Original Article

http://dx.doi.org/10.1590/1678-7757-2018-0671

Clinical attachment loss and molecular

profile of inflamed sites before
treatment

Abstract

Cristine D’Almeida BORGES1 Objective: To monitor early periodontal disease progression and to

Milla Sprone RICOLDI1 investigate clinical and molecular profile of inflamed sites by means of
crevicular fluid and gingival biopsy analysis. Methodology: Eighty-one
Michel Reis MESSORA1
samples of twenty-seven periodontitis subjects and periodontally healthy
Daniela Bazan PALIOTO1
individuals were collected for the study. Measurements of clinical parameters
Sérgio Luís Scombatti de SOUZA1 were recorded at day -15, baseline and 2 months after basic periodontal
Arthur Belém NOVAES JÚNIOR1 treatment aiming at monitoring early variations ofthe clinical attachment
Mario TABA JR1 level. Saliva, crevicular fluid and gingival biopsies were harvested from
clinically inflamed and non-inflamed sites from periodontal patients and
from control sites of healthy patients for the assessment of IL-10, MMP-8,
VEGF, RANKL, OPG and TGF-β1 protein and gene expression levels. Results:
Baseline IL-10 protein levels from inflamed sites were higher in comparison
to both non-inflamed and control sites (p<0.05). Higher expression of mRNA
for IL-10, RANK-L, OPG, e TGF-β1 were also observed in inflamed sites at
day -15 prior treatment (p<0.05). After the periodontal treatment and the
resolution of inflammation, seventeen percent of evaluated sites still showed
clinically detectable attachment loss without significant differences in the
molecular profile. Conclusions: Clinical attachment loss is a negative event
that may occur even after successful basic periodontal therapy, but it is small
and limited to a small percentage of sites. Elevated inflammation markers
of inflamed sites from disease patients reduced to the mean levels of those
observed in healthy subjects after successful basic periodontal therapy.
Significantly elevated both gene and protein levels of IL-10 in inflamed sites
prior treatment confirms its modulatory role in the disease status.

Keywords: Periodontal attachment loss. Biological markers. Gingival

crevicular fluid. Biopsy. Gene expression.

Submitted: November 20, 2018

Modification: February 15, 2019
Accepted: February 26, 2019

Corresponding address: 1
Universidade de São Paulo, Faculdade de Odontologia de Ribeirão Preto, Departamento de Cirurgia
Mario Taba Jr e Traumatologia Bucomaxilofacial e Periodontia, Ribeirão Preto, São Paulo, Brasil.
Departamento de Cirurgia Oral e Periodontia
- Faculdade de Odontologia de Ribeirão Preto -
Universidade de São Paulo.
Avenida do Café - s/n - 14040-904 -
Ribeirão Preto - SP - Brasil.
Phone: 55 16 36024135 -
Fax number: 55 16 3602 4788
e-mail: [email protected]

J Appl Oral Sci. 1/10 2019;27:e20180671

 Clinical attachment loss and molecular profile of inflamed sites before treatment

Introduction clinical and molecular features of progressive sites

through saliva, gingival crevicular fluid and gingival

Periodontal disease is a chronic microbial infection tissue samples.

characterized by the inflammation of supportive

tissues and alveolar bone loss. Particularly in chronic
periodontitis, the presence of local irritants is compatible Methodology
with the severity of the disease. Although bacteria are
1

essential in the onset and maintenance of periodontitis, Patient population

susceptibility and disease progression are determined Twenty-seven participants were selected; amongst
by a complex interaction driven by the modulation of them eighteen presented periodontitis stage II grade
an immune-inflammatory host response.2,3 Locally, B16 (periodontitis group) and nine were healthy (control
bacterial lipopolysaccharides induce inflammatory group). Post hoc power analysis was made through
cells to release pro-inflammatory mediators that G*Power 3.1.9.2 using mean and standard deviations
seem to act in the destruction of periodontal tissues. 3
of the total amount level of IL-10 in inflamed and
The presence of inflammatory cells and lymphocytes control sites, and 99% of power was obtained in this
infiltration, chemotactic factors involved in recruiting study.
these cells and cytokines involved in the pathogenesis Participants were chosen from the dental clinics
and progression of the periodontal disease.4 of the Ribeirão Preto School of Dentistry and
The activation of a local immune response by T were invited to take part in the study. All enrolled
helper cells would determine the stability or progression patients gave written consent on a form approved
of the periodontal disease. Th1 lymphocytes are by the Ethics Committee Protocol of the Ribeirão
characterized by the secretion of cytokines involved Preto School of Dentistry - USP (approval number
in eradicating intracellular pathogens, whereas Th2 # 02841912.0.0000.5419). Participants underwent
cells are responsible for secreting cytokines involved in anamnesis, clinical and radiographic examination.
eliminating extracellular micro-organisms. Also, Th17 5
Included participants had at least 14 natural teeth
and T regulatory (Treg) cells are involved in disease and posterior occlusion stability. Participants in the
progression. Th17 subset presents pro-inflammatory chronic periodontitis group were at least 35 years old
and pro-resorptive activities, especially for secretion of with 5 teeth presenting probing depth (PD) of ≥5 mm
IL-17 and RANKL, both involved in the differentiation and clinical attachment loss of ≥3 mm.17 Participants
and activation of osteoclasts. On the other hand, Treg in the control group had PD ≤3 mm in all teeth and
cells subset displays suppressor functions producing plaque index and bleeding on probing values ≤20%.
IL-10 and transforming growth factors (TGF-β1).6,7 In Participants presenting any disorder or ongoing
this context, IL-10 seems to have a modulatory role medication usage were excluded. Also, they could not
on inflamed and progressive sites. have received periodontal treatment in the past six
Host modulatory effects of specific cytokines such months.
as IL-10, IL-13, OPG and TGF-β1 are responsible
for the selective recruitment of different cells, Clinical parameters
cytokines production and may determine the disease Clinical examinations and data collections were
progression. These cytokines associated to host
8
performed at day -15, baseline and two months after
defense have been identified in saliva,3,9 blood,10,11 basic periodontal therapy. Figure 1 illustrates the
gingival crevicular fluid 8,11
and gingival tissues. 12,13
timeline of the study. Probing pocket depth (PPD),
Elevated levels of these molecules may be related to relative clinical attachment level (rCAL) and bleeding
periodontal disease condition, allowing identification on probing (BOP) were recorded at six sites per tooth
and controlling patients with periodontal disease. 14
(mesio-buccal, buccal, disto-buccal, mesio-lingual,
Studies that aim to analyze cytokines host lingual and disto-lingual) with the aid of a computerized
modulatory effect during disease progression seem to periodontal probe (Florida Probe Corporation,
be promising in periodontal diagnostic. 15
Therefore, Gainsville, FL, USA). The presence or absence of
in this study, we aimed to monitor early changes in biofilm at four sites per tooth (plaque index - PI)
attachment levels of progressive sites and investigate were also recorded.18 It was also verified the furcation

J Appl Oral Sci. 2/10 2019;27:e20180671

 BORGES CD, RICOLDI MS, MESSORA MR, PALIOTO DB, SOUZA SL, NOVAES JÚNIOR AB, TABA JR M

involvement with the aid of a manual periodontal probe according to the method described by Navazesh22
(Hu-Friedy, Chicago, IL, USA). (1993), by one calibrated examiner the day after the
After clinical examinations of day -15 and baseline, initial diagnosis and on the day after post-therapy
sites were categorized according to the presence or periodontal evaluation. Saliva samples were placed
absence of inflammation: (i) inflamed sites (PD ≥ 5 mm on ice immediately and aliquoted prior to freezing at
and recurrent BOP after clinical exams at -15 days and -80°C.
baseline); (ii) non-inflamed sites (PD ≤4 mm without The salivary inflammatory protein levels were
BOP after clinical exams at -15 days and baseline); identified simultaneously using Multiplex Cytokine
(iii) and control sites (PD ≤3 mm without BOP after Profiling Assay in the Luminex platform (Luminex
clinical exams at -15 days and baseline). For matching Corporation, Austin, TX, USA). The following proteins
comparison purpose, inflamed sites and non-inflamed were analyzed: IL-10, MMP-8, VEGF, RANKL, OPG
sites were from the same participant (periodontitis and TGF-β1. The assay was performed according to
group) for gingival crevicular fluid and gingival biopsy the manufacturer’s protocol. Briefly, ten microliters
analysis. of the diluted sample (proteins) were added to a 50
Scaling and root planning sessions were performed µl cocktail of capture beads and an antibody detector,
by the same operator in two to four sessions within and the mixture was incubated for 4 hours at room
24- to 48-hour interval 19
using hand instruments (Hu- temperature. Excess unbound antibody detector was
Friedy, Chicago, IL, USA) and an ultrasonic (Dentsply, washed off and flow cytometric analysis were performed
York, PA, USA) device. Oral hygiene was reviewed using the appropriate CMA analysis software.
after a week and repeated 30 days after periodontal
disinfection, followed by dental prophylaxis. After two Gingival crevicular fluid sampling and analysis
months, a new periodontal examination was performed Gingival crevicular fluid samples were collected
to evaluate PI, BOP, PD and rCAL using a computerized at baseline, 15 days and 2 months after therapy. In
probe to detect progressive sites. periodontitis group patients, gingival crevicular fluid
Progressive sites categorization was based on the samples were collected from three inflamed sites
tolerance method.20,21 In brief, progressive sites were and one non-inflamed site. In control patients, fluid
those that presented clinical attachment loss of ≥1 mm samples were collected from one control site. First, the
after two months considering the average error of 0.3 supragengival plaque was removed, sites were isolated
mm of the electronic probe multiplied by 3. with cotton rolls and gently air dried. Fluid samples were
Scaling and root planning sessions, clinical collected with sterile Periopaper strips (Oraflow Inc.,
examinations and data collections were made by only Planviwe, NT, USA) that were inserted into the gingival
one examiner, who is an experienced Periodontist crevice until mild resistance was felt and left in place
(Borges, C.D.). for 30 seconds. After gingival crevicular fluid collection,
strips were placed in Eppendorf vials and immediately
Saliva collection and analysis frozen at -80°C until use.
The patients were instructed not to drink or eat for Gingival crevicular fluid samples were placed into
at least 60 min before the saliva sample collection. 60 µl of sodium phosphate buffer (Life Technologies,
Non-stimulated whole expectorated saliva was Carlsbad, California, USA) and 0.01 ml of Tween® 20
collected (~3 ml) from each subject into sterile tubes, (USB Corporation, Cleveland, USA). Protein levels

Figure 1- Timeline of the study

J Appl Oral Sci. 3/10 2019;27:e20180671

 Clinical attachment loss and molecular profile of inflamed sites before treatment

of IL-10 and VEGF were identified simultaneously Statistical analysis

using multiplex cytokine profiling assay (Luminex Data were grouped by average and their respective
Corporation, Austin, TX, USA). MMP-8 levels were standard deviation. Specific sites and individuals were
analyzed by ELISA and carried out according to the considered for parametric or non-parametric statistical
manufacturer’s instructions. analysis when appropriated after Lilliefors normality
test.
Gingival biopsy
For collecting gingival tissue samples (containing Clinical parameters
both epithelial and connective tissues), all patients For intra-group comparison, before and after
received local anesthesia. In periodontitis group treatment, Wilcoxon test or t test was applied. For
patients, samples were harvested from one inflamed intergroup comparison, Mann-Whitney test or t test
and one non-inflamed site. In Control patients, samples was applied. A significance level of 5% was adopted
were removed from one control site. The gingival for all statistical analyses (P<0.05).
biopsies were harvested from the same site that had
the gingival crevicular fluid collected. Two incisions Gingival crevicular fluid proteins
were made for samples collection. First, the initial For intra- and intergroup comparison, at baseline,
incision was made 1.5 mm away from the tooth with 15 days and 2 months, Kruskal-Wallis test or ANOVA
a scalpel, until bone crest. Then, an intracrevicular was applied.
incision was made for gingival tissue removal that
consists of periodontal pocket/gingival sulcus wall. Real Time PCR arrays
Incisions were made around the selected sites, not Differential expression calculation was done by a
around the tooth. In patients from periodontitis group, specific software for data analysis (SABiosciences,
samples were removed during periodontal treatment, Frederick, MD, USA). Relative gene expression
before scaling and root planning. In patients from normalization and quantification were performed by
control group, samples were removed during surgical 2-ΔΔCT method.23 This software also performed pair-
procedures as root coverage. These samples were wise comparisons between groups of experimental
immediately flash frozen in liquid nitrogen then replicates and defined fold-change and statistical
preserved under -80°C for posterior RNA extraction significant thresholds. Therefore, data were presented
and gene expression analysis of IL-10, MMP-8, VEGF, as a difference (fold regulation) in gene expression,
RANKL, OPG e TGF-β1. which would be normalized by the geometric mean
value of actin-beta (ACTB). Significance level was set
RNA extraction and Real-time PCR at p<0.05.
Total RNA from biopsies was extracted using the
Trizol reagent (Invitrogen, Milan, Lombardy, Italy)
method. The aqueous phase was transferred to a
Results
new tube, to which 0.25 ml of 95% ethanol (Sigma,
St Louis, MO, USA) was added. The suspension was
Clinical findings
transferred to the spin basket assembly of the kit
The subjects’ demographic data are displayed in
(Promega, Madison, WI, EUA) and centrifuged at
Table 1. There was a higher prevalence of women and
10,500 rpm for 1 min at 4°C. From 1 µg of total RNA, a
Caucasians in our sample. At baseline, periodontitis
strand of complementary DNA (cDNA) was synthesized
and control groups had different mean values of clinical
through a reverse transcription reaction (SABioscience,
parameters (Table 1). After basic periodontal therapy,
Frederick, MD, USA).
periodontitis group showed a significant improvement
Reactions were carried out in triplicate for each
in the clinical parameters (p<0.05).
sample (inflamed sites, non-inflamed sites and control
Significant differences between inflamed and non-
sites). The reactions were performed on a real-time
inflamed sites for PD, rCAL and BOP, and between
thermocycler (Life Applied Biosystems, Carlsbad,
inflamed and control sites for PD and PI (p<0.05)
California, USA), according to the directions supplied by
(Table 2) were also observed. 2,436 sites from
the manufacturer. Following sample amplification and
periodontitis group were analyzed and after periodontal
calculations, the expression levels were determined.

J Appl Oral Sci. 4/10 2019;27:e20180671

 BORGES CD, RICOLDI MS, MESSORA MR, PALIOTO DB, SOUZA SL, NOVAES JÚNIOR AB, TABA JR M

therapy, 17% of total sites showed progressive clinical group 1.2 pg/mL (p=0.0313) was observed. OPG
attachment loss (p<0.05). protein expression was higher in periodontitis group
Comparisons of clinical measurements between before therapy. After 2 months, a 40% reduction was
-15 days and baseline, without any interventional observed (p=0.0002).
therapy, showed difference in PD (5.6±0.85 and
5.9±1.30, respectively) in inflamed sites, but not Gingival crevicular fluid proteins
significant (p=0.37). For non-inflamed sites (2.7±0.6 Eighty-one samples were included for the gingival
and 2.5±0.9, respectively), difference was also not crevicular fluid analyses. IL-10, VEGF and MMP-8
significant (p=0.39). were detected in gingival crevicular fluid collected at
baseline, 15 days and 2 months (Figure 2). Our data
Salivary proteins showed a higher total amount of VEGF in inflamed
In the baseline, higher expression of RANK-L in sites in comparison to non-inflamed sites at all times.
periodontitis group 2.99 pg/mL in comparison to control There were no differences between baseline and 2

Table 1- Demographic and clinical data from Control and CP groups. NS - non significant (p>0.05). Mean ± standard deviation. * Wilcoxon
test for intragroup comparisons; ** t test for intragroup comparisons and between two groups; ¥ Mann-Whitney test for comparisons
between two groups at baseline and at 2-month evaluation. PD: Probing depth; rCAL: Relative attachment level; PI: Plaque index; BOP:
Bleeding on probe

Control (n = 9) Periodontitis (n = 18) *P Value

Age (years; mean ± SD) 33.2 ± 7.82 48.1 ± 7.82 p = 0.001¥
Female (%) 66,70% 72,20% _
Caucasian (%) 100% 83,30% _
Non-Caucasian (%) 0% 16,70% _
N. teeth 27.4 ± 4.2 23.7 ± 2.6 0,007
PPD (mm)
initial 2.2 ± 0.1 3.1 ± 0.6 p < 0.0001**
2 months 2.1 ± 0.1 2.4 ± 0.3 p = 0.0035**
p value NS* < 0.0001** -
Delta (Δ) 0.1 ± 0.2 0.7 ± 0.4 p < 0.0001**
rCAL (mm)
initial 8.3 ± 1.2 10.4 ± 1.2 p = 0.004¥
2 months 8.0 ± 1.0 9.5 ± 0.9 p = 0.0007¥
p value NS** 0.0002* -
Delta (Δ) 0.3 ± 0.3 0.9 ± 0.5 p = 0.0012**
PI (%)
initial 11.1 ± 6.3 68.9 ± 21.5 p < 0.0001**
2 months 10.6 ± 6.0 31.8 ± 22.7 p = 0.0007¥
p value NS** < 0.0001** -
Delta (Δ) 0.5 ± 4.8 37.1 ± 25 p < 0.0001**
BOP (%)
initial 16.7 ± 10.3 49.3 ± 12.8 p < 0.0001¥
2 months 13.7 ± 7.7 27.2 ± 7.3 p = 0.0001**
p value NS* < 0.0001** -
Delta (Δ) 3.1 ± 7.2 22.1 ± 13.3 p = 0.0005**

Table 2- Mean difference after periodontal therapy of inflamed, non-inflamed and control sites. Mean ± standard deviation. Kruskal-Wallis
test for comparison between inflammation, non-inflammation and control sites. a: significantly lower than inflammation sites (p<0.05)

PD (mm) rCAL (mm) PI (%) BOP (%)

Inflamed sites 1.93 ± 0.72 1.3 ± 0.82 46.3 ± 41.44 38.89 ± 32.84
Non-inflamed sites 0.6 ± 0.7a 0.1 ± 1.0a 22.2 ± 73.2 - 22.2 ± 42.8
Control sites 0.2 ± 0.4 a
0.4 ± 0.7 -11.1 ± 33.3a
0.0a

J Appl Oral Sci. 5/10 2019;27:e20180671

 Clinical attachment loss and molecular profile of inflamed sites before treatment

months in all sites. The total amount of MMP-8 had a reduction 15

The total amount of IL-10 was higher in inflamed days after periodontal therapy, but not statistically
sites in comparison to non-inflamed sites at all times significant, and the total amount was higher in inflamed
(p<0.05). Also, non-inflamed sites showed higher sites after two months (p<0.05). Also, it was higher in
amounts of IL-10 in comparison to control sites at all control sites in comparison to non-inflamed sites after
times (p<0.05). 15 days (p<0.05).

Figure 2- Total amount of VEGF, IL-10 and MMP-8 in gingival crevicular fluid of inflamed, non-inflamed and control sites, at baseline,
15 days and 2 months. Kruskal-Wallis test and ANOVA test. *: difference between inflamed sites at baseline and 2 months; €: difference
between inflamed sites at 15 days and 2 months; a: difference between inflamed and non-inflamed sites at 15 days; b: difference between
inflamed and control sites; c: difference between non-inflamed and control sites; VEGF: Vascular endothelial growth factor; IL-10:
Interleukin-10; MMP-8: Matrix metalloproteinase-8

J Appl Oral Sci. 6/10 2019;27:e20180671

 BORGES CD, RICOLDI MS, MESSORA MR, PALIOTO DB, SOUZA SL, NOVAES JÚNIOR AB, TABA JR M

Figure 3- Relative mRNA expression of RANKL, OPG, MMP-8, VEGF, IL-10 and TGF-β1 in inflamed, non-inflamed and control sites.
Kruskal-Wallis test and ANOVA test. a: significant difference between inflamed and non-inflamed sites b: significant difference between
inflamed and control sites c: significant difference between control and non-inflamed sites. RANKL: Receptor activator of nuclear factor
ĸB; OPG: Osteoprotegerin; VEGF: Vascular endothelial growth factor; IL-10: Interleukin-10; MMP-8: Matrix metalloproteinase-8; TGF-β1:
Transforming growth factor- β1

mRNA expression in order to express significant clinical differences

We examined the expression of IL-10, RANKL, measured by periodontal parameters (PD a rCAL) and
OPG, MMP-8, VEGF, and TGF-β1 in inflamed, non- inflammation (BOP). Additionally, early changes in the
inflamed and control sites after periodontal therapy. clinical attachment levels were used to investigate the
Comparisons between inflamed sites and non-inflamed role of inflammatory markers in disease modulation.
sites, showed increased expression of IL-10 (p=0.03), Samples were collected at baseline, 15 days
RANKL (p<0.001) OPG (p=0.02), and TGF-β1 (p<0.05) and 2 months after basic periodontal therapy. As
in inflamed sites. Control sites demonstrated higher expected, our data showed significant difference in
expression of OPG (p<0.001) and TGF-β1 (p<0.05) clinical parameters between periodontitis group and
when compared to non-inflamed sites. Inflamed sites control group at baseline. After periodontal therapy,
had higher expression of IL-10 when compared to data showed significant improvements on clinical
control sites (p=0.026). MMP-8 and VEGF showed no parameters in periodontitis group. It was observed
differences (Figure 3). reduction in PD (0.7 mm ±0.4), BOP (37.1%±5.0), PI
(27.2%±7.3), and rCAL gain (0.9 mm ±0.5). These
results confirm the short-term beneficial effect of the
therapy and are in accordance with previous data
Discussion
that showed better clinical conditions after full mouth
disinfection24, 25 or scaling and root planning over a 3-
In the present study, we monitored inflammation
to 4- week period.26, 27
and progressive periodontal sites to investigate
Inflamed sites showed higher amount of IL-10
potential differences in the molecular profile of gingival
(0.29 pg ±0.09) in comparison to control sites (0.21
crevicular fluid and gingival biopsies from inflamed and
pg ±0.08) before treatment (p<0.05). Furthermore,
non-inflamed sites. Groups and sites were categorized

J Appl Oral Sci. 7/10 2019;27:e20180671

 Clinical attachment loss and molecular profile of inflamed sites before treatment

IL-10 mRNA expression was higher in inflamed sites in periodontitis patients compared to healthy patients,
comparison to non-inflamed and control sites. This is as well as higher expression of IL-10 mRNA. The
in accordance to some previous results. 28-30
Goutoudi, expression of OPG was also higher, but not significant.
et al. (2004) using a different methodology observed
31
According to the authors, higher expression of OPG
a similar amount of IL-10 when compared diseased could control the alveolar bone loss driven by RANKL,
and non-diseased sites instead of the inflamed sites attenuating the progression and severity of the disease.
classification of our study. The expression of RANKL and MMPs may result in tissue
Periodontal disease activity is accepted as bone and destruction and disease progression, whereas the
attachment loss related to variations in inflammatory
32
higher expression of TIMPs and OPG possibly induced
cells, migration of monocytes/macrophage33 and has by IL-10, could be responsible for the control of tissue
been associated to inflammatory biomarkers. 7, 34, 35
destruction.29 Indeed, these results are in agreement
Our results found that 17% of total sites could be to ours and suggest that, in higher amounts, IL-10
classified as progressive, according to the tolerance could control bone resorption and moderate periodontal
method. 20, 21, 36
However, we did not find differences destruction.
in the protein levels of MMP-8, VEGF and IL-10 in We also found higher expression of TGF-β1 mRNA
gingival crevicular fluid of progressive sites compared in inflamed sites compared to non-inflamed sites
to inactive sites after therapy. Indeed, no association (p<0.05). Dutzan, et al.41 (2012) observed higher
was observed between bleeding on probe and disease expression of TGF-β1 in healthy sites compared to
progression. A previous study observed a relationship periodontitis sites, which in our study showed no
between bleeding on probe and disease activity, but it difference. Unlikely, we found higher expression of
is yet controversial and other authors showed similar TGF-β1 mRNA in inflamed and control sites compared
results to ours. 37
to non-inflamed sites, probably indicating the anti-
Interestingly, the higher expression of MMP-8 in inflammatory characteristic and modulatory role of
inflamed sites observed in our study may explain TGF-β1 in inflamed sites, possibly promoting modulation
why progressive sites also demonstrated higher IL-10 of pro-inflammatory cytokines and stimulating the
levels. The anti-inflammatory effect of IL-10 decreases production of IL-1 receptor antagonist, which regulates
the expression of pro-inflammatory cytokines, like anti-inflammatory and immunesupressor activities.39
TNF-alfa, IL-1β and matrix metalloproteinases (MMPs). Regarding VEGF, we found significant difference
Because of its protective function against bone loss, IL- between inflamed sites and control sites at all times.
10 inhibits MMPs29 through the up-regulation of Tissue This result is subject to bias given gingival tissue
Inhibitor of Metalloproteinase (TIMPs) that are capable samples collected from sites that received periodontal
of inhibiting almost every member of the MMP family 38
therapy. Besides, the presence of VEGF in gingival
Thus, the higher expression of IL-10 in inflamed sites fluid of healthy patients may reflect sub-clinical levels
may have moderated the destructive effect of Th1 of inflammation, healing following bacterial assault
response and may have been accounted for lowering or physiological angiogenesis in periodontal tissues.40
the expression of MMP-8.28 Although clinical results Despite having some sites with periodontal disease
demonstrated periodontal pocket reduction after progression, our site-specific analysis also showed
periodontal therapy, some sites remained with probing considerable levels of anti-inflammatory markers,
depth >4 mm. This can explain the increase in MMP- possibly reducing risk for more attachment loss.
8 levels in 2 months, although its reduction after 15 In conclusion, in spite of data analysis limitations
days. Remaining periodontal pocket could increase and the short follow-up period to appreciate major
inflammatory cytokines. disease breakdown, this preliminary study stressed
Furthermore, our site-specific analysis presented out that progressive disease activity is a possible
higher expression of RANKL mRNA in inflamed sites occurrence even after basic periodontal therapy, but is
compared to non-inflamed sites. Inflamed sites also limited to a small percentage of sites. Also, periodontal
had higher expression of OPG mRNA compared to treatment reduces elevated inflammation markers
non-inflamed sites and, consequently, relative ratio of inflamed sites from disease patients to levels of
RANKL/OPG mRNA was higher. Garlet, et al. 32
(2004) those observed in healthy subjects, but these levels
observed higher expression of RANKL mRNA in chronic could not be sustained in case of residual periodontal

J Appl Oral Sci. 8/10 2019;27:e20180671

 BORGES CD, RICOLDI MS, MESSORA MR, PALIOTO DB, SOUZA SL, NOVAES JÚNIOR AB, TABA JR M

pockets. However, as elevated gene and protein anti- 15- Ozmeric N. Advances in periodontal disease markers. Clin Chim
Acta. 2004;343(1-2):1-16.
inflammatory marker levels in inflamed sites prior
16- Caton JG, Armitage G, Berglundh T, Chapple IL, Jepsen S, Komman
treatment could suggest its modulatory role, it does KS, et al. A new classification scheme for periodontal and peri-implant
not seem to discriminate future progressive sites. diseases and conditions - Introduction and key changes from the 1999
classification. J Periodontol 2018;89 Suppl 1:S1-S8.
Predictors of future attachment loss are still a challenge
17- Hernandez M, Valenzuela MA, Lopez-Otin C, Alvarez J,
in periodontal diagnosis. Lopez JM, Vernal R, et al. Matrix metalloproteinase-13 is highly
expressed in destructive periodontal disease activity. J Periodontol.

Aknowledgments 2006;77(11):1863-70.
18- O’Leary TJ, Drake RB, Naylor JE. The plaque control record. J
Authors thank the State of São Paulo Foundation
Periodontol. 1972;43(1):38.
(FAPESP) for financial support, grant number 19- Mongardini C, van Steenberghe D, Dekeyser C, Quirynen M. One

12/15265-7 to MTJ; Coordenação de Nível Superior stage full- versus partial-mouth disinfection in the treatment of chronic
adult or generalized early-onset periodontitis. I. Long-term clinical
– Brasil (CAPES) – Finance code 001 and Viviane
observations. J Periodontol. 1999;70(6):632-45.
Mariguela for assisting with laboratory methods. 20- Haffajee AD, Socransky SS, Goodson JM. Comparison of different
data analyses for detecting changes in attachment level. J Clin
Periodontol. 1983;10(3):298-310.
21- Hernández M, Dutzan N, García-Sesnich J, Abusleme L, Dezerega

References A, Silva N, et al. Host-pathogen interactions in progressive chronic

periodontitis. J Dent Res. 2011;90(10):1164-70.
22- Navazesh M. Methods for collecting saliva. Ann N Y Acad Sci.
1- Armitage GC. Development of a classification system for periodontal
1993;694:72-7.
diseases and conditions. Ann Periodontol. 1999;4:1-6.
23- Livak KJ, Schmittgen TD. Analysis of relative gene expression data
2- Socransky SS, Haffajee AD. The bacterial etiology of destructive
using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.
periodontal disease: current concepts. J Periodontol. 1992;63(4
Methods 2001;25(4):402-8.
Suppl):322-31.
24- Konopka L, Pietrzak A, Brzezínska-Blaszczyk E. Effect of scaling
3- Taba M Jr, Kinney J, Kim AS, Giannobile WV. Diagnostic biomarkers for
and root planing on interleukin-1β, interleukin-8 and MMP-8 levels in
oral and periodontal diseases. Dent Clin North Am. 2005;49:551-71, vi.
gingival crevicular fluid from chronic periodontitis patients. J Periodontal
4- Gamonal J, Acevedo A, Bascones A, Jorge O, Silva A. Levels of
Res. 2012;47(6):681-8.
interleukin-1 beta, -8, and -10 and RANTES in gingival crevicular fluid
25- Preus HR, Gunleiksrud TM, Sandvik L, Gjermo P, Baelum
and cell populations in adult periodontitis patients and the effect of
V. A randomized, double-masked clinical trial comparing four
periodontal treatment. J Periodontol. 2000;71(10):1535-45.
periodontitis treatment strategies: 1-year clinical results. J Periodontol.
5- Mosmann TR, Coffman RL. TH1 and TH2 cells: different patterns of
2013;84(8):1075-86.
lymphokine secretion lead to different functional properties. Annu Rev
26- Chen HY, Cox SW, Eley BM, Mäntylä P, Rönkä H, Sorsa T. Matrix
Immunol. 1989;7:145-73.
metalloproteinase-8 levels and elastase activities in gingival crevicular
6- Vernal R, Garcia-Sanz JA. Th17 and Treg cells, two new lymphocyte
fluid from chronic adult periodontitis patients. J Clin Periodontol.
subpopulations with a key role in the immune response against
2000;27(5):366-9.
infection. Infect Disord Drug Targets. 2008;8(4):207-20.
27- Marcaccini AM, Meschiari CA, Zuardi LR, Sousa TS, Taba JM Jr,
7- Vernal R, Chaparro A, Graumann R, Puente J, Valenzuela MA,
Teofilo JM, et al. Gingival crevicular fluid levels of MMP-8, MMP-9,
Gamonal J. Levels of cytokine receptor activator of nuclear factor
TIMP-2, and MPO decrease after periodontal therapy. J Clin Periodontol.
kappaB ligand in gingival crevicular fluid in untreated chronic
2010;37(2):180-90.
periodontitis patients. J Periodontol. 2004;75(12):1586-91.
28- Garlet GP, Martins W Jr, Ferreira BR, Milanezi CM, Silva JS. Patterns
8- Kumar MS, Vamsi G, Sripriya R, Sehgal PK. Expression of matrix
of chemokines and chemokine receptors expression in different forms
metalloproteinases (MMP-8 and -9) in chronic periodontitis patients
of human periodontal disease. J Periodontal Res. 2003;38(2):210-7.
with and without diabetes mellitus. J Periodontol. 2006;77(11):1803-8.
29- Garlet GP, Martins W Jr, Fonseca BA, Ferreira BR, Silva JS. Matrix
9- Sexton WM, Lin Y, Kryscio RJ, Dawson DR 3 , Ebersole JL, Miller CS.
rd

metalloproteinases, their physiological inhibitors and osteoclast factors

Salivary biomarkers of periodontal disease in response to treatment.
are differentially regulated by the cytokine profile in human periodontal
J Clin Periodontol. 2011;38(5):434-41.
disease. J Clin Periodontol. 2004;31(8):671-9.
10- Takayanagi H. Mechanistic insight into osteoclast differentiation in
30- Napimoga MH, Nunes LH, Maciel AA, Demasi AP, Benatti BB, Santos
osteoimmunology. J Mol Med (Berl). 2005;83(3):170-9.
VR, et al. Possible involvement of IL-21 and IL-10 on salivary IgA levels
11- Kinney JS, Morelli T, Oh M, Braun TM, Ramseier CA, Sugai JV, et
in chronic periodontitis subjects. Scand J Immunol. 2011;74(6):596-
al. Crevicular fluid biomarkers and periodontal disease progression. J
602.
Clin Periodontol. 2014;41(2):113-20.
31- Goutoudi P, Diza E, Arvanitidou M. Effect of periodontal therapy on
12- Guneri P, Unlü F, Yeşilbek B, Bayraktar F, Kokuludağ A, Hekimgil
crevicular fluid interleukin-1beta and interleukin-10 levels in chronic
M, et al. Vascular endothelial growth factor in gingival tissues and
periodontitis. J Dent. 2004;32(7):511-20.
crevicular fluids of diabetic and healthy periodontal patients. J
32- Haffajee AD, Socransky SS, Goodson JM. Clinical parameters as
Periodontol. 2004;75(1):91-7.
predictors of destructive periodontal disease activity. J Clin Periodontol.
13- César-Neto JB, Duarte PM, Oliveira MC, Tambeli CH, Sallum EA,
1983;10(3):257-65.
Nociti FH Jr. Smoking modulates interleukin-6:interleukin-10 and
33- Zappa U, Simona C, Schäppi P, Graf H, Espeland M. Episodic probing
RANKL:osteoprotegerin ratios in the periodontal tissues. J Periodontal
attachment loss in humans: histologic associations. J Periodontol.
Res. 2007;42(2):184-91.
1990;61(7):420-6.
14- Armitage GC. Periodontal diagnoses and classification of periodontal
diseases. Periodontology 2000. 2004;34:9-21.

J Appl Oral Sci. 9/10 2019;27:e20180671

 Clinical attachment loss and molecular profile of inflamed sites before treatment

34- Reinhardt RA, Stoner JA, Golub LM, Lee HM, Nummikoski PV, 37- Sorsa T, Tjäderhane L, Konttinen YT, Lauhio A, Salo T, Lee HM, et al.
Sorsa T, et al. Association of gingival crevicular fluid biomarkers during Matrix metalloproteinases: contribution to pathogenesis, diagnosis and
periodontal maintenance with subsequent progressive periodontitis. J treatment of periodontal inflammation. Ann Med. 2006;38(5):306-21.
Periodontol. 2010;81(2):251-9. 38- Dutzan N, Vernal R, Vaque JP, García-Sesnich J, Hernandez M,
35- Hernandez M, Gamonal J, Tervahartiala T, Mäntylä P, Rivera O, Abusleme L, et al. Interleukin-21 expression and its association with
Dezerega A, et al. Associations between matrix metalloproteinase-8 and proinflammatory cytokines in untreated chronic periodontitis patients.
-14 and myeloperoxidase in gingival crevicular fluid from subjects with J Periodontol. 2012;83(7):948-54.
progressive chronic periodontitis: a longitudinal study. J Periodontol. 39- Turner M, Chantry D, Katsikis P, Berger A, Brennan FM, Feldmann
2010;81(11):1644-52. M. Induction of the interleukin 1 receptor antagonist protein by
36- Timmerman MF, Van der Weijden GA, Abbas F, Arief EM, Armand transforming growth factor-beta. Eur J Immunol. 1991;21(7):1635-9.
S, Winkel EG, et al. Untreated periodontal disease in Indonesian 40- Booth V, Young S, Cruchley A, Taichman NS, Paleolog E. Vascular
adolescents. Longitudinal clinical data and prospective clinical and endothelial growth factor in human periodontal disease. J Periodontal
microbiological risk assessment. J Clin Periodontol. 2000;27(12):932- Res. 1998;33(8):491-9.
42.

J Appl Oral Sci. 10/10 2019;27:e20180671