Keywords
periodontal diseases - saliva - salivary glands - trefoil factor
Introduction
Trefoil factor family (TFF) peptides are composed of TFF1, TFF2, and TFF3. Members
of TFF peptides share a common molecular structure known as a trefoil domain.[1] It was reported that an interaction between a free cysteine residue in the C-terminal
of this TFF peptide and other proteins would alter biological properties and activities
of the TFF molecules.[2] The distribution and localization of TFF peptides vary according to organs, tissues,
and body fluids.[3]
[4] These peptides have several biological functions including cell migration[5] and wound healing.[6] Results from animal studies have shown that TFF3 modulated inflammation by interfering
the production and secretion of inflammatory cytokines, such as interleukin-1beta
(IL-1β, IL-6, and IL-8.[7]
[8] Recombinant human TFF3 peptides also inhibit the production of toll-like receptor
4, nuclear factor kappa B (NF-KB), and tumor necrosis factor alpha (TNF-α in epithelia
of colitis mice.[9] In contrast, it was demonstrated that NF-κB signaling pathway was related with the
downregulation of TFF expression.[10] The TFF peptides have been intensively investigated in the gastrointestinal tract
and are considered to exert their functions in maintenance and protection of mucosal
tissues.[11] However, information on TFF peptides in the oral cavity is limited.
TFF peptides are identified in different oral tissues including salivary glands,[12] oral mucosa,[13] gingiva,[14] and saliva.[4] They are mainly produced from the salivary glands[15] with some addition from the parotid duct and oral mucosal epithelia.[16] Among salivary TFF peptides, TFF3 is the most prominent, followed by TFF1 and TFF2.[4] The previous report explained that TFF3 was a modifying factor involved in oral
keratinocytes signaling pathways, such as cell survival, cell proliferation, and cell
migration.[17] Therefore, presence of TFF peptides in saliva may be crucial for the protection
of oral mucosal against tissue damage. Previously, our cross-sectional study demonstrated
reduced salivary TFF3 peptides in chronic periodontitis (CP) subjects, and the levels
of TFF3 negatively correlated with the severity of periodontitis.[14] Additionally, our in vitro study has revealed that TFF peptides could be digested
by major proteolytic enzymes produced by periopathogenic bacteria.[18] Taking these findings into account, periodontal inflammation mediated by periodontopathic
bacteria may be a downregulating factor in the production of salivary TFF3 peptides
in patients with CP. We hypothesized that reduction of periodontal inflammation by
nonsurgical periodontal treatment would elevate the production of salivary TFF3 peptides.
The present study was aimed to verify our hypothesis by examining salivary TFF3 peptide
levels in gingivitis and periodontitis subjects prior to and following completion
of nonsurgical periodontal treatment.
Materials and Methods
Study Population and Clinical Examination
This prospective study was performed at the Dental Hospital, Khon Kaen University,
Thailand during 2014 to 2016. All procedures were approved by Khon Kaen University
Ethics committee (HE551372). Eighty-seven systemically healthy volunteers including
26 CP patients, 31 dental plaque induced gingival diseased (GD) patients, and 30 clinically
periodontally healthy (PH) individuals participated in this study. Written informed
consents were obtained from all participants. All subjects were nonsmokers who had
at least 15 remaining teeth. A general periodontal examination periodontal parameters
including bleeding on probing (BOP), probing depth (PD), and clinical attachment level
(CAL) on six sites of each remaining tooth, except third molar, were measured and
recorded by one periodontist. Diagnosis of periodontal diseases was based on the 1999
International Workshop for classification of Periodontal Diseases and Conditions.[19] Two weeks following completion of the conventional periodontal treatment, a second
full-mouth general periodontal examination was performed and the parameters were recorded.
Saliva Collection
Saliva collection was performed as previously described.[14] Each volunteer was asked to refrain from eating or drinking for at least 1 hour
prior to saliva collection. After rinsing the mouth thoroughly with water, unstimulated
whole saliva sample (3–5 mL) was obtained in a 50 mL centrifuge tube. The first saliva
collection was done in the morning during 9 to 11 am, followed by the first periodontal
examination for each volunteer. Two weeks post-treatment, second saliva collection
and periodontal examination were performed with the GD and CP groups. Saliva samples
were immediately placed in an ice container, followed by centrifugation at 10,000
× g at 4°C for 10 minutes. Saliva samples were stored at–80°C until further measurements.
Total protein concentrations from each saliva sample were determined employing a commercially
available protein assay kit (Qubit Protein Assay kit; Thermo Fisher Scientific, Invitogen,
United States) to test whether any differences in salivary TFF3 concentrations could
be referred to the differences in the total salivary protein concentrations.
Measurement of Salivary TFF3 Concentrations
For the quantification of salivary TFF3 concentrations, we used our generated monoclonal
antibody (mAb) clones 286 and 116 for a modified sandwich enzyme-linked immunosorbent
assay (ELISA) technique.[20] To develop the sensitivity, fluorescein isothiocyanate (FITC) and anti-FITC identification
methods were used in this sandwich ELISA technique. An antihuman TFF3 mAb clone 286
was used as the plate-coating antibody to capture the peptides in saliva samples.
The antihuman TFF3 mAb clone 116 labeled with FITC was applied to detect the captured
TFF3 peptides. Horseradish peroxidase (HRP)-conjugated sheep anti-FITC antibodies
along with TMB substrate were used as colored markers. The color intensity was measured
using a microplate reader at 450 nm. A standard curve revealing salivary TFF3 between
0.5 and 32 ng/mL was observed. All details of salivary TFF3 concentrations were normalized
by total salivary protein concentrations and reported as TFF3 concentrations (ng)/salivary
protein concentration (mg).
Nonsurgical Periodontal Treatments
Nonsurgical mechanical treatments were provided for gingivitis and periodontitis patients
in the periodontal clinic, Faculty of Dentistry, Khon Kaen University. The treatment
included plaque control by providing oral hygiene instructions and mechanical debridement
by full-mouth scaling and root planing.[21]
[22]
[23] Periodontal treatments were performed in single or multiple visits, utilizing manual
curettes (Hu-Friedy, Chicago, Illinois, United States) and ultrasonic instruments
(P5 Newtron, Acteon, France). Two weeks following completion of nonsurgical periodontal
treatment, the periodontal health was re-examined by the same periodontist.
Statistical Analysis
IBM SPSS software version 19.0 was used for the statistical analyses. Student's t-test was used to evaluate normally distributed data between the two groups, whereas
Mann–Whitney U test was used when the investigated data were not normally distributed.
Chi-squared test was used when dealing with categorical data. Kruskal–Wallis test
with post-hoc corrections was used to compare data among the three investigated groups.
Spearman's rank correlation was used to evaluate correlations between salivary TFF3
peptides levels and periodontal parameters. Two-tailed p < 0.05 was considered as statistically significant.
Results
Demographic and Clinical Characteristics of the Study Population
Demographic and clinical characteristics of three investigated groups were as shown
in [Tables 1]
[2]. The mean age of PH individuals and GD patients was similar. In contrast, the mean
age of CP patients was significantly higher than those in the PH and GD groups. Females
were predominant in all the three groups. Prior to the nonsurgical periodontal treatment,
the total salivary protein concentrations were not significantly different among the
three groups. Following treatment, salivary protein concentrations in the GD group
were significantly different than those in the CP group (p < 0.001). In regard to the periodontal parameters prior to the treatments, the mean
BOP value in the PH group was significantly lower than the GD and CP groups (p < 0.001). PD and CAL measures from the CP group were significantly greater than those
from PH and GD groups (p < 0.001). Improvement in periodontal health status at 2 weeks following the treatments
was observed in GD and CP groups.
Table 1
Demographic data of the study population
|
Investigated groups
|
|
Healthy
|
Gingivitis
|
Periodontitis
|
p-Value
|
|
Abbreviations: ANOVA, analysis of variance; SD, standard deviation.
aKruskal–Wallis one-way ANOVA.
bChi-squared test.
|
|
Number
|
30
|
31
|
26
|
|
|
Age in years (mean ± SD)
|
32.19 ± 1.61
|
32.13 ± 1.81
|
44.04 ± 2.74
|
0.001a
|
|
Male
|
5
|
5
|
9
|
|
|
Female
|
25
|
26
|
14
|
0.085b
|
Table 2
Clinical parameters and salivary characteristic data of the investigated groups prior
to and 2 weeks following nonsurgical periodontal treatment
|
Clinical parameters
|
Investigated groups
|
|
Healthy (n = 30)
|
Gingivitis (n = 31)
|
Periodontitis (n = 26)
|
p-Values
|
|
Abbreviations: ANOVA, analysis of variance; BOP, bleeding on probing; PD, probing
depth; CAL, clinical attachment level; TFF3, trefoil factor family 3; SD, standard
deviation.
aKruskal–Wallis one-way ANOVA.
bChi-squared test.
cMann–Whitney U test.
dStudent's t-test.
|
|
PD (mm; mean ± SD)
|
Before treatment
|
1.89 ± 0.19
|
1.94 ± 0.16
|
2.64 ± 0.59
|
<0.001a
|
|
After treatment
|
–
|
1.86 ± 0.14
|
1.98 ± 0.25
|
<0.001c
|
|
CAL (mm; mean ± SD)
|
Before treatment
|
0.52 ± 1.77
|
0.55 ± 0.12
|
2.35 ± 1.07
|
<0.001a
|
|
After treatment
|
–
|
0.54 ± 0.14
|
2.09 ± 0.92
|
0.358d
|
|
BOP (mean ± SD)
|
Before treatment
|
15.42 ± 3.57
|
38.28 ± 9.98
|
51.7 ± 15.29
|
<0.001a
|
|
After treatment
|
–
|
23.02 ± 6.93
|
30.47 ± 12.65
|
0.015d
|
|
Salivary characteristics
|
|
Total salivary protein concentrations (mg/mL: mean ± SD)
|
Before treatment
|
0.59 ± 0.28
|
0.57 ± 0.31
|
0.73 ± 0.76
|
0.775a
|
|
After treatment
|
–
|
0.49 ± 0.16
|
0.75 ± 0.29
|
<0.001c
|
|
pH
|
Before treatment
|
6.99 ± 0.24
|
6.98 ± 0.29
|
6.79 ± 0.4
|
0.083a
|
|
After treatment
|
–
|
6.89 ± 0.3
|
6.76 ± 0.36
|
0.153d
|
|
Normalized salivary TFF3 concentrations (ng/mg protein)
|
Before treatment
|
228.46 ± 149.0
|
93.05 ± 83.33
|
156.73 ± 147.23
|
<0.001a
|
|
After treatment
|
–
|
252.73 + 171.17
|
40.74 + 41.77
|
<0.001d
|
Levels of Salivary TFF3 Peptides in GD and CP Groups before and after Nonsurgical
Periodontal Treatments
The median, mean, upper, and lower quartiles values of normalized salivary TFF3 concentrations
were as shown in [Fig. 1]. The trend of alterative salivary TFF3 in the GD and CP groups following nonsurgical
periodontal treatment was as shown in [Fig. 2]. Prior to the periodontal treatment, mean normalized salivary TFF3 concentrations
in PH, GD, and CP groups were 228.46, 93.05, and 156.73 ng/mg, respectively. Normalized
salivary TFF3 concentrations in the PH group were significantly higher than the GD
and CP groups (p < 0.05). Two weeks following completion of periodontal treatment in the GD group,
there was a significant increase in the salivary TFF3 (252.73 ng/mg) (p < 0.001). The level of salivary TFF3 peptides in the GD group after receiving the
treatment was comparable with those in the PH group. On the other hand, salivary TFF3
peptides in the CP group after receiving the treatment significantly decreased as
compared with those before the treatment (p < 0.001).
Fig. 1 Normalized salivary Trefoil factor family 3 (TFF3) concentrations (ng/mg) among the
three investigated groups including clinically periodontal healthy (healthy, n = 30); gingival diseased (gingivitis, n = 31); and chronic periodontitis (periodontitis, n = 26) individuals prior to and 2 weeks following nonsurgical periodontal treatment.
Middle line indicates median, cross indicates mean, box indicates the upper and lower
quartiles, and whiskers indicate minimum and maximum values. The asterisk represents
the p-value <0.05 (Mann–Whitney U test). The sharp represents the p-value <0.001 (Wilcoxon signed-rank test).
Fig. 2 A trend of alterative salivary trefoil factor family 3 (TFF3) in the gingival disease
(gingivitis, n = 31) and chronic periodontitis (periodontitis, n = 26) patients prior to and 2 weeks following nonsurgical periodontal treatment.
Correlations between Salivary TFF3 Concentrations and Periodontal Parameters
Spearman's rank correlation analysis was performed on all the subjects with periodontal
diseases. Prior to the periodontal treatment, significantly negative correlations
were observed between salivary TFF3 levels and BOP (correlation coefficient =-0.379,
p = 0.005). On the other hand, no correlation was observed between salivary TFF3 levels
and other periodontal parameters. Likewise, following periodontal treatment, no correlation
was demonstrated between salivary TFF3 levels and periodontal parameters.
Discussion
According to our observations, the levels of salivary TFF3 peptides in gingivitis
and periodontitis patients prior to the periodontal treatment were lower than those
in PH subjects. These results were in agreement with our previous study[14] and suggested that decreased levels of salivary TFF3 peptides could be due to local
and systemic effects of periodontal diseases. Regarding the local effects, our previous
in vitro study demonstrated proteolytic activity of gingipains, one of the major proteolytic
enzymes produced by Porphyromonas gingivalis, on digesting TFF peptides.[18] These findings imply that periodontopathic enzymes may locally reduce TFF3 peptides
in saliva. As TFF3 peptides are mainly produced by salivary glands, decreased levels
of salivary TFF3 peptides may reflect the systemic impact of periodontal diseases
on the functions of the salivary gland. Several investigations have demonstrated the
association between periodontal diseases and biological functions of salivary glands.[24]
[25] One study demonstrated hyposalivation in experimental periodontitis model rats.[24] Similarly, alterations of inflammatory microRNA in salivary glands of rats infected
with periodontal bacteria have also been observed.[25] Our findings provide new implication that periodontal diseases may be a downregulating
factor in the production of TFF3 from the salivary glands. Regarding the pathological
effects of periodontal diseases on salivary gland dysfunctions, further studies to
validate correlations between periodontal diseases and protein synthesis from salivary
glands would be of importance.
The effects of nonsurgical periodontal treatments on salivary compositions were investigated
in patients with periodontitis, and outcomes were varied.[26]
[27]
[28]
[29] Some studies reported changes in salivary components,[26]
[27]
[30] whereas others demonstrated no significant impact of nonsurgical periodontal treatment
on salivary components.[25]
[26] Nonsurgical periodontal treatments have been observed to improve the levels of proinflammatory
cytokines, including TNF-α and endothelial growth factor A, in serum and gingival
crevicular fluid.[31] In our results, nonsurgical periodontal treatment increased levels of salivary TFF3
peptides in gingivitis patients to the same levels as in PH individuals. Taking these
findings into account, it could be possible that gingival inflammation could be a
temporary downregulator of salivary TFF3 production, and the reduction in proinflammatory
cytokines related to gingivitis by nonsurgical periodontal treatments would help to
regain salivary TFF3 production. In contrast, the periodontal therapy had no effect
on the recovery of salivary TFF3 peptides in periodontitis patients. These results
are indicative of the suppressive effects of periodontitis on the functioning of salivary
glands and reflect differential effects of nonsurgical periodontal treatment on the
production of salivary TFF3 peptides in periodontitis patients as compared with those
in gingivitis patients. However, it should be noted that the follow-up period in this
study was 2 weeks following periodontal treatment. It is possible that at this time
point the ongoing inflammatory response due to CP may still remain in effect and could
interrupt the recovery of salivary glands’ functions. Thus, an extended follow-up
study should be conducted to re-examine the levels of salivary TFF3 peptides.
Previous studies demonstrated that TFF3 peptides had a protective effect on mucosal
tissues by the downregulation of proinflammatory cytokines.[7]
[8]
[9] It was reported that recombinant human TFF3 administered as a topical oral spray
could reduce oral mucositis in colorectal cancer patients who received fluorouracil-based
chemotherapy.[32] Thus, the presence of salivary TFF3 peptides may be an important protective factor
against oral mucosal tissue damage. However, it remains unclear whether salivary TFF3
plays any role in periodontal diseases. Our previous observations[14] combined with the present results demonstrated inverse correlations between levels
of salivary TFF3 peptides and periodontal parameters in patients with periodontal
diseases. It would be of interest to further investigate whether administration of
topical TFF3 peptides could help to reduce periodontal inflammation.
In conclusion, the present study demonstrated the impact of periodontal diseases on
the production of salivary TFF3 peptides, and confirmed our previous findings of decreased
levels of salivary TFF3 peptides in patients with periodontal diseases. Nonsurgical
periodontal treatments had varied results on the recovery of salivary TFF3 peptides
between gingivitis and periodontitis patients. The treatment elevated salivary TFF3
peptides in gingivitis patients but the treatment had no effect on the recovery of
salivary TFF3 peptides in periodontitis patients. Our findings provide a new implication
that periodontal diseases may be a downregulator of salivary glands in the context
of TFF3 production.
