demand for dental implant therapy. In gen-eral, the literature reports long-term cumulative survival/success rates ranging from 90.6 to 100% for treatments with dental implants including single-unit or partial/com-plete restorations (Setzer and Kim 2014; Moraschini et al. 2015; Thoma et al. 2017). Treatment success is fairly high even for implants placed in individuals with a previous history of periodontal disease (survival rates ranging from 79.22% up to 100%). However, previously contaminated sites are associated with a higher incidence of biological complications, which may result in implant loss (Sousa et al. 2016; Veitz-Keenan and Keenan 2017).

Although satisfactory follow-up data have been reported for dental implants, implant failure due to bio-film-associated infections can occur in healthy sites even

after osseointegration (Lang et al. 2000). The oral biofilm is normally colonized by a polymicrobial community in which different bacterial species coexist synergistically in a structured extracellular matrix. Implants and their components are immediately colonized by oral biofilm bacteria when exposed to the oral cavity and, in a few days, the subgingival microbiota is similar to that found on the remaining teeth (van Winkelhof et al. 2000). Bacterial species have been found in implants as early as 30 min after their placement in the oral cavity (Fürst et al. 2007). It is a well-established consensus that microorganisms are the primary etiological factor in the development of peri-implantitis. If early bacterial colonization of dental implants is not controlled, it may result in suppuration and bleeding, loss of alveolar bone, and formation of pockets around dental implants (Salvi et al. 2017).

An inherent problem of misfit between implant and abutment in the two-stage dental implant systems result in microgaps that act as a reservoir for the adhesion and growth of oral microorganisms (Passos et al. 2013). Typical coloniz-ers found in the early formation of oral biofilm are members of the genera Streptococcus, Actinomyces and Veillonella.

KEYWORDSMicrobiology; prosthodontics; bacterial adhesion; clinical assessment

ARTICLE HISTORYReceived 18 August 2017 Accepted 5 December 2017

© 2018 Informa UK Limited, trading as Taylor & Francis Group

CONTACT Cássio do Nascimento cassionasc@forp.usp.br

174 F. C. RAFFAINI ET AL.

study was performed in compliance with the Good Clinical Practice, the Declaration of Helsinki and local legal and regulatory requirements. The study was con-ducted in the dental clinic of the Faculty of Dentistry of Ribeirão Preto (University of São Paulo, Brazil). Approval for implant surgery and sampling was obtained from the local Research Ethics Committee (CAAE 0066.0.138.000–10) and all procedures were done after informed and written consent was signed by each subject according to ethical principles.

Twenty partially edentate individuals recommended for cement-retained single-unit implant-supported res-toration were included in this investigation. Individuals were invited to participate if they were at least 18 years old, selected for an immediate implant placement in the anterior or posterior region of maxilla or mandible and possessed contra-lateral and antagonist teeth. Exclusion criteria were pregnancy, lactation, periodontal or antibi-otic therapy in the previous three months, and any sys-temic condition that could influence periodontal status.

Surgical and prosthetic procedures and sampling were performed by a certified clinician. Participants (17 women and three men; mean age 45.5 years) were enrolled into two groups of 10 participants each, according to the inves-tigated abutments. All the participants received a two-part dental implant with a morse taper connection (Ankylos C/X, Dentsply, USA). One group was comprised of eight women and two men (mean age 47 years) who received implants in the anterior area of the maxilla and were reha-bilitated using esthetic pre-machined zirconia abutments (Ankylos Cercon Balance, Dentsply). The other group included nine women and one man (mean age 48 years) who received implants in the posterior area of maxilla or mandible and were rehabilitated using pre-machined titanium abutments (Ankylos Regular Abutment C/X, Dentsply). Titanium abutments are the gold standard for implant rehabilitation of the posterior area of the oral cavity due to their mechanical properties, biocompatibility and stability. However, they may cause an unnatural appear-ance due to the metal showing through the soft tissue in esthetic areas. Zirconia abutments are indicated for the anterior maxilla due to their esthetic properties, as their color can be selected according to the color of neighboring teeth. In addition, they cause less gingival discoloration than titanium abutments and result in an esthetically pleas-ing gingival smile line. Implants were inserted following a strictly aseptic two-stage open-flap surgical technique. Briefly, implants were placed at the level of the alveolar bone crest followed by the insertion of transmucosal heal-ing abutments. The healing abutments had different lengths so that the occlusal surface ended 1.0 mm above the gingi-val margin level. After 75 days, the implants were exposed and a single unit cement-retained restoration was placed.

These bacteria are capable of co-aggregating with other species, creating attachment sites favoring adhesion and growth of other microorganisms. As the biofilm matures, species are able to develop mixed communities with late col-onizers, including periodontopathogenic species (Diaz et al. 2006). Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia and Treponema denticola are associated with periodontitis and frequently found at peri-implantitis sites (Padial-Molina et al. 2016; Pérez-Chaparro et al. 2016).

Since the impact of implant loss on patient lives is also high, the improvement in the performance of implant- retained restorations remains a challenge. Strategies include the development of substratum materials, abut-ment surface coatings, or mechanical barriers to prevent or minimize biofilm formation and bacterial leakage through the implant–abutment interface and the optimization of antimicrobial therapies. Implants with smooth surfaces have been shown to present a greater incidence of early failure when compared with rougher surfaces in which failure rates increase over time (Fürst et al. 2007). The potential advantage of zirconia compared with titanium abutments have been related to the electric conductivity of zirconia and its surface free energy that disfavors bacterial adhesion. Some studies have reported less biofilm accu-mulation and less pronounced inflammation around zir-conia abutments (Scarano et al. 2004; Decidi et al. 2006). However, the results are not conclusive and the peri-im-plant microbiota have not been completely characterized.

Investigations on bacterial adhesion and biofilm for-mation in dental implants are clinically relevant as these results can contribute to the maintenance of healthy peri-implant tissues and the treatment of established peri-implantitis. Data on the microbial profile of the early biofilm formed in different implant abutments and its impact on clinical outcomes are still not conclusive. Thus, the aim of the present study was to characterize the differences between titanium- and zirconia-related microbiomes in the very early stage of implant coloni-zation and relate these findings to clinical outcomes. The checkerboard DNA–DNA hybridization method was used to identify and quantify up to 42 microbial species after seven and 30 days of implant loading. The null hypoth-esis was that there are no differences in the subgingival microbiota of different substratum materials and that the microbiota do not impact clinical outcomes at the very early stage of implant function.

Material and methods

Participant enrolment and study design

This cross sectional study aimed to compare the microbio-logical profile of the subgingival sulci of implant- supported restorations using titanium or zirconia abutments. The

BIOFOULING 175

Titanium abutments were restored with metal-ceramic crowns (cobalt-chrome alloy veneered with feldspathic porcelain) while zirconia abutments were restored with all-ceramic crowns (CAD-CAM Lava Frame framework ceramic and Lava Ceram veneer ceramic, 3 M ESPE, USA).

Follow-up and data collection

Microbiological sampling and clinical data collection were conducted at three different time points: T0 (baseline – implant loading), T1 (after seven days of function), and T2 (after 30 days of function). The contra-lateral tooth of the implant was set as the control site. First, supragin-gival biofilm samples were recovered from restorations and teeth crowns using sterile microbrushes. Afterwards, subgingival biofilm samples from peri-implant and perio-dontal sulci were collected with sterile endodontic paper points. Each subgingival sample was a pool of six paper points inserted for 30 s in the peri-implant or periodon-tal sulcus, three in the buccal and three in the palatal/lingual aspects, respectively in mesial, medial and distal positions. Biofilm samples from the internal parts of the implants and abutments surfaces were also collected with sterile microbrushes. Biofilm samples from the mucosa surrounding the implant were also taken at the pre-sur-gical time point. All the microbiological sampling was performed after isolating the sampling area with cotton rolls and gentle drying with an air syringe. All the bio-film samples were transferred to individual microtubes containing 150 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 7.6) followed by the addition of 150 ml of 0.5 M NaOH for cell lysis and DNA extraction. Microtubes were stored at –20°C prior to genome-based identification.

The electronic periodontal Florida Probe (FP32, Florida Probe, USA) was used to record the clinical parameters (probing depth, clinical attachment level and bleeding on probing) at each evaluation time point. Mesial, medial and distal aspects of implants and teeth were probed twice to reduce the potential error in probing angulation, and the final measurement for probing depth and clinical attachment level was the mean of the two evaluations. Radiographic examinations were also performed to assess the marginal bone level around dental implants over time. A parallel technique was conducted by means of an oral device for standardized positioning of the film in all par-ticipants. Radiographs were digitized using computerized scanning and images were evaluated using the software Image J Tool (Version 3.00 for Windows, University of Texas Health Sciences Center, USA). Vertical and hori-zontal marginal bone loss was measured from the most coronal point of the implant to the marginal bone level around the implant. Bone loss was measured on the mesial and distal sides of the implants.

Identification and quantification of biofilm

The microbial profile of the investigated samples was assessed by DNA checkerboard hybridization analysis. Thirty-seven bacterial species, including putative per-iodontal pathogens (Aggregatibacter actinomycetem-comitans a and b, Bacteroides fragillis, Campylobacter rectus, Capynocitophaga gingivalis, Eikenella corrodens, Enterococcus faecalis, Escherichia coli, Fusobacterium nucleatum, F. periodonticum, Klebisella pneumoniae, Lactobacillus casei, Mycoplasma salivarium, Parvimonas micra, Peptostreptococcus anaerobius, Porphyromonas aeruginosa, P. endodontalis, P. gingivalis, Prevotella inter-media, P. melaninogenica, P. nigrescens, Pseudomonas putida, Solobacterium moorei, Staphylococcus aureus, S. pasteuri, Streptococcus constellatus, S. gordonii, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. salivarius, S. san-guinis, S. sobrinus, Tannerella forsythia, Treponema den-ticola, and Veillonella parvula) and five Candida species (C. albicans, C. dublinienses, C. glabrata, C. krusei, and C. tropicalis) were investigated. Biofilm samples were pro-cessed as described by do Nascimento et al. (2010). Briefly, after thawing at room temperature, the samples were vor-texed for 2 min and the paper points were removed. The samples were boiled for 5 min and after cooling, 800 μl of 5 M ammonium acetate were added to each microtube. The contents were applied into the extended slots of a MiniSlot apparatus (Immunetics, Cambridge, MA, USA), and then concentrated onto a 15 × 15 cm nylon membrane (Hybond N + Amersham Biosciences) followed by bak-ing at 80°C for 2 h. For standard references, mixtures of genomic DNA corresponding to either 105 or 106 bacterial cells of each target species were assembled, denatured, precipitated and applied into two standard comparative lanes. The number of microorganisms recovered from clinical samples are reported in counts by comparing the chemiluminescent intensity signals against standard lanes. Chemoluminescent hybridization signals were detected by exposing the membrane to ECL Hyperfilm-MP (GE Healthcare, UK). The Hyperfilm images were digitized and quantification of microbial counts was analyzed with TotalLab Quant software (TotalLab Ltd, Newcastle upon Tyne, UK).

Data analysis

The outcome variables, and microbiological and clinical parameters were summarized as means and standard devi-ations (SDs) or medians and quartile values. Due to the dependence structure of longitudinal data and cluster-ing within participants, data were compared by nonpar-ametric mixed model regression analysis. The R-library ‘nparLD 2.1’ (Noguchi et al. 2012) was used to perform

176 F. C. RAFFAINI ET AL.

Figure 1 illustrates the median, maximum and mini-mum values, and the upper, and lower quartiles of the total number of microorganisms harvested from the experi-mental sites. Significant differences were found between the medians over time (p < 0.0001). Genome counts of either implant-related sites or subgingival/supragingival samples were significantly reduced at the implant loading time point (T0) for both abutment materials. Conversely, the values recorded after seven and 30 days of function were similar to those found at the pre-surgical time point in both groups. Data from contralateral teeth presented a similar distribution to the implants. Titanium, zirconia and teeth presented a similar number of genome counts over time. The tested materials did not appear to influ-ence the total amount of microorganisms adhered to the investigated surfaces. Subgingival samples showed lower genome counts than supragingival and implant-related samples.

Figures 2 and 3 illustrate the microbial profile of the 42 target species detected on titanium and zirconia-related sites and teeth surfaces. All the investigated species were found colonizing implant surfaces or subgingival/suprag-ingival sulci over time. Most species were detected on both teeth and implant-related surfaces. As shown in Figures 2 and 3, the distribution of the data confirms that the micro-bial profile of early-colonized implants is quite similar to that of the remaining teeth. All the implant-related sites (inner parts of implants, abutment surface, peri-implant sulcus and supragingival biofilm of implant-supported restorations) harbored microorganisms upon exposure to the oral cavity (implant loading). Low to moderate counts of target species were recorded for both implants and teeth. Wald-type, ANOVA-type, and modified ANOVA-type statistics for whole-plot factors indicate a significant interaction between relative effects (species, time point, and sampling site; p < 0.0001). The main dif-ferences between different abutment materials could be found at implant loading; only nine target species were found colonizing subgingival biofilm of implants (Table 3). The lowest genome counts were found at implant load-ing (T0), increasing over time with no significant differ-ence between seven (T1) and 30 days (T2) of function for most of the species. As the biofilm matured, the data indicated that most of the species seemed to remain con-stant over time while a few species seemed to increase or decrease at specific sites but with no significant difference. S. sobrinus and S. parasanguinis were not detected in the peri-implant sulci of zirconia abutments after 30  days, while peri-implant sulci of titanium harbored all target species. Periodontopathogenic species such as P. gingivalis, T. forsythia and T. denticola, as well as Aggregatibacter spp. and Prevotella spp., were found colonizing both materials after seven days of implant function.

the Brunner–Langer nonparametric analysis of longitu-dinal data in factorial experiments (Brunner and Langer 1999). The global effect of participant, substratum, time point, and their interaction were first tested. The calcu-lated effects resulting from the regressions were adjusted for multiple comparisons using Bonferroni’s correction. Statistical significance was set at 0.05 probability level. Analyses were performed using R Statistical Software Package with the Stats library 3.4.0 (R Foundation for Statistical Computing, Vienna, Austria).

Results

Clinical outcomes

Primary stability was achieved for all the implants after surgery and no further complication was reported after loading. A total of 720 sampling sites were assessed over time. They consisted of the peri-implant or periodontal samples collected from implant-related sites and con-tralateral teeth. The mean values of probing depth (mm, ±SD), clinical attachment level (mm, ±SD) and bleeding on probing (%) of all sampling sites are displayed in Table 1. Overall, the peri-implant and periodontal status remained unchanged over time. Few differences were found between the titanium and zirconia groups. Regarding probing depth, peri-implant sulci from zirconia abutments had higher mean values at baseline (T0) but reduced over time (p < 0.05). Also, implants restored with zirconia pre-sented higher values of probing depth when compared with their contra-lateral teeth (p  <  0.05). Bleeding on probing increased significantly over time for both tested materials and contralateral teeth. However, no relationship was found between bleeding on probing and deep sulci. Clinical attachment level did not change over time.

Marginal bone level reduction (mm, mean  ±  SD) around dental implants restored with titanium or zirconia abutments after seven and 30 days of loading is shown in Table 2. Factorial model ANOVA showed no significant difference among medians from different groups over time (Brunner–Langer nonparametric mixed model approach; ANOVA-type statistic).

Genome-based identification

Data from checkerboard DNA–DNA hybridization anal-ysis were reported in two different ways: (1) by providing the microbial counts after assessing a pool of all 42 target microorganisms without discriminating between different species (median and interquartile range of all species) and (2) by providing the individual microbial counts of each target species (median and interquartile range of each species).

BIOFOULING 177

Tabl

e 1.

 Clin

ical

out

com

es fr

om p

erio

dont

al o

r per

i-im

plan

t rel

ated

site

s of i

ndiv

idua

ls ov

er ti

me

afte

r im

plan

t loa

ding

.

Not

es: D

iffer

ent u

pper

case

lett

ers i

n th

e ro

ws m

ean

signi

fican

t diff

eren

ces b

etw

een

inte

rgro

ups d

etec

ted

by n

onpa

ram

etric

mul

tiple

com

paris

ons b

y th

e Br

unne

r–La

nger

mod

el; p

 < 0

.05;

A <

 B.

Diff

eren

t low

erca

se le

tter

s in

the

row

s mea

n sig

nific

ant d

iffer

ence

s bet

wee

n in

trag

roup

det

ecte

d by

non

para

met

ric m

ultip

le co

mpa

rison

s by

the

Brun

ner–

Lang

er m

odel

; p <

 0.0

5; A

 > B

.* D

iffer

ence

s sou

ght b

y no

npar

amet

ric m

ultip

le co

mpa

rison

s by

the

Brun

ner–

Lang

er m

odel

; * p

 < 0

.05;

**

p <

 0.0

1; T

(0):

Impl

ant l

oadi

ng; T

(1):

seve

n da

ys; T

(2):

30 d

ays.

Tita

nium

Zirc

onia

impl

ant

Cont

ra-la

tera

l too

thim

plan

tCo

ntra

-late

ral t

ooth

T(0)

T(1)

T(2)

T(0)

T(1)

T(2)

T(0)

T(1)

T(2)

T(0)

T(1)

T(2)

Part

icip

ants

1010

1010

1010

1010

1010

1010

Age

(yea

rs,

mea

n ±

SD)

48 ±

3.6

A48

± 3

.6A

48 ±

3.6

A48

± 3

.6A

48 ±

3.6

A48

± 3

.6A

47 ±

4.2

A47

± 4

.2A

47 ±

4.2

A47

± 4

.2A

47 ±

4.2

A47

± 4

.2A

Gend

er

(fem

ale/

mal

e)

9/1

9/1

9/1

9/1

9/1

9/1

8/2

8/2

8/2

8/2

8/2

8/2

Sam

plin

g sit

es60

6060

6060

6060

6060

6060

60

Prob

ing

dept

h (m

m,

mea

n ±

SD)

2.06

± 1

.52A a

2.26

± 1

.44A a

1.66

± 0

.79A a

2.15

± 0

.79A a

1.86

± 0

.81

A a

1.76

±

0.72

A a

3.18

± 1

.84B a

2.31

±

1.11

A a

2.22

±

0.90

A a

2.07

± 0

.77A b

1.64

±

0.61

A b

1.66

±

0.75

A b

Clin

ical

at

tach

men

t le

vel (

mm

, m

ean

±SD

)

0.13

± 0

.38A a

0.30

± 0

.56A a

0.33

± 0

.60A a

0.30

± 0

.64

A a

0.13

± 0

.43

A a

0.25

± 0

.57

A a

0.12

± 0

.61

A a

0.22

± 0

.46

A a

0.16

± 0

.37

A a

0.12

± 0

.33

A a

0.14

± 0

.45

A a

0.03

± 0

.19

A a

Blee

ding

on

prob

ing

(%)

1.66

13.3

3 *

20.0

0 **

10.0

0 *

6.66

21.6

6 **

6.66

13.3

3 *

35.0

0 **

16.6

6 *

25.0

0 **

33.3

3 **

178 F. C. RAFFAINI ET AL.

implants, whereas zirconia harbored only S. mitis, A. actinomycetemcomitans serotype a, P. anaerobius, P. gin-givalis and V. parvula. These findings are of clinical rele-vance since both materials harbored both non-pathogenic and pathogenic species upon their exposure in the oral cavity. Currently, there is a consensus in the literature that the microbiota in dental implants is mainly influenced by the implant/abutment surface and the peri-implant environment (Subramani et al. 2009; de Avila et al. 2014; Esposito et al. 2014). Variations in the microbiota have been observed depending on the implant material used, the roughness of the surface, the surface free energy, and implant design (Lang and Berglundh 2011). Likewise, there is enough evidence that bacterial load and micro-bial profile of implant biofilms are similar to the biofilm on the remaining teeth (Larsen and Fiehn 2017). Thus, the present results confirm that colonization of implant com-ponents and associated supra-structures share similarities with biofilm from natural teeth even in the very first stages of colonization. However, the differences in selective bac-terial adhesion on titanium and zirconia after investiga-tion for 30 days could not be confirmed. Differences were found only at the implant loading time point, which may have been due to the difficulty of microbiological sam-pling around dental implants after surgical reopening for

Discussion

This investigation compared the microbial profile of early biofilm in dental implants restored with titanium or zirconia abutments and its impact on the initial clini-cal outcomes of implant-supported restorations. The data presented support the null hypothesis. Clinical findings showed that 30 days after prosthetic loading, the peri-im-plant mucosal status of implants restored with titanium or zirconia abutments were comparable and consistent with a healthy condition. Peri-implant bone loss surrounding implants was similar for both groups, suggesting that the type of abutment substratum is not clinically relevant at this stage of osseointegration. In terms of microbiologi-cal data, both materials presented a similar distribution of target species in the biofilms over time, demonstrat-ing a higher number of bacteria as the biofilm matured. Also, the total number of adhered microorganisms was similar. Indeed, after 30 days of function, the microbial profiles of implant and teeth samples were quite similar. Significant differences could be found only at the implant loading time point. Both titanium and zirconia showed low genome counts at this stage but with significant dif-ferences regarding identified bacterial species. S. gordonii, S. salivarius, S. sanguinis, T. denticola and V. parvula were the only species detected in the inner parts of titanium

Table 2. Marginal bone level reduction (in mm, mean ± SD) around dental implants after seven and 30 days of loading.

*No significant differences were detected by nonparametric multiple comparisons by the Brunner–Langer model (p > 0.05).

    Follow-up0 to 7 days 7 to 30 days   Total bone loss

Titanium 0.02 0.06 0.08Zirconia 0.03 0.04 0.07

Figure 1. Median, minimum, maximum, lower, and upper quartiles of the total genome counts detected in implant-related sites (titanium and zirconia) and tooth surfaces over time.

BIOFOULING 179

resulting in multiple microgaps (Passos et al. 2013). Studies have indicated that bacterial leakage into and from the implant–abutment interface is enhanced by mechan-ical loading (Steinebrunner et al. 2005; do Nascimento et al. 2012). Other factors associated with bacterial leakage through the implant–abutment interface are the precision of the fit between different connections and the size of the microgaps (Assenza et al. 2012). In the present investiga-tion, different connective systems were not assessed; only morse taper implants, which have been shown to be more effective in reducing bacterial contamination (Scarano et al. 2016) were used.

The selected target probes are part of a panel of spe-cies from different stages of biofilm formation. Probes included primary (eg Streptococcus spp., Veillonella spp.), secondary (eg Fusobacterium spp.) and late colonizers (eg Porphyromonas spp., Aggregatibacter spp.). These species are representative of groups associated with periodontal health or disease (Pérez-Chaparro et al. 2016). The pres-ence of the early colonizer Streptococcus spp. was not sur-prising since this microbial species is able to proliferate

installation of the prosthetic system. Sampling the bottom of the sulci of peri-implant tissues is difficult because of the irregularities caused by the surgery.  Therefore, the findings suggest that titanium or zirconia abutments do not have a relevant impact on the composition of early bacterial biofilm formation. However, the results must be interpreted with caution since we only selected target species were tested.

With regards to quantitative analysis, both groups showed the lowest microbial counts at implant loading, although no significant difference was found between groups. Despite the difficulty of biofilm sampling men-tioned above, these results were expected since the depth of the peri-implant sulcus is shallower at this stage. The low counts may also be credited to the position of implants at the level of the alveolar bone crest which has been shown to influence microbial counts (Degidi et al. 2008; Donovan et al. 2010). Another expected result was the increase in bacterial load over time due to functional load-ing and biofilm maturation. All two-part implant systems are retained to the fixture using a mechanical attachment

(a)

(b)

Figure 2.  Area chart showing the microbial profile of the sub- and supra-gingival biofilm from implants and contralateral teeth by genome count (median, ×105) of the 42 target species over time. (a) Titanium-related sites; (b) zirconia-related sites. T(-1): presurgical time point; T(0): implant loading; T(1): seven days; T(2): 30 days of function.

180 F. C. RAFFAINI ET AL.

finding of this study was the presence of pathogenic anaer-obic species (T. denticola, P. anaerobius and P. gingivalis) colonizing implant-related sites at the loading time point. These species are considered late colonizers and probably came from the remaining teeth since they are commonly found in reduced levels in healthy implant sites (Zipprich

under aerobic conditions, a characteristic of the early bio-film environment. Recently, these species were found in dental surfaces after biofilm formation for 6 h (Heller et al. 2016). Streptococci form a structured architecture and act as a substratum facilitating biofilm development and mat-uration (Kolenbrander et al. 2002). Another interesting

(a)

(b)

Figure 3. Area chart showing the microbial profile of the implant-related surfaces and contralateral teeth by genome count (median, ×105) of the 42 target species over time. (a) Titanium-related sites; (b) zirconia-related sites. T(-1): presurgical time point; T(0): implant loading; T(1): seven days; T(2): 30 days of function.

Table 3. Median, lower, and upper quartiles of microbial counts (×105) and respective p-values of subgingival biofilm assessment from titanium- or zirconia-related sites at implant loading (T0).

*Significant differences detected by Brunner–Langer nonparametric analysis of longitudinal data followed by Bonferroni multiple comparisons (p˂0.05).; Aa: Aggregatibacter actinomycetemcomitans.

 

  Titanium Zirconia  

Lower Upper Lower Upper

quartile Median quartile quartile Median quartile p-value

Aa serotype a* 0 0 0 0 1.78 1.91 0.022P. anaerobius* 0 0 0 1.23 1.99 2.17 0.007P. gingivalis* 0 0 0 1.34 2.11 2.17 0.007S. gordonii * 1.23 1.86 2.12 0 0 0 0.014S. mitis* 0 0 0 2.14 2.62 3.26 0.039S. salivarius * 1.34 2.01 2.25 0 0 0 0.007S. sanguinis* 0 1.87 2.14 0 0 0 0.015T. denticola * 0 1.95 2.12 0 0 0 0.015V. parvula   0 1.86 2.09   0 2.90 3.53 0.375

BIOFOULING 181

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et al. 2016). If not controlled, these species may lead to an inflammatory state and induce peri-implant bone loss under functional loading.

The literature is still not clear on the possible effects of different abutment materials and connections on bio-film formation and maturation. Laboratory and clinical studies have shown conflicting results, mainly due to dif-ferent experimental models and assessment methods. In the present study, biofilm formation was similar for both titanium and zirconia abutments with regards to bacte-rial load and microbial diversity after 30 days of function and no difference was found in the clinical implications. However, further studies are needed to monitor biofilm maturation over time and to determine its impact on long-term success of dental implants. Potential microbial profile changes leading to a more complex biofilm may cause peri-implant tissues to become more susceptible to disease development.

Conclusions

In conclusion, this investigation has demonstrated that implants with titanium or zirconia abutments did not show differences in microbial counts and diversity after 30 days of functional loading and that the microbiota was quite similar to that found on the remaining teeth. The clinical findings were consistent with healthy conditions. No significant difference was found for marginal bone loss, suggesting that the abutment material might not be clinically relevant at this stage of osseointegration.

Disclosure statementNo potential conflict of interest was reported by the authors.

FundingThis work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo [FAPESP – Process 2010/12830-0 and 2014/22876-8] and Conselho Nacional de Desenvolvimento Científico e Tecnológico [CNPq - Process 457941/2014-6].

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