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The effect of supragingival biofilm re-development on the subgingival microbiota in chronic periodontitis
Archives of Oral Biology 85 (2018) 51–57
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Research Paper
The effect of supragingival biofilm re-development on the subgingival microbiota in chronic periodontitis
T
Fátima Aparecida Rocha Resende Hartenbacha,b, Carina Maciel Silva-Boghossiana,b,c, ⁎ Ana Paula Vieira Colomboa,b, a b c
School of Dentistry, Department of Clinics, Rio de Janeiro, RJ, Brazil Institute of Microbiology, Department of Medical Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Department of Periodontics, and Postgraduate Program in Translational Biomedicine, University of Grande Rio, Duque de Caxias, RJ, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Dental plaque Gingivitis Chronic periodontitis Oral hygiene Periodontal microbiota DNA probe
Objective: In this study, we hypothesized that in the absence of oral hygiene, re-growth of the climax microbial communities of supra and subgingival biofilm happens in a faster and more intense fashion in individuals with chronic periodontitis (CP) compared to periodontally healthy controls (PH). Design: Thirty patients (PH = 15 and CP = 15) received professional supragingival prophylaxis, and were asked to refrain from oral hygiene for 7 days. Supra and subgingival biofilm samples and GCF were collected from randomly selected quadrants at baseline (before prophylaxis), immediately after prophylaxis, 2 h, 6 h, 24 h, and 7 days after prophylaxis. The composition of the biofilm was determined by the checkerboard method. Results: All subjects developed gingivitis at the end of 7 days without oral hygiene. GCF mean volumes were significantly higher in CP than PH patients at baseline, but they started decreasing 2 h after prophylaxis, returning to baseline levels after 24 h in both groups. Significant increases in mean counts for most of the species evaluated were observed in both groups and biofilms over time (p < 0.05). Few hours after prophylaxis, a more marked reduction in microbial counts happened in the supragingival biofilm of the CP group, and re-development of biofilm started later than in the PH group. At 7 days, no differences were seen between groups. Significant differences in kinetics of re-colonization between groups were observed only in the subgingival biofilm for T. denticola and F. nucleatum ss vicentii (increased in the CP), and N. mucosa (increased in the PH group; p < 0.05). Conclusions: Biofilm re-development was very similar between CP and PH individuals, although microbial regrowth occurred few hours earlier in PH than PC. Only 3 species in the subgingival biofilm differed in recolonization between groups. Thus, we reject the hypothesis that re-colonization of biofilm in CP patients is more intense and faster than in individuals with PH.
1. Introduction Dental plaque or biofilm is a sophisticated structure comprising a large variety of inter-related oral species that develops on the teeth surface. Depending on several local and/or systemic modulator factors, these structures may acquire pathogenic features such as a cariogenic or a periodontal pathogenic profile (Kolenbrander et al., 2006; Marsh, 2006; Socransky & Haffajee, 2002). Accumulation and persistence of a biofilm comprising high proportions of periodontal pathogens on teeth will lead to the development of gingivitis (Loe, Theilade, & Jensen, 1965), an inflammation of the marginal gingiva characterized by edema, redness and gingival bleeding (Armitage, 1999). Adequate
biofilm removal results in resolution of inflammation and re-establishment of periodontal health (Chapple et al., 2015; Loe et al., 1965; Needleman, Suvan, Moles, & Pimlott, 2005). However, long-term biofilm accumulation combined to chronic inflammation may progress to periodontitis, an irreversible more severe periodontal inflammation that will lead to periodontal attachment loss and alveolar bone resorption (Marsh, 2006; Marsh & Devine, 2011; Page, Offenbacher, Schroeder, Seymour, & Kornman, 1997). The mechanisms involved in dental biofilm pathogenicity and induction of periodontal inflammation and destruction are complex and not fully understood. Studies on dental biofilm development and maturation have shown an orchestrated microbial colonization that is closely associated with specific micro-
⁎ Corresponding author at: UFRJ/CCS – Instituto de Microbiologia Paulo de Góes – Bloco I, lab. I2-03; Av. Carlos Chagas Filho, 373 Cidade Universitária – Rio de Janeiro, RJ, CEP: 21941-902, Brazil. E-mail address: [email protected] (A.P.V. Colombo).
http://dx.doi.org/10.1016/j.archoralbio.2017.10.007 Received 30 July 2017; Received in revised form 8 October 2017; Accepted 9 October 2017 0003-9969/ © 2017 Elsevier Ltd. All rights reserved.
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environmental changes and host susceptibility (Marsh & Devine, 2011; Page et al., 1997). During biofilm formation, early colonizers adhesion provides substrates for subsequent colonizers to co-aggregate in this structure (Kolenbrander et al., 2006; Li et al., 2004; Marsh, 2006). Increase in biofilm microbial density will elicit a marginal gingival inflammation and a significant increase in gingival crevicular fluid (GCF) volume and flow (Goodson, 2003; Grant et al., 2010; Ngo et al., 2010). In turn, the elevated levels of inflammatory mediators and serum proteins in GCF will favor the overgrowth and establishment of more pathogenic species, including the orange and red complexes (Marsh & Devine, 2011; Socransky & Haffajee, 2002, 2005). Although the main stages of biofilm formation and gingival inflammation are observed in most people, the rate of microbial remodeling, host response and tissue destruction may vary (Marsh & Devine, 2011). For instance, in the experimental gingivitis model in humans (Loe et al., 1965), the onset of gingivitis varied among individuals, indicating that biofilm composition and host-related factors may determine biofilm pathogenicity and disease progression. In individuals with periodontitis, periodontal pockets constitute reservoirs of periodontal pathogens that may promptly colonize other sites of the oral cavity, including healthy sulci (Colombo et al., 2002; Riviere et al., 1996; Socransky & Haffajee, 2005). Thus, increased intraoral microbial transmission associated to a probable host immune susceptibility may explain in part the higher risk of periodontitis patients for further attachment loss compared to individuals with periodontal health. In fact, studies have indicated that differences in microbial colonization during biofilm formation between subjects with periodontitis and periodontal health do exist (Socransky & Haffajee, 2005; Teles et al., 2012; Uzel et al., 2011). Therefore, we hypothesized that biofilm re-development and composition, as well as the establishment of gingival inflammation occurs earlier and faster in individuals with chronic periodontitis (CP) compared to periodontally healthy (PH) individuals during withdraw of oral hygiene and supragingival plaque accumulation.
patients was required in each arm of the trial. To allow for 15% drop out, 15 patients were recruited in each clinical group. Secondary outcome variable were gingival bleeding (GI) and mean volume of GCF at day 7.
2. Material and methods
2.4. Supra and subgingival biofilm sampling
2.1. Sample population The study protocol was approved by the Human Research Ethics Committee of the Hospital of the Federal University of Rio de Janeiro (UFRJ), Brazil (CEP approval n° 707.355). Participants were recruited from the Division of Graduate Periodontics of the School of Dentistry at UFRJ, between February and December of 2014. Patients were individually informed about the nature of the study, its risks and benefits, and signed an informed consent form. To participate in the study, subjects had to have at least 18 years of age and 16 teeth (at least 4 in each quadrant). Patients were diagnosed as having CP or PH according to Da Silva-Boghossian et al. (2011). Briefly, CP was defined as ≥10% of teeth with PD and/or CAL ≥5 mm or ≥15% of teeth with PD and/or CAL ≥4 mm and BOP, and PH was defined as < 10% of sites with BOP, no PD or CAL > 3 mm, although PD or CAL = 4 mm in up to 5% of the sites without BOP was allowed. Exclusion criteria included smoking, diagnosed inflammatory systemic diseases, autoimmune diseases, aggressive periodontitis, use of topical or systemic antimicrobials in the last 6 months, periodontal therapy in the last year, orthodontic treatment, antibiotic prophylaxis, pregnancy or nursing.
At baseline (-T1), 2 individual supragingival biofilm samples (from disto-buccal sites of one anterior and one posterior tooth) and 2 individual subgingival biofilm samples (from the same sites and teeth) were collected per quadrant, providing a total of 8 supra and 8 subgingival biofilm samples per patient. For biofilm re-development analysis, supra and subgingival samples were collected from the distalbuccal sites of all teeth of contralateral quadrants at T0 (immediately after prophylaxis) and T1 (2 h), T2 (6 h), T3 (24 h) and T4 (7 days) after professional prophylaxis and no oral hygiene (Fig. 1S). For instance, if quadrant 2 was drawn for sampling at T0/T1, all distal-buccal sites from anterior teeth and from posterior teeth would be pooled into 2 pools (1 anterior and 1 posterior) for supra and subgingival biofilm (total of 4 pooled samples). Then, at T2, the contralateral quadrant 4 would be sampled likewise. At T3, quadrant 1 or 3 would be drawn and sampled, followed by the contralateral quadrant at T4. Thus, at time from T0 to T4 a patient would provide 4 samples (2 supra and 2 subgingival) per quadrant. During sampling, the area was dried and isolated with cotton rolls. Supragingival samples were always collected first, followed by subgingival samples with sterile curettes (Hu-Friedy®). Samples were placed into microtubes containing 150 μl of TE buffer.
2.2. Sample size calculation
2.5. GCF sampling
In a previous analysis of our microbial database, we computed the mean total counts of oral bacteria in 120 PH and 290 CP individuals. Considering total mean counts as the primary outcome variable, and assuming that CP would presented higher mean counts than PH patients, to detect a difference of 2.5 × 107 total mean counts between groups at 7 days with a one-sided significance level of 5% and power of 85% with equal allocation to two groups, a minimum sample size of 13
Before removal of biofilm from distal-buccal sites, GCF was collected from the mesio-buccal sites of the same 8 teeth selected for biofilm sampling at baseline (-T1), following the same sequence of quadrants at T0 to T4. For post-prophylaxis sampling, GCF samples were obtained individually from the mesio-buccal sites of all teeth in that particular quadrant selected, and samples were not pooled. GCF samples were collected with paper strips (Periopaper, Oraflow Inc.,
2.3. Clinical monitoring and biofilm development At the screening visit, participants answered a questionnaire, and information about demographic features, medical and dental health history was obtained. For diagnosis of periodontal status, clinical measurements were performed by a single calibrated examiner (F.A.R.R.H) using a North Carolina probe (UNC-15, Hu-Friedy, Chicago, IL, USA). The intra-class correlation coefficients for probing depth (PD) and clinical attachment level (CAL) were 0.94 and 0.88, respectively. The periodontal parameters evaluated included PD and CAL (mm), and presence of supragingival plaque (PL), bleeding on probing (BOP), GI, suppuration (SUP) and calculus (CA), at 6 sites per tooth of all teeth, except third molars. Ninety-seven recruited patients who agreed to participate in the study (signed the consent form) were clinically examined. Of those, 67 did not meet the inclusion criteria. Therefore, 30 individuals, 15 with PH and 15 with CP entered and finished the study. Patients selected for the study based on the inclusion/exclusion criteria and consent returned within one week at time -T1 (baseline) for sampling (described in Sections 2.5 and 2.6), and for full mouth measurements of PD, CAL, BOP, PL, GI, SUP and CA. Following baseline sampling and clinical evaluation (-T1), participants were submitted to supragingival ultrasonic and manual debridement, followed by dental polishing with rubber cup and prophylactic paste. At this moment, individuals were instructed to refrain brushing and flossing of their teeth for 7 days. GI was again measured at the seventh day of refraining oral hygiene procedures, after sampling (Fig. 1S). Patients were then referred to dental care in the different clinical specialties according to their treatment needs.
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New York, USA) to volumetric analysis in spectrophotometer (Periotron 8000, Oraflow Inc.). Teeth were isolated with cotton rolls, gently dried and a periopaper was inserted into gingival sulcus during 30 s. The strip was removed and GCF volume measured by Periotron 8000. The volumes obtained for each strip/sample were averaged for each patient at each time point.
Table 1 Demographic and clinical periodontal data of the sample population. Parameters
Gender (%) Females Males Race (%) White Afro-American Others Mean (SD) Age(years) N of missing teeth Mean% of sites Probing depth < 4 mm Probing depth 4–6 mm Probing depth > 6 mm Clinical attachment level < 4 mm Clinical attachment level 4–6 mm Clinical attachment level > 6 mm Bleeding on probing Supragingival biofilm Calculus Suppuration Gingival inflammation Gingival inflammation at day 7 without oral hygiene#
2.6. Microbiological assessment Microbiological analyses were carried out by the checkerboard DNA–DNA hybridization technique (Socransky et al., 1994), with modifications (Heller et al., 2011). Briefly, pooled biofilm samples were placed in microtubes containing 150 μl of TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.6). Samples were lysed by adding 150 μl of 0.5 M NaOH and boiling for 10 min. Denatured DNA was neutralized with 800 μl of 5 M C2H3O2NH4 and fixed in individual lanes on a nylon membrane (Hybond-N+, GE Healthcare Life Sciences, Piscataway, NJ, USA) using the Minislot 30 (Immunetics, Cambridge, MA). The membrane was placed in a Miniblotter 45 (Immunetics) with the lanes of fixed DNA perpendicular to the lanes of the device, and hybridized against 39 digoxigenin-labeled (“Random Primer Digoxigenin Labeling Kit”, Roche Molecular Systems, Inc., Alameda, CA) whole genomic DNA probes (Table 1S). DNA from Aggregatibacter actinomycetemcomitans (Aa) serotypes a, b and c was grouped into one probe, as was DNA from Propionibacterium acnes I and II. After hybridization, the membranes were washed at high stringency and bound probes incubated with phosphatase-conjugated antibody to digoxigenin at 1:15.000 (Roche Molecular Systems). Signals on the membranes were detected by fluorescence (AttoPhos®, PromegaCorporation, Madison, WI), and captured by an imaging system (Storm TM 860 and ImageQuant® version 5.2, Molecular Dynamics, GE Healthcare Life Sciences). Signals were evaluated visually by comparison with the standards at 105 and 106 cells for the tested species on the same membrane, and recorded as: 0 = not detected; 1 = < 105 cells; 2 = ∼105; 3 = 105–106 cells; 4 = ∼106; 5 = > 106 cells. The sensitivity of the assay was adjusted to permit the detection of 104 cells of a given species by adjusting the concentration of each DNA probe. Failure to detect a signal was recorded as zero, although conceivably, counts in the 1 to 1000 range could have been present.
PH group (n = 15)
CP group (n = 15)
P*
80.0 20.0
53.3 46.7
0.121†
80 0 20
53.3 20.0 26.7
0.139†
23.3 (0.8) 0.7 (0.3)
49.8 (2.9) 4.5 (0.7)
< 0.001* < 0.001*
99.1 0.9 0 99.0 1.0 0 5.8 (0.5) 21.4 (2.3) 8.8 (1.9) 0 4.3 (0.9) 19.5 (2.5)
79.4 20.1 0.6 73.1 25.2 1.6 19.3 (2.8) 48.5 (4.1) 30.0 (4.0) 0.1 (0.1) 19.5 (2.5) 23.5 (2.8)
< 0.001*
< 0.001*
< 0.001* < 0.001* < 0.001* 0.150§ < 0.001* > 0.05*
PH: Periodontal Health; CP: Chronic Periodontitis; SD: standard deviation. * Student T test. † Chi-square test. § Mann–Whitney test. # Refers to significant increase in inflammation from baseline to 7 days in both groups (p = 0.006, GLM).
3. Results 3.1. Demographic and clinical features of the study population Demographic and clinical data of the population are presented in Table 1. No statistical differences in gender or race between groups were seen, but PH patients were younger than CPpatients (p < 0.001). The CP group presented more signals and symptoms of periodontal inflammation and destruction than the PH group (p < 0.001). After 7 days of no oral hygiene, all PH individuals developed gingivitis, and CP individuals presented an extension of the previous gingivitisindicated by a significant increase in% of sites with GI (p = 0.006), with no difference between groups (p > 0.05).
2.7. Statistical analysis All analyses were performed using the SPSS 21.0 (IBM Brazil, SP, Brazil). Data entry was carried out by one investigator (F.A.R.R.H) and error proofed by a senior investigator (A.P.V.C.). All variables were tested for normality by the Kolmogorov–Smirnov test. Demographic data were computed for each individual and compared between groups by Student t and Chi-square tests. Periodontal measurements were averaged for each patient and within groups at baseline, and for GI at day 7. Differences between groups in clinical parameters were tested by the Student t-test, whereas difference in GI change from baseline to day 7 was examined by the Generalized Linear Model of Repeated Measures (GLM). GCF volumes were averaged for each patient and time, and differences between groups were sought by Mann–Whitney and Friedman tests. Microbial data were presented as mean counts of the tested species and calculated in each individual and within groups at different time points. Differences in mean counts of species between groups at each time of evaluation were examined by the Mann–Whitney test. Differences in microbiological changes over time in supra and subgingival biofilm between groups were evaluated by Friedman and GLM tests. Comparisons between mean counts of species between supra and subgingival biofilm were analyzed by Wilcoxon test. Adjustments for multiple comparisons were carried out (Socransky, Haffajee, Smith, & Dibart, 1991) so that the adjusted p-value was 0.0013 for microbiological analysis. For other parameters, the level of significance was 5%.
3.2. Changes in GCF volume over time The mean volumes of GCF measured over the 7 days without oral hygiene in both groups are shown in Fig. 1. At baseline, the CP group presented a significantly higher mean volume of GCF when compared to the PH group (p = 0.017). Although the volumes were still higher in the CP patients at all time points after prophylaxis, these differences were not significant. Right after prophylaxis, there was a marked reduction in GCF volume, particularly in the PH group at T2, 6 h after prophylaxis (p = 0.001). The decrease in GCF volume in the CP group was modest and no significant. After 24 h without oral hygiene, the GCF volumes increased in both groups to baseline levels in the PH group and to levels slightly lower than baseline in the CP. It is interesting to note that even though the GI and supra and subgingival microbial mean counts increased significantly in both groups at day 7 (Table 1), the GCF volume was kept at baseline values at this time point evaluation. 3.3. Total microbial counts Changes in mean total countsin supra and subgingival biofilm over 53
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Fig. 1. Mean (SD) volumes of GCF before prophylaxis (baseline) and up to 7 days without oral hygiene.T0, immediately after prophylaxis; T1, 2 h after; T2, 6 h after; T3, 24 h after; and T4, 7 days after prophylaxis. *Refers to significant difference between groups at baseline (p = 0.017, Mann–Whitney test). †Refers to significant reduction over time in GCF volume in the PH group (p = 0.001, Friedman test).
time in both groups are shown in Fig. 2. At baseline, the CP group presented significantly higher mean counts of microorganisms in both biofilms than the PH group (p < 0.05). A modest reduction in mean counts in supra and subgingival biofilm is observed in periodontally healthy individuals until 2 h after prophylaxis, increasing significantly at 6 h to levels greater than baseline values at 7 days without oral hygiene (p < 0.01). In the CP group, the reduction in counts is more marked after prophylaxis, and the microbial levels start to go up later than the PH group (p < 0.01). Nevertheless, both groups presented similar total mean counts at 24 h and 7 days without oral hygiene (Fig. 2). 3.4. Supra and subgingival biofilm composition At baseline, most species were detected at higher levels in the supra and subgingival biofilm of the CP group compared to PH patients (Fig. 1S). However, after adjusting for multiple comparisons,nosignificantdifferences between groups were observed for any species (adjusted p = 0.0013). Comparisons between supra and subgingival biofilm in each clinical group showed that only A. actinomycetemcomitans was detected in significantly higher counts in the subgingival biofilm compared to the supragingival biofilm in the PH group (p < 0.05). 3.5. Supra and subgingival biofilm Re-development Changes in mean counts in supra and subgingival biofilm after prophylaxis and during suspension of oral hygiene in both groups are depicted in Fig. 3. As observed, there was a significant increase in counts in most of the species evaluated in both groups and biofilms over time (p < 0.05). This increase occurred earlier in the PH group, while in the CP group the increase was more evident from T2. The only species that did not present significant changes over time in both groups were A. actinomycetemcomitans, A. naeslundii, E. nodatum, and S. constellatus. Significant differences in kinetics of re-colonization between groups were observed only in the subgingival biofilm (Fig. 4) for T. denticola and F. nucleatumssvicentii that increased more in the CP than PH group, and N. mucosa that showed greater increase in the PH compared to the CP group (p < 0.05). Comparisons in mean counts between groups at each time point indicated few significant differences. In the supragingival biofilm, differences were seen for L. buccalis at T0 and T1, N. mucosa, E. corrodens and C. sputigena at T1, C. showae and P. melaninogenica at T3, P. micra and E. saburreum at T4 (p < 0.05,
Fig. 2. Mean (SD) total counts of microorganisms in supragingival and subgingivalbiofilm from both groups (PH: Periodontal Health; CP: Chronic Periodontitis) before prophylaxis (baseline) and up to 7 days without oral hygiene. T0, immediately after prophylaxis; T1, 2 h after; T2, 6 h after; T3, 24 h after; and T4, 7 days after prophylaxis.*Refers to significant difference between groups at baseline (p = 0.037, Mann–Whitney test).†Refers to significant increase over time in mean counts in supra and subgingival biofilm for both groups (p < 0.01, Friedman test).
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Fig. 3. Changes in mean counts in supragingival and subgingival biofilm of individuals with periodontal health and chronic periodontitis after prophylaxis and during suspension of oral hygiene.T0, immediately after prophylaxis; T1, 2 h after; T2, 6 h after; T3, 24 h after; and T4, 7 days after prophylaxis.*Refers to statistical differences in counts of each species over time in each clinical group (p < 0.05; Friedman test).†Refers to statistical differences over time without differences between clinical groups, and ‡ with differences between groups (p < 0.05, GLM test). Bacterial species were grouped by the microbial complexes according to Socransky, Haffajee, Cugini, Smith, & Kent (1998).
(p < 0.05, Wilcoxon test). 4. Discussion Given that there are marked differences in biofilm composition and periodontal inflammation between CP and PH individuals, we hypothesized that the re-development of supra and subgingival biofilm during short term suspension of oral hygiene, as well as the clinical manifestations of gingival inflammation were distinct among these individuals. As expected, a significant greater number of sites with supragingival biofilm and gingival inflammation was detected in the CP than the PH group at baseline. However, all individuals presented high levels of biofilm accumulation associated to clinical signs of gingivitis on the seventh day of oral hygiene withdrawn, and no differences between groups were observed. Despite the short period of biofilm accumulation used in this study, the clinical data confirmed that this period was enough to induce gingivitis, even in individuals with PH (Loe et al., 1965). In fact, based on histological analysis in animal model, Page & Schroeder (1976) demonstrated that after 1week of dental biofilm accumulation, an early lesion developed characterized by increased vascular permeability, inflammatory infiltrate, and GCF flow, as well as degradation of connective tissue. Fransson, Mooney, Kinane, and Berglundh (1999) have also demonstrated that there were no significant changes in the composition of the gingival inflammatory infiltrate from 7 to 21 days of experimental gingivitis, concluding that the inflammatory response is well established within 1 week. For biofilm development, even a 3-day period of biofilm accumulation has been shown to result in a complex and organized mature microbial community (Listgarten, Mayo, & Tremblay, 1975). Although there was a significant increase in gingival bleeding in
Fig. 4. Species that showed significant differences in kinetics of re-colonization in the subgingival biofilm between periodontally healthy (PH) and chronic periodontitis (CP) individuals over time (p < 0.05, GLM). T0, immediately after prophylaxis; T1, 2 h after; T2, 6 h after; T3, 24 h after; and T4, 7 days after prophylaxis.
Mann–Whitney test). In the subgingival biofilm, differences were observed for P. melaninogenica at T2 and T3, F. periodonticum at T0, T. denticola at T2, and S. intermedius at T4 (p < 0.05, Mann–Whitney test). However, no significant differences were found after adjusting for multiple comparisons (data no shown). When the composition of the supragingival biofilm was compared to the subgingival biofilm in each group at every time points, significant differences were detected for S. constellatus at T0 in the PH group, and for T. denticola(T0), L. buccalis (T1) and P. gingivalis(T2) in the CP group 55
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performed in the same tooth site at every time of evaluation but at different teeth and quadrants. Analysis of the composition of the biofilm was carried out in pooled samples and it was limited to 37 oral species. Finally, the short period of time (7 days) chosen for oral hygiene withdrawn and biofilm re-development may have been insufficient to induce a significant long lasting disturbance in the periodontal microenvironment and microbial colonization/maturation of biofilm. Due to the large number of biological samples (biofilm and GCF) at several time points of evaluation, we had to select different tooth surfaces for sampling. Repeated sampling at the same tooth site in different moments would require repeated steps of prophylaxis, oral hygiene suppression and supragingival biofilm accumulation. Extending the time of biofilm accumulation was a critical issue in choosing the time span for the experimental gingivitis. Initially, we had set up a 14-day period of oral hygiene withdrawn. However, the large rate of dropouts during the screening phase of the population led us to reduce this period to 7 days. Despite these limitations, most of our findings are in agreement with data reported in the literature (Diaz et al., 2006; Heller et al., 2016; Li et al., 2004; Offenbacher et al., 2007; Ramberg et al., 1995; Ramberg et al., 2003; Uzel et al., 2011; Weidlich et al., 2001). In summary, a 1-week withdraw of oral hygiene and supragingival biofilm accumulation led to an increase in gingival inflammation in both clinical groups, although no major changes in GCF volume were observed. Re-development of supra and subgingival biofilm was similar between PH and CP individuals during this period of time. Supragingival prophylaxis had a greater short term impact in delaying biofilm re-colonization in periodontitis patients compared to healthy individuals. These changes were more evident in the supragingival biofilm. In contrast to supragingival biofilm, few species (N. mucosa, T. denticola, F. nucleatumssvicentii) differed between groups in kinetics of re-colonization in the subgingival biofilm. Based on these data, we reject the hypothesis that supra and subgingival biofilm re-development, as well as gingival inflammation during a short period of oral hygiene withdrawn happens earlier and more intensively in individuals with chronic periodontitis in relation to individuals with periodontal health.
both clinical groups at 7 days without oral hygiene, no major increase in GCF volume was observed at this time point. As expected, CP patients presented higher volumes of GCF than PH patients (Goodson, 2003; Lamster, Vogel, Hartley, DeGeorge, & Gordon, 1985; Teles et al., 2010; Zhang, Kashket, & Lingstrom, 2002) at all evaluations. Hours after prophylaxis there was a reduction in these volumes which was more marked in the PH group. After the establishment of gingivitis, however, the GCF volumes return to baseline values in both groups. It is important to consider here that the total volume of GCF and not flow rate was measured. The resting volume is more relatively constant, whereas GCF flow is expected to increase significantly with inflammation (Lamster, Vogel, Hartley, DeGeorge, & Gordon, 1985). Moreover, GCF was sampled from different sites and teeth at each time point, so that significant increases in mean volumes may have been underestimated. The relationship between supragingival biofilm accumulation and gingival inflammation has been studied for over decades (Loe et al., 1965; Ramberg, Axelsson, & Lindhe, 1995; Ramberg, Sekino, Uzel, Socransky, & Lindhe, 2003; Socransky & Haffajee, 2002; Weidlich, de Souza, & Oppermann, 2001; Zhang et al., 2002). Increase in biofilm density results in increased GCF flow and inflammatory mediators, which in turn function as important microbial nutrients and growth factors for the fast development and complex maturation of this biofilm (Socransky & Haffajee, 2005). The kinetics of re-colonization of dental biofilm by specific microorganisms during gingival inflammation has been investigated in more detail by few studies (Diaz et al., 2006; Heller et al., 2016; Li et al., 2004; Offenbacher et al., 2007; Ramberg et al., 2003; Teles et al., 2012; Uzel et al., 2011). In general, these studies demonstrated a hierarchical microbial succession in the re-colonization of supra and subgingival biofilm, with initial increase of first colonizers (yellow, blue, and purple complexes, N. mucosa), followed latter by the overgrowth of more pathogenic species, more specifically members of the orange complex (Fusobacterium spp., Prevotella spp., Campylobacter spp.). Similar data were also observed in our study for supra and subgingival biofilm in both PH and CP groups. In the supragingival biofilm, no differences in biofilm re-development were detected between groups for any particular species, corroborating those studies (Teles et al., 2012; Uzel et al., 2011). However, it is possible to note a tendency for greater mean counts of the blue, purple and green complexes in the PH group compared to the CP group at 7 days, whereas the CP individuals presented higher counts of the orange complex at this time point.In the subgingival biofilm, we did find significant differences in re-colonization between groups for T. denticola and F. nucleatumssvincentii (significant increase in CP) and N. mucosa (significant increase in PH). Of interest, after prophylaxis, re-development of supra and subgingival biofilm happened few hours earlier in PH than CP individuals. These findings suggested that supragingival prophylaxis seems to have a greater impact in sites/individuals with periodontitis than PH, more precisely in reducing periodontal inflammation and consequently delaying early biofilm re-colonization. On the other hand, the disturbance of prophylaxis in healthy sites/individuals with already low inflammation and biofilm accumulation may be less expressive. Other authors have shown that an adequate supragingival biofilm removal have a significant long-term effect on diminishing inflammation and total counts of subgingival microorganisms, mainly pathogenic species in patients with periodontal diseases (Cugini, Haffajee, Smith, Kent, & Socransky, 2000; Ximenez-Fyvie et al., 2000). Here, supragingival prophylaxis led to a decrease in total counts of subgingival biofilm but the impact on the supragingival biofilm in CP patients was more evident. These subtle distinct initial changes in biofilm re-development between groups disappear after 24 h without oral hygiene, and no significant differences in microbial composition were detected between supra and subgingival biofilms in any group at 7 days of follow up. The overall similarity in biofilm re-development in PH and CP individuals observed here should be considered carefully given that limitations in the study design do exist. First, biofilm sampling was not
Conflict of interest The authors declare that they have no conflict of interests. Acknowledgments This study was supported in part by National Council for Scientific and Technological Development (CNPq) Grant/Award Number: 302685/2013-8; Coordination of Improvement of Higher Education Personnel (CAPES), Brasilia, Brazil; and Foundation for Research Financial Support in the State of Rio de Janeiro (FAPERJ)Grant/Award Number: E-26/201.162/2014, Rio de Janeiro, Brazil. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.archoralbio.2017.10.007. References Armitage, G. C. (1999). Development of a classification system for periodontal diseases and conditions. Annal of Periodontology, 4(1), S1–S6. Chapple, I. L., Van der Weijden, F., Doerfer, C., Herrera, D., Shapira, L., Polak, D., et al. (2015). Primary prevention of periodontitis: Managing gingivitis. Journal of Clinical Periodontology, 42(Suppl. 16), S71–S76. Colombo, A. P., Teles, R., Torres, M. C., Souto, R., Rosalém, W. J., Mendes, M. C., et al. (2002). Subgingival microbiota of Brazilian subjects with untreated chronic periodontitis. Journal of Periodontology, 73, 360–369. Cugini, M. A., Haffajee, A. D., Smith, C., Kent, R. L., Jr., & Socransky, S. S. (2000). The effect of scaling and root planing on the clinical and microbiological parameters of periodontal diseases: 12-month results. Journal of Clinical Periodontology, 27, 30–36. Da Silva-Boghossian, C. M., Souto, R. M., Luiz, R. R., & Colombo, A. P. (2011). Association
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Periodontal disease at the biofilm-gingival interface. Journal of Periodontology, 78. Page, R. C., & Schroeder, H. E. (1976). Pathogenesis of inflammatory periodontal disease. A summary of current work. Laboratory Investigation, 34(Suppl. 3), S235–S249. Page, R. C., Offenbacher, S., Schroeder, H. E., Seymour, G. J., & Kornman, K. S. (1997). Advances in the pathogenesis of periodontitis: Summary of developments, clinical implications and future directions. Periodontology 2000, 14, 216–248. Ramberg, P., Axelsson, P., & Lindhe, J. (1995). Plaque formation at healthy and inflamed gingival sites in young adults. Journal of Clinical Periodontology, 22, 85–88. Ramberg, P., Sekino, S., Uzel, N. G., Socransky, S., & Lindhe, J. (2003). Bacterial colonization during de novo plaque formation. Journal of Clinical Periodontology, 30, 990–995. Riviere, G. R., Smith, K. S., Tzagaroulaki, E., Kay, S. L., Zhu, X., DeRouen, T. A., et al. (1996). Periodontal status and detection frequency of bacteria at sites of periodontal health and gingivitis. Journal of Periodontology, 67, 109–115. Socransky, S. S., & Haffajee, A. D. (2002). Dental biofilms: Difficult therapeutics target. Periodontology 2000, 28, 12–55. Socransky, S. S., & Haffajee, A. D. (2005). Periodontal microbial ecology. Periodontology 2000, 38, 135–187. Socransky, S. S., Haffajee, A. D., Smith, C., & Dibart, S. (1991). Relation of counts of microbial species to clinical status at the sampled site. Journal of Clinical Periodontology, 18(Suppl. 10), S766–S775. Socransky, S. S., Smith, C., Martin, L., Paster, B. J., Dewhirst, F. E., & Levin, A. E. (1994). Checkerboard DNA–DNA hybridization. Biotechnique, 17, 788–792. Socransky, S. S., Haffajee, A. D., Cugini, M. A., Smith, C., & Kent, R. L., Jr. (1998). Microbial complexes in subgingival plaque. Journal of Clinical Periodontology, 25, 134–144. Teles, R. P., Sakellari, D., Teles, F. R., Konstantinidis, A., Kent, R., Socransky, S., et al. (2010). Relationships among gingival crevicular fluid biomarkers, clinical parameters of periodontal disease, and the subgingival microbiota. Journal of Periodontology, 81, 89–98. Teles, F. R., Teles, R. P., Uzel, N. G., Song, X. Q., Torresyap, G., Socransky, S. S., et al. (2012). Early microbial succession in redeveloping dental biofilms in periodontal health and disease. Journal of Periodontal Research, 47, 95–104. Uzel, N. G., Teles, F. R., Teles, R., Song, X. Q., Torresyap, G., Socransky, S. S., et al. (2011). Microbial shifts during dental biofilm re-development in the absence of oral hygiene in periodontal health and disease. Journal of Clinical Periodontology, 38, 612–620. Weidlich, P., de Souza, M. A. L., & Oppermann, R. V. (2001). Evaluation of the dentogingival area during early plaque formation. Journal of Periodontology, 72, 901–910. Ximenez-Fyvie, L. A., Haffajee, A. D., Som, S., Thompson, M., Torresyap, G., & & Socransky, S. S. (2000). The effect of repeated professional supragingival plaque removal on the composition of the supra- and subgingival microbiota. Journal of Clinical Periodontology, 27, 637–647. Zhang, J., Kashket, S., & Lingstrom, P. (2002). Evidence for the early onset of gingival inflammation following short-term plaque accumulation. Journal of Clinical Periodontology, 29, 1082–1085.
of red complex, A. actinomycetemcomitans and non-oral bacteria with periodontal diseases. Archives of Oral Biology, 56, 899–906. Diaz, P. I., Chalmers, N. I., Rickard, A. H., Kong, C., Milburn, C. L., Palmer, R. J., Jr., et al. (2006). Molecular characterization of subject-specific oral microflora during initial colonization of enamel. Applied Environmental Microbiology, 72, 2837–2848. Fransson, C., Mooney, J., Kinane, D. F., & Berglundh, T. (1999). Differences in the inflammatory response in young and old human subjects during the course of experimental gingivitis. Journal of Clinical Periodontology, 26, 453–460. Goodson, J. M. (2003). Gingival crevice fluid flow. Periodontology 2000, 31, 43–54. Grant, M. M., Creese, A. J., Barr, G., Ling, M. R., Scott, A. E., Matthews, J. B., et al. (2010). Proteomic analysis of a noninvasive human model of acute inflammation and its resolution: The twenty-one day gingivitis model. Journal of Proteome Research, 9, 4732–4744. Heller, D., Varela, V. M., Silva-Senem, M. X., Torres, M. C., Feres-Filho, E. J., & Colombo, A. P. (2011). Impact of systemic antimicrobials combined to anti-infective mechanical debridement on the microbiota of generalized aggressive periodontitis: A 6month RCT. Journal of Clinical Periodontology, 38, 355–364. Heller, D., Helmerhorst, E. J., Gower, A. C., Siqueira, W. L., Paster, B. J., & Oppenheim, F. G. (2016). Microbial diversity in the early in vivo-formed dental biofilm. Applied Environmental Microbiology, 82, 1881–1888. Kolenbrander, P. E., Palmer, R. J., Jr, Rickard, A. H., Jakubovics, N. S., Chalmers, N. I., & Diaz, P. I. (2006). Bacterial interactions and successions during plaque development. Periodontology 2000, 42, 47–79. Lamster, I. B., Vogel, R. I., Hartley, L. J., DeGeorge, C. A., & Gordon, J. M. (1985). Lactate dehydrogenase, beta-glucuronidase and arylsulfa-tase activity in gingival crevicular fluid associated with experimentalgingivitis in man. Journal of Periodontology, 56, 139–147. Li, J., Helmerhorst, E. J., Leone, C. W., Troxler, R. F., Yaskell, T., Haffajee, A. D., et al. (2004). Identification of early microbial colonizers in human dental biofilm. Journal of Applied Microbiology, 97, 1311–1318. Listgarten, M. A., Mayo, H. E., & Tremblay, R. (1975). Development of dental plaque on epoxy resin crowns in man: A light and electron microscopic study. Journal of Periodontology, 46, 10–26. Loe, H., Theilade, E., & Jensen, S. B. (1965). Experimental gingivitis in man. Journal of Periodontology, 36, 177–187. Marsh, P. D., & Devine, D. A. (2011). How is the development of dental biofilms influenced by the host? Journal of Clinical Periodontology, 38(Suppl. 11), S28–S35. Marsh, P. D. (2006). Dental plaque as biofilm and a microbial community- implication for health and disease. BioMedCentral Oral Health, 6(Suppl. 1), S14. Needleman, I., Suvan, J., Moles, D. R., & Pimlott, J. (2005). A systematic review of professional mechanical plaque removal for prevention of periodontal diseases. Journal of Clinical Periodontology, 32(Suppl. 6), S229–S282. Ngo, L. H., Veith, P. D., Chen, Y. Y., Chen, D., Darby, I. B., & Reynolds, E. C. (2010). Mass spectrometric analyses of peptides and proteins in human gingival crevicular fluid. Journal of Proteome Research, 9, 1683–1693. Offenbacher, S., Barros, S. P., Singer, R. E., Moss, K., Williams, R. C., & Beck, J. D. (2007).
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Research Paper
The effect of supragingival biofilm re-development on the subgingival microbiota in chronic periodontitis
T
Fátima Aparecida Rocha Resende Hartenbacha,b, Carina Maciel Silva-Boghossiana,b,c, ⁎ Ana Paula Vieira Colomboa,b, a b c
School of Dentistry, Department of Clinics, Rio de Janeiro, RJ, Brazil Institute of Microbiology, Department of Medical Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Department of Periodontics, and Postgraduate Program in Translational Biomedicine, University of Grande Rio, Duque de Caxias, RJ, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords: Dental plaque Gingivitis Chronic periodontitis Oral hygiene Periodontal microbiota DNA probe
Objective: In this study, we hypothesized that in the absence of oral hygiene, re-growth of the climax microbial communities of supra and subgingival biofilm happens in a faster and more intense fashion in individuals with chronic periodontitis (CP) compared to periodontally healthy controls (PH). Design: Thirty patients (PH = 15 and CP = 15) received professional supragingival prophylaxis, and were asked to refrain from oral hygiene for 7 days. Supra and subgingival biofilm samples and GCF were collected from randomly selected quadrants at baseline (before prophylaxis), immediately after prophylaxis, 2 h, 6 h, 24 h, and 7 days after prophylaxis. The composition of the biofilm was determined by the checkerboard method. Results: All subjects developed gingivitis at the end of 7 days without oral hygiene. GCF mean volumes were significantly higher in CP than PH patients at baseline, but they started decreasing 2 h after prophylaxis, returning to baseline levels after 24 h in both groups. Significant increases in mean counts for most of the species evaluated were observed in both groups and biofilms over time (p < 0.05). Few hours after prophylaxis, a more marked reduction in microbial counts happened in the supragingival biofilm of the CP group, and re-development of biofilm started later than in the PH group. At 7 days, no differences were seen between groups. Significant differences in kinetics of re-colonization between groups were observed only in the subgingival biofilm for T. denticola and F. nucleatum ss vicentii (increased in the CP), and N. mucosa (increased in the PH group; p < 0.05). Conclusions: Biofilm re-development was very similar between CP and PH individuals, although microbial regrowth occurred few hours earlier in PH than PC. Only 3 species in the subgingival biofilm differed in recolonization between groups. Thus, we reject the hypothesis that re-colonization of biofilm in CP patients is more intense and faster than in individuals with PH.
1. Introduction Dental plaque or biofilm is a sophisticated structure comprising a large variety of inter-related oral species that develops on the teeth surface. Depending on several local and/or systemic modulator factors, these structures may acquire pathogenic features such as a cariogenic or a periodontal pathogenic profile (Kolenbrander et al., 2006; Marsh, 2006; Socransky & Haffajee, 2002). Accumulation and persistence of a biofilm comprising high proportions of periodontal pathogens on teeth will lead to the development of gingivitis (Loe, Theilade, & Jensen, 1965), an inflammation of the marginal gingiva characterized by edema, redness and gingival bleeding (Armitage, 1999). Adequate
biofilm removal results in resolution of inflammation and re-establishment of periodontal health (Chapple et al., 2015; Loe et al., 1965; Needleman, Suvan, Moles, & Pimlott, 2005). However, long-term biofilm accumulation combined to chronic inflammation may progress to periodontitis, an irreversible more severe periodontal inflammation that will lead to periodontal attachment loss and alveolar bone resorption (Marsh, 2006; Marsh & Devine, 2011; Page, Offenbacher, Schroeder, Seymour, & Kornman, 1997). The mechanisms involved in dental biofilm pathogenicity and induction of periodontal inflammation and destruction are complex and not fully understood. Studies on dental biofilm development and maturation have shown an orchestrated microbial colonization that is closely associated with specific micro-
⁎ Corresponding author at: UFRJ/CCS – Instituto de Microbiologia Paulo de Góes – Bloco I, lab. I2-03; Av. Carlos Chagas Filho, 373 Cidade Universitária – Rio de Janeiro, RJ, CEP: 21941-902, Brazil. E-mail address: [email protected] (A.P.V. Colombo).
http://dx.doi.org/10.1016/j.archoralbio.2017.10.007 Received 30 July 2017; Received in revised form 8 October 2017; Accepted 9 October 2017 0003-9969/ © 2017 Elsevier Ltd. All rights reserved.
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environmental changes and host susceptibility (Marsh & Devine, 2011; Page et al., 1997). During biofilm formation, early colonizers adhesion provides substrates for subsequent colonizers to co-aggregate in this structure (Kolenbrander et al., 2006; Li et al., 2004; Marsh, 2006). Increase in biofilm microbial density will elicit a marginal gingival inflammation and a significant increase in gingival crevicular fluid (GCF) volume and flow (Goodson, 2003; Grant et al., 2010; Ngo et al., 2010). In turn, the elevated levels of inflammatory mediators and serum proteins in GCF will favor the overgrowth and establishment of more pathogenic species, including the orange and red complexes (Marsh & Devine, 2011; Socransky & Haffajee, 2002, 2005). Although the main stages of biofilm formation and gingival inflammation are observed in most people, the rate of microbial remodeling, host response and tissue destruction may vary (Marsh & Devine, 2011). For instance, in the experimental gingivitis model in humans (Loe et al., 1965), the onset of gingivitis varied among individuals, indicating that biofilm composition and host-related factors may determine biofilm pathogenicity and disease progression. In individuals with periodontitis, periodontal pockets constitute reservoirs of periodontal pathogens that may promptly colonize other sites of the oral cavity, including healthy sulci (Colombo et al., 2002; Riviere et al., 1996; Socransky & Haffajee, 2005). Thus, increased intraoral microbial transmission associated to a probable host immune susceptibility may explain in part the higher risk of periodontitis patients for further attachment loss compared to individuals with periodontal health. In fact, studies have indicated that differences in microbial colonization during biofilm formation between subjects with periodontitis and periodontal health do exist (Socransky & Haffajee, 2005; Teles et al., 2012; Uzel et al., 2011). Therefore, we hypothesized that biofilm re-development and composition, as well as the establishment of gingival inflammation occurs earlier and faster in individuals with chronic periodontitis (CP) compared to periodontally healthy (PH) individuals during withdraw of oral hygiene and supragingival plaque accumulation.
patients was required in each arm of the trial. To allow for 15% drop out, 15 patients were recruited in each clinical group. Secondary outcome variable were gingival bleeding (GI) and mean volume of GCF at day 7.
2. Material and methods
2.4. Supra and subgingival biofilm sampling
2.1. Sample population The study protocol was approved by the Human Research Ethics Committee of the Hospital of the Federal University of Rio de Janeiro (UFRJ), Brazil (CEP approval n° 707.355). Participants were recruited from the Division of Graduate Periodontics of the School of Dentistry at UFRJ, between February and December of 2014. Patients were individually informed about the nature of the study, its risks and benefits, and signed an informed consent form. To participate in the study, subjects had to have at least 18 years of age and 16 teeth (at least 4 in each quadrant). Patients were diagnosed as having CP or PH according to Da Silva-Boghossian et al. (2011). Briefly, CP was defined as ≥10% of teeth with PD and/or CAL ≥5 mm or ≥15% of teeth with PD and/or CAL ≥4 mm and BOP, and PH was defined as < 10% of sites with BOP, no PD or CAL > 3 mm, although PD or CAL = 4 mm in up to 5% of the sites without BOP was allowed. Exclusion criteria included smoking, diagnosed inflammatory systemic diseases, autoimmune diseases, aggressive periodontitis, use of topical or systemic antimicrobials in the last 6 months, periodontal therapy in the last year, orthodontic treatment, antibiotic prophylaxis, pregnancy or nursing.
At baseline (-T1), 2 individual supragingival biofilm samples (from disto-buccal sites of one anterior and one posterior tooth) and 2 individual subgingival biofilm samples (from the same sites and teeth) were collected per quadrant, providing a total of 8 supra and 8 subgingival biofilm samples per patient. For biofilm re-development analysis, supra and subgingival samples were collected from the distalbuccal sites of all teeth of contralateral quadrants at T0 (immediately after prophylaxis) and T1 (2 h), T2 (6 h), T3 (24 h) and T4 (7 days) after professional prophylaxis and no oral hygiene (Fig. 1S). For instance, if quadrant 2 was drawn for sampling at T0/T1, all distal-buccal sites from anterior teeth and from posterior teeth would be pooled into 2 pools (1 anterior and 1 posterior) for supra and subgingival biofilm (total of 4 pooled samples). Then, at T2, the contralateral quadrant 4 would be sampled likewise. At T3, quadrant 1 or 3 would be drawn and sampled, followed by the contralateral quadrant at T4. Thus, at time from T0 to T4 a patient would provide 4 samples (2 supra and 2 subgingival) per quadrant. During sampling, the area was dried and isolated with cotton rolls. Supragingival samples were always collected first, followed by subgingival samples with sterile curettes (Hu-Friedy®). Samples were placed into microtubes containing 150 μl of TE buffer.
2.2. Sample size calculation
2.5. GCF sampling
In a previous analysis of our microbial database, we computed the mean total counts of oral bacteria in 120 PH and 290 CP individuals. Considering total mean counts as the primary outcome variable, and assuming that CP would presented higher mean counts than PH patients, to detect a difference of 2.5 × 107 total mean counts between groups at 7 days with a one-sided significance level of 5% and power of 85% with equal allocation to two groups, a minimum sample size of 13
Before removal of biofilm from distal-buccal sites, GCF was collected from the mesio-buccal sites of the same 8 teeth selected for biofilm sampling at baseline (-T1), following the same sequence of quadrants at T0 to T4. For post-prophylaxis sampling, GCF samples were obtained individually from the mesio-buccal sites of all teeth in that particular quadrant selected, and samples were not pooled. GCF samples were collected with paper strips (Periopaper, Oraflow Inc.,
2.3. Clinical monitoring and biofilm development At the screening visit, participants answered a questionnaire, and information about demographic features, medical and dental health history was obtained. For diagnosis of periodontal status, clinical measurements were performed by a single calibrated examiner (F.A.R.R.H) using a North Carolina probe (UNC-15, Hu-Friedy, Chicago, IL, USA). The intra-class correlation coefficients for probing depth (PD) and clinical attachment level (CAL) were 0.94 and 0.88, respectively. The periodontal parameters evaluated included PD and CAL (mm), and presence of supragingival plaque (PL), bleeding on probing (BOP), GI, suppuration (SUP) and calculus (CA), at 6 sites per tooth of all teeth, except third molars. Ninety-seven recruited patients who agreed to participate in the study (signed the consent form) were clinically examined. Of those, 67 did not meet the inclusion criteria. Therefore, 30 individuals, 15 with PH and 15 with CP entered and finished the study. Patients selected for the study based on the inclusion/exclusion criteria and consent returned within one week at time -T1 (baseline) for sampling (described in Sections 2.5 and 2.6), and for full mouth measurements of PD, CAL, BOP, PL, GI, SUP and CA. Following baseline sampling and clinical evaluation (-T1), participants were submitted to supragingival ultrasonic and manual debridement, followed by dental polishing with rubber cup and prophylactic paste. At this moment, individuals were instructed to refrain brushing and flossing of their teeth for 7 days. GI was again measured at the seventh day of refraining oral hygiene procedures, after sampling (Fig. 1S). Patients were then referred to dental care in the different clinical specialties according to their treatment needs.
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New York, USA) to volumetric analysis in spectrophotometer (Periotron 8000, Oraflow Inc.). Teeth were isolated with cotton rolls, gently dried and a periopaper was inserted into gingival sulcus during 30 s. The strip was removed and GCF volume measured by Periotron 8000. The volumes obtained for each strip/sample were averaged for each patient at each time point.
Table 1 Demographic and clinical periodontal data of the sample population. Parameters
Gender (%) Females Males Race (%) White Afro-American Others Mean (SD) Age(years) N of missing teeth Mean% of sites Probing depth < 4 mm Probing depth 4–6 mm Probing depth > 6 mm Clinical attachment level < 4 mm Clinical attachment level 4–6 mm Clinical attachment level > 6 mm Bleeding on probing Supragingival biofilm Calculus Suppuration Gingival inflammation Gingival inflammation at day 7 without oral hygiene#
2.6. Microbiological assessment Microbiological analyses were carried out by the checkerboard DNA–DNA hybridization technique (Socransky et al., 1994), with modifications (Heller et al., 2011). Briefly, pooled biofilm samples were placed in microtubes containing 150 μl of TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.6). Samples were lysed by adding 150 μl of 0.5 M NaOH and boiling for 10 min. Denatured DNA was neutralized with 800 μl of 5 M C2H3O2NH4 and fixed in individual lanes on a nylon membrane (Hybond-N+, GE Healthcare Life Sciences, Piscataway, NJ, USA) using the Minislot 30 (Immunetics, Cambridge, MA). The membrane was placed in a Miniblotter 45 (Immunetics) with the lanes of fixed DNA perpendicular to the lanes of the device, and hybridized against 39 digoxigenin-labeled (“Random Primer Digoxigenin Labeling Kit”, Roche Molecular Systems, Inc., Alameda, CA) whole genomic DNA probes (Table 1S). DNA from Aggregatibacter actinomycetemcomitans (Aa) serotypes a, b and c was grouped into one probe, as was DNA from Propionibacterium acnes I and II. After hybridization, the membranes were washed at high stringency and bound probes incubated with phosphatase-conjugated antibody to digoxigenin at 1:15.000 (Roche Molecular Systems). Signals on the membranes were detected by fluorescence (AttoPhos®, PromegaCorporation, Madison, WI), and captured by an imaging system (Storm TM 860 and ImageQuant® version 5.2, Molecular Dynamics, GE Healthcare Life Sciences). Signals were evaluated visually by comparison with the standards at 105 and 106 cells for the tested species on the same membrane, and recorded as: 0 = not detected; 1 = < 105 cells; 2 = ∼105; 3 = 105–106 cells; 4 = ∼106; 5 = > 106 cells. The sensitivity of the assay was adjusted to permit the detection of 104 cells of a given species by adjusting the concentration of each DNA probe. Failure to detect a signal was recorded as zero, although conceivably, counts in the 1 to 1000 range could have been present.
PH group (n = 15)
CP group (n = 15)
P*
80.0 20.0
53.3 46.7
0.121†
80 0 20
53.3 20.0 26.7
0.139†
23.3 (0.8) 0.7 (0.3)
49.8 (2.9) 4.5 (0.7)
< 0.001* < 0.001*
99.1 0.9 0 99.0 1.0 0 5.8 (0.5) 21.4 (2.3) 8.8 (1.9) 0 4.3 (0.9) 19.5 (2.5)
79.4 20.1 0.6 73.1 25.2 1.6 19.3 (2.8) 48.5 (4.1) 30.0 (4.0) 0.1 (0.1) 19.5 (2.5) 23.5 (2.8)
< 0.001*
< 0.001*
< 0.001* < 0.001* < 0.001* 0.150§ < 0.001* > 0.05*
PH: Periodontal Health; CP: Chronic Periodontitis; SD: standard deviation. * Student T test. † Chi-square test. § Mann–Whitney test. # Refers to significant increase in inflammation from baseline to 7 days in both groups (p = 0.006, GLM).
3. Results 3.1. Demographic and clinical features of the study population Demographic and clinical data of the population are presented in Table 1. No statistical differences in gender or race between groups were seen, but PH patients were younger than CPpatients (p < 0.001). The CP group presented more signals and symptoms of periodontal inflammation and destruction than the PH group (p < 0.001). After 7 days of no oral hygiene, all PH individuals developed gingivitis, and CP individuals presented an extension of the previous gingivitisindicated by a significant increase in% of sites with GI (p = 0.006), with no difference between groups (p > 0.05).
2.7. Statistical analysis All analyses were performed using the SPSS 21.0 (IBM Brazil, SP, Brazil). Data entry was carried out by one investigator (F.A.R.R.H) and error proofed by a senior investigator (A.P.V.C.). All variables were tested for normality by the Kolmogorov–Smirnov test. Demographic data were computed for each individual and compared between groups by Student t and Chi-square tests. Periodontal measurements were averaged for each patient and within groups at baseline, and for GI at day 7. Differences between groups in clinical parameters were tested by the Student t-test, whereas difference in GI change from baseline to day 7 was examined by the Generalized Linear Model of Repeated Measures (GLM). GCF volumes were averaged for each patient and time, and differences between groups were sought by Mann–Whitney and Friedman tests. Microbial data were presented as mean counts of the tested species and calculated in each individual and within groups at different time points. Differences in mean counts of species between groups at each time of evaluation were examined by the Mann–Whitney test. Differences in microbiological changes over time in supra and subgingival biofilm between groups were evaluated by Friedman and GLM tests. Comparisons between mean counts of species between supra and subgingival biofilm were analyzed by Wilcoxon test. Adjustments for multiple comparisons were carried out (Socransky, Haffajee, Smith, & Dibart, 1991) so that the adjusted p-value was 0.0013 for microbiological analysis. For other parameters, the level of significance was 5%.
3.2. Changes in GCF volume over time The mean volumes of GCF measured over the 7 days without oral hygiene in both groups are shown in Fig. 1. At baseline, the CP group presented a significantly higher mean volume of GCF when compared to the PH group (p = 0.017). Although the volumes were still higher in the CP patients at all time points after prophylaxis, these differences were not significant. Right after prophylaxis, there was a marked reduction in GCF volume, particularly in the PH group at T2, 6 h after prophylaxis (p = 0.001). The decrease in GCF volume in the CP group was modest and no significant. After 24 h without oral hygiene, the GCF volumes increased in both groups to baseline levels in the PH group and to levels slightly lower than baseline in the CP. It is interesting to note that even though the GI and supra and subgingival microbial mean counts increased significantly in both groups at day 7 (Table 1), the GCF volume was kept at baseline values at this time point evaluation. 3.3. Total microbial counts Changes in mean total countsin supra and subgingival biofilm over 53
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Fig. 1. Mean (SD) volumes of GCF before prophylaxis (baseline) and up to 7 days without oral hygiene.T0, immediately after prophylaxis; T1, 2 h after; T2, 6 h after; T3, 24 h after; and T4, 7 days after prophylaxis. *Refers to significant difference between groups at baseline (p = 0.017, Mann–Whitney test). †Refers to significant reduction over time in GCF volume in the PH group (p = 0.001, Friedman test).
time in both groups are shown in Fig. 2. At baseline, the CP group presented significantly higher mean counts of microorganisms in both biofilms than the PH group (p < 0.05). A modest reduction in mean counts in supra and subgingival biofilm is observed in periodontally healthy individuals until 2 h after prophylaxis, increasing significantly at 6 h to levels greater than baseline values at 7 days without oral hygiene (p < 0.01). In the CP group, the reduction in counts is more marked after prophylaxis, and the microbial levels start to go up later than the PH group (p < 0.01). Nevertheless, both groups presented similar total mean counts at 24 h and 7 days without oral hygiene (Fig. 2). 3.4. Supra and subgingival biofilm composition At baseline, most species were detected at higher levels in the supra and subgingival biofilm of the CP group compared to PH patients (Fig. 1S). However, after adjusting for multiple comparisons,nosignificantdifferences between groups were observed for any species (adjusted p = 0.0013). Comparisons between supra and subgingival biofilm in each clinical group showed that only A. actinomycetemcomitans was detected in significantly higher counts in the subgingival biofilm compared to the supragingival biofilm in the PH group (p < 0.05). 3.5. Supra and subgingival biofilm Re-development Changes in mean counts in supra and subgingival biofilm after prophylaxis and during suspension of oral hygiene in both groups are depicted in Fig. 3. As observed, there was a significant increase in counts in most of the species evaluated in both groups and biofilms over time (p < 0.05). This increase occurred earlier in the PH group, while in the CP group the increase was more evident from T2. The only species that did not present significant changes over time in both groups were A. actinomycetemcomitans, A. naeslundii, E. nodatum, and S. constellatus. Significant differences in kinetics of re-colonization between groups were observed only in the subgingival biofilm (Fig. 4) for T. denticola and F. nucleatumssvicentii that increased more in the CP than PH group, and N. mucosa that showed greater increase in the PH compared to the CP group (p < 0.05). Comparisons in mean counts between groups at each time point indicated few significant differences. In the supragingival biofilm, differences were seen for L. buccalis at T0 and T1, N. mucosa, E. corrodens and C. sputigena at T1, C. showae and P. melaninogenica at T3, P. micra and E. saburreum at T4 (p < 0.05,
Fig. 2. Mean (SD) total counts of microorganisms in supragingival and subgingivalbiofilm from both groups (PH: Periodontal Health; CP: Chronic Periodontitis) before prophylaxis (baseline) and up to 7 days without oral hygiene. T0, immediately after prophylaxis; T1, 2 h after; T2, 6 h after; T3, 24 h after; and T4, 7 days after prophylaxis.*Refers to significant difference between groups at baseline (p = 0.037, Mann–Whitney test).†Refers to significant increase over time in mean counts in supra and subgingival biofilm for both groups (p < 0.01, Friedman test).
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Fig. 3. Changes in mean counts in supragingival and subgingival biofilm of individuals with periodontal health and chronic periodontitis after prophylaxis and during suspension of oral hygiene.T0, immediately after prophylaxis; T1, 2 h after; T2, 6 h after; T3, 24 h after; and T4, 7 days after prophylaxis.*Refers to statistical differences in counts of each species over time in each clinical group (p < 0.05; Friedman test).†Refers to statistical differences over time without differences between clinical groups, and ‡ with differences between groups (p < 0.05, GLM test). Bacterial species were grouped by the microbial complexes according to Socransky, Haffajee, Cugini, Smith, & Kent (1998).
(p < 0.05, Wilcoxon test). 4. Discussion Given that there are marked differences in biofilm composition and periodontal inflammation between CP and PH individuals, we hypothesized that the re-development of supra and subgingival biofilm during short term suspension of oral hygiene, as well as the clinical manifestations of gingival inflammation were distinct among these individuals. As expected, a significant greater number of sites with supragingival biofilm and gingival inflammation was detected in the CP than the PH group at baseline. However, all individuals presented high levels of biofilm accumulation associated to clinical signs of gingivitis on the seventh day of oral hygiene withdrawn, and no differences between groups were observed. Despite the short period of biofilm accumulation used in this study, the clinical data confirmed that this period was enough to induce gingivitis, even in individuals with PH (Loe et al., 1965). In fact, based on histological analysis in animal model, Page & Schroeder (1976) demonstrated that after 1week of dental biofilm accumulation, an early lesion developed characterized by increased vascular permeability, inflammatory infiltrate, and GCF flow, as well as degradation of connective tissue. Fransson, Mooney, Kinane, and Berglundh (1999) have also demonstrated that there were no significant changes in the composition of the gingival inflammatory infiltrate from 7 to 21 days of experimental gingivitis, concluding that the inflammatory response is well established within 1 week. For biofilm development, even a 3-day period of biofilm accumulation has been shown to result in a complex and organized mature microbial community (Listgarten, Mayo, & Tremblay, 1975). Although there was a significant increase in gingival bleeding in
Fig. 4. Species that showed significant differences in kinetics of re-colonization in the subgingival biofilm between periodontally healthy (PH) and chronic periodontitis (CP) individuals over time (p < 0.05, GLM). T0, immediately after prophylaxis; T1, 2 h after; T2, 6 h after; T3, 24 h after; and T4, 7 days after prophylaxis.
Mann–Whitney test). In the subgingival biofilm, differences were observed for P. melaninogenica at T2 and T3, F. periodonticum at T0, T. denticola at T2, and S. intermedius at T4 (p < 0.05, Mann–Whitney test). However, no significant differences were found after adjusting for multiple comparisons (data no shown). When the composition of the supragingival biofilm was compared to the subgingival biofilm in each group at every time points, significant differences were detected for S. constellatus at T0 in the PH group, and for T. denticola(T0), L. buccalis (T1) and P. gingivalis(T2) in the CP group 55
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performed in the same tooth site at every time of evaluation but at different teeth and quadrants. Analysis of the composition of the biofilm was carried out in pooled samples and it was limited to 37 oral species. Finally, the short period of time (7 days) chosen for oral hygiene withdrawn and biofilm re-development may have been insufficient to induce a significant long lasting disturbance in the periodontal microenvironment and microbial colonization/maturation of biofilm. Due to the large number of biological samples (biofilm and GCF) at several time points of evaluation, we had to select different tooth surfaces for sampling. Repeated sampling at the same tooth site in different moments would require repeated steps of prophylaxis, oral hygiene suppression and supragingival biofilm accumulation. Extending the time of biofilm accumulation was a critical issue in choosing the time span for the experimental gingivitis. Initially, we had set up a 14-day period of oral hygiene withdrawn. However, the large rate of dropouts during the screening phase of the population led us to reduce this period to 7 days. Despite these limitations, most of our findings are in agreement with data reported in the literature (Diaz et al., 2006; Heller et al., 2016; Li et al., 2004; Offenbacher et al., 2007; Ramberg et al., 1995; Ramberg et al., 2003; Uzel et al., 2011; Weidlich et al., 2001). In summary, a 1-week withdraw of oral hygiene and supragingival biofilm accumulation led to an increase in gingival inflammation in both clinical groups, although no major changes in GCF volume were observed. Re-development of supra and subgingival biofilm was similar between PH and CP individuals during this period of time. Supragingival prophylaxis had a greater short term impact in delaying biofilm re-colonization in periodontitis patients compared to healthy individuals. These changes were more evident in the supragingival biofilm. In contrast to supragingival biofilm, few species (N. mucosa, T. denticola, F. nucleatumssvicentii) differed between groups in kinetics of re-colonization in the subgingival biofilm. Based on these data, we reject the hypothesis that supra and subgingival biofilm re-development, as well as gingival inflammation during a short period of oral hygiene withdrawn happens earlier and more intensively in individuals with chronic periodontitis in relation to individuals with periodontal health.
both clinical groups at 7 days without oral hygiene, no major increase in GCF volume was observed at this time point. As expected, CP patients presented higher volumes of GCF than PH patients (Goodson, 2003; Lamster, Vogel, Hartley, DeGeorge, & Gordon, 1985; Teles et al., 2010; Zhang, Kashket, & Lingstrom, 2002) at all evaluations. Hours after prophylaxis there was a reduction in these volumes which was more marked in the PH group. After the establishment of gingivitis, however, the GCF volumes return to baseline values in both groups. It is important to consider here that the total volume of GCF and not flow rate was measured. The resting volume is more relatively constant, whereas GCF flow is expected to increase significantly with inflammation (Lamster, Vogel, Hartley, DeGeorge, & Gordon, 1985). Moreover, GCF was sampled from different sites and teeth at each time point, so that significant increases in mean volumes may have been underestimated. The relationship between supragingival biofilm accumulation and gingival inflammation has been studied for over decades (Loe et al., 1965; Ramberg, Axelsson, & Lindhe, 1995; Ramberg, Sekino, Uzel, Socransky, & Lindhe, 2003; Socransky & Haffajee, 2002; Weidlich, de Souza, & Oppermann, 2001; Zhang et al., 2002). Increase in biofilm density results in increased GCF flow and inflammatory mediators, which in turn function as important microbial nutrients and growth factors for the fast development and complex maturation of this biofilm (Socransky & Haffajee, 2005). The kinetics of re-colonization of dental biofilm by specific microorganisms during gingival inflammation has been investigated in more detail by few studies (Diaz et al., 2006; Heller et al., 2016; Li et al., 2004; Offenbacher et al., 2007; Ramberg et al., 2003; Teles et al., 2012; Uzel et al., 2011). In general, these studies demonstrated a hierarchical microbial succession in the re-colonization of supra and subgingival biofilm, with initial increase of first colonizers (yellow, blue, and purple complexes, N. mucosa), followed latter by the overgrowth of more pathogenic species, more specifically members of the orange complex (Fusobacterium spp., Prevotella spp., Campylobacter spp.). Similar data were also observed in our study for supra and subgingival biofilm in both PH and CP groups. In the supragingival biofilm, no differences in biofilm re-development were detected between groups for any particular species, corroborating those studies (Teles et al., 2012; Uzel et al., 2011). However, it is possible to note a tendency for greater mean counts of the blue, purple and green complexes in the PH group compared to the CP group at 7 days, whereas the CP individuals presented higher counts of the orange complex at this time point.In the subgingival biofilm, we did find significant differences in re-colonization between groups for T. denticola and F. nucleatumssvincentii (significant increase in CP) and N. mucosa (significant increase in PH). Of interest, after prophylaxis, re-development of supra and subgingival biofilm happened few hours earlier in PH than CP individuals. These findings suggested that supragingival prophylaxis seems to have a greater impact in sites/individuals with periodontitis than PH, more precisely in reducing periodontal inflammation and consequently delaying early biofilm re-colonization. On the other hand, the disturbance of prophylaxis in healthy sites/individuals with already low inflammation and biofilm accumulation may be less expressive. Other authors have shown that an adequate supragingival biofilm removal have a significant long-term effect on diminishing inflammation and total counts of subgingival microorganisms, mainly pathogenic species in patients with periodontal diseases (Cugini, Haffajee, Smith, Kent, & Socransky, 2000; Ximenez-Fyvie et al., 2000). Here, supragingival prophylaxis led to a decrease in total counts of subgingival biofilm but the impact on the supragingival biofilm in CP patients was more evident. These subtle distinct initial changes in biofilm re-development between groups disappear after 24 h without oral hygiene, and no significant differences in microbial composition were detected between supra and subgingival biofilms in any group at 7 days of follow up. The overall similarity in biofilm re-development in PH and CP individuals observed here should be considered carefully given that limitations in the study design do exist. First, biofilm sampling was not
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