Clin Oral Invest DOI 10.1007/s00784-014-1195-4
ORIGINAL ARTICLE
Association study between clinicopathological variables and periodontal breakdown in gingival pyogenic granuloma Leonor V. González-Pérez & Diana M. Isaza-Guzmán & Sergio I. Tobón-Arroyave
Received: 25 May 2013 / Accepted: 17 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Objective The aim was to investigate a possible association between the immunoexpression of interleukin (IL)-4 and clinicopathological parameters with the periodontal breakdown observed in gingival pyogenic granuloma (PG). Materials and methods Paraffin-embedded samples of gingival PG (n=46) were prepared for histological and immunohistochemical assessment. Demographic and clinical parameters were assessed by criteria based on age stratum, gender, smoking habit, evolution course, location, lesion size, macroscopic appearance, predisposing factors, recurrence, and periodontal breakdown. Histological assessment included the appearance of epithelial lining, microvessel density, inflammatory infiltrate density, interstitial fibrosis, and histological arrangement. A staining intensity distribution (SID) score was used to assess IL-4 immunoreactivity. The association between candidate predictor variables and periodontal breakdown was analyzed individually and adjusted for confounding using a bivariate binary logistic regression model. Results Mean IL-4 SID values were significantly increased for long-standing and large lesions, presence of periodontal breakdown, high microvessel density, and moderate-to-severe inflammatory infiltrate density. While bivariate and univariate analyses revealed a positive association of the evolution course ≥12 months, lesion size >1 cm, high microvessel density, moderate-to-severe inflammatory infiltrate density, and IL-4 SID score ≥8.04 with periodontal breakdown, after
bivariate logistic regression analysis, only the evolution course ≥12 months, moderate-to-severe inflammatory infiltrate density, and IL-4 SID score ≥8.04 remained as robust predictors of periodontal damage. Confounding and interaction effects between candidate predictor variables were also noted. Conclusion These findings suggest that while evolution course, inflammatory infiltrate density, and the overexpression of IL-4 may act as predictors of periodontal breakdown in gingival PG, there are mutual confounding and synergistic biological interactive effects with respect to the lesion size and microvessel density in the susceptible host that may be also associated with the bone resorption and tissue destruction. Clinical relevance Although the first-line therapy of gingival PG continues to be the surgical excision, this approach poses unwanted complications such as severe mucogingival defects and recurrence. Hence, early diagnosis and detection of these three significant predictor variables as well as timely surgical excision might help prevent the periodontal tissue destruction observed in some of these lesions.
L. V. González-Pérez : D. M. Isaza-Guzmán : S. I. Tobón-Arroyave (*) POPCAD Research Group, Laboratory of Immunodetection and Bioanalysis, Faculty of Dentistry, University of Antioquia, Calle 64 N° 52-59, Medellín, Colombia e-mail: [email protected]
Pyogenic granuloma (PG) is one of the inflammatory hyperplasias seen in the oral cavity that occurs mainly in the gingiva between two teeth (almost 75 %) and less frequently at other sites like the tongue, lips, palate, and cheeks [1, 2]. However, this term is a misnomer because the condition is not associated with pus production and does not represent a granulomatous inflammation histologically [3, 4]. Notwithstanding, the term continues to be widely used in the literature even though it
S. I. Tobón-Arroyave e-mail: [email protected]
Keywords Clinicopathological features . IL-4 . Immunoregulatory cytokines . Immunopathogenesis . Gingival pyogenic granuloma . Periodontal breakdown
Introduction
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causes confusion. It usually arises in response to various stimuli such as chronic low-grade local irritation, traumatic injury, hormonal factors, or certain kinds of drugs, and reactions to grafts [3]. Clinically, PG occurs as a smooth or lobulated exophytic lesion manifesting as small, red, erythematous papules on a pedunculated or sometimes sessile base, which is usually hemorrhagic [5]. Microscopically, PG has been classified into two subtypes: (a) lobular capillary hemangioma (LCH) type, characterized by proliferating smalland medium-sized blood vessels lined by discrete endothelial cells that are organized in lobular aggregates separated by a fibrous stroma with mild or no inflammation, and (b) nonLCH type, represented by a highly vascular proliferation similar to the granulation tissue which is more infiltrated by chronic inflammatory cells as compared to the LCH type [3, 6, 7]. The inflammatory cell infiltration is composed predominantly of mature lymphocytes admixed with neutrophils, and plasma cells [3, 4, 7]. The epithelial lining may be intact, atrophic, or may show areas of ulceration covered by a thick fibrin membrane or even exhibiting acanthosis [3, 8]. Although over time, some of the lesions may undergo fibrous maturation [7], large and long-standing gingival lesions may lead to cupping erosions and/or interseptal resorption of the underlying bone [9], thus generating a localized periodontal breakdown. It has been recognized that the formation of hyperplasic granulation tissue, which includes the migration of inflammatory cells, migration and proliferation of vascular endothelial cells and fibroblasts, and synthesis of extracellular matrix (ECM), is one of the most distinct histological finding of PG [10, 11]. Hence, it would be possible to assume that such process could be resultant of pro- and antiinflammatory pathways controlled by various kinds of inflammatory angiogenesis-associated factors. In this sense, the authors’ group recently demonstrated that the deregulation between cyclooxygenase (COX)-2 and interleukin (IL)-10 immunoexpression may be an important event contributing to the onset and evolution of oral PG [8]. However, other factors might be modulating different cell-cell and/or cell-matrix interactions within the lesional tissue and could offer an interesting model for immunological evaluation of granulation tissue organization. Among these factors, IL-4, a member of the CD132dependent cytokines [12], has been regarded to possess both anti- and proinflammatory functions [13]. This cytokine is secreted mainly by T-helper 2 (Th2) cells and also by macrophages, monocytes, mast cells, basophils, and nonimmune cells such as fibroblasts and endothelial cells [14]. The antiinflammatory effect of IL-4 results partly from its efficient inhibition of the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), IL-1α, IL-1β, IL-6, and IL-8 by monocytes/ macrophages [15–17]. Additionally, it significantly inhibits
the expression of COX-2 both at mRNA and protein levels [18–21] as well as the production of other proinflammatory mediators such as reactive oxygen species, reactive nitrogen species, and prostaglandins in monocytes/macrophages [22]. On the contrary, IL-4 has also been shown to have other proinflammatory functions, such as the recruitment of inflammatory cells by increasing the expression of vascular cell adhesion molecule (VCAM)-1 on the endothelial surface [23] and increasing neutrophil, eosinophil, macrophage, and B cell chemoattraction [13, 24–26] and antibody production [13, 27, 28]. Because there is evidence suggesting that IL-4 plays an important role in the regulation of the immunoinflammatory process by inducing a Th2-biased local host response [13, 29, 30], it would possible to speculate that the alteration in the mechanisms regulating the synthesis of this inflammatory and angiogenic mediator might have a potential impact on the onset, evolution, and destructive behavior of gingival PG. Therefore, the aim of the present study was to investigate a possible association between the immunohistochemical expression of IL-4 and clinicopathological variables and the periodontal breakdown observed in the cases of gingival PG.
Materials and methods Tissue samples and inclusion/exclusion criteria Tissue samples consisted of cases of gingival PG which were followed up until 1.5 years after surgical excision, so that long-term data were not available. The cases were treated in the Postgraduate Periodontics Clinic and diagnosed histologically in the Laboratory of Immunodetection and Bioanalysis of the Faculty of Dentistry, University of Antioquia between 2006 and 2011. The specimens were obtained during periodontal surgery or biopsy for pathological diagnosis. All cases represented excisional biopsies, fixed in 10 % buffered formalin, and embedded in paraffin wax. Informed consent for use of material in the study was obtained from each patient according to the Helsinki Declaration before the collection of samples. Ethical approval for the study was obtained from the Ethics Committee for Human Studies of the Faculty of Dentistry of the University of Antioquia. All of the cases were assessed and confirmed by an experienced researcher (L.V.G-P.) following previous defined criteria [6]. To obtain a more homogeneous sample material, only gingival PG cases with initial surgical treatment were included. None of the patients had received any treatment with a therapeutic agent prior to this study. Patients with PGs developed at other sites in the oral cavity, as well as selective lesions with clinical resemblance to PGs, but with different histological features such as peripheral giant cell granuloma, peripheral ossifying fibroma, peripheral
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odontogenic fibroma, hemangioma, hyperplastic gingival inflammation, and Kaposi’s sarcoma [3], were excluded.
addition, the cases were stratified as LCH and non-LCH PG subtypes according to histological arrangement [6, 31].
Demographic, clinical, and radiographic data collection
Immunohistochemical staining and evaluation of IL-4-bearing cells
All of the clinical and demographic data were investigated retrospectively from review of preexisting medical records. Consequently, the study included only cases with detailed clinical and radiographic information, which were analyzed and categorized focusing on age stratum (i.e., ≤30 vs. >30 years), gender (male vs. female), smoking habit (i.e., smokers vs. nonsmokers), evolution course (i.e., 1 cm), macroscopic appearance (i.e., pedunculated vs. sessile), predisposing factors (i.e., local irritants, pregnancy, and unknown), recurrence (i.e., recurrent vs. nonrecurrent), and periodontal breakdown demonstrated radiographically by an interseptal resorption of the underlying bone >2 mm from the cemento-enamel junction and/or widening of the periodontal ligament space, along with evidence of periodontal pocket formation, clinical attachment loss, localized destruction of cortical plates, and/or tooth mobility, as described within the intraoperative findings of the operation reports (i.e., present vs. absent). Histological assessment Histological assessment was performed on 4-μm-thick sections mounted on common glass slides and stained with hematoxylin and eosin (H&E) by a routine protocol. Ten high-power nonoverlapping fields (HPFs, ×400 magnification) were observed randomly on each slide by a single rater (L.V.G-P.) using an image analyzer system (ZEN Pro, Carl Zeiss, Oberkochen, Germany) coupled to a Zeiss Axiolab light microscope (Carl Zeiss). Measurements of captured images were calibrated using a microscope stage micrometer (Carl Zeiss) as the reference. Histological evaluation included the record of appearance of epithelial lining which was classified based on the predominant morphological aspect as ulcerated (necrotic), atrophic (thinned), and acanthotic (thickened). In addition, the microvessel density of the lesions was determined based on the average of microvessel count (MVC) per HPF and was categorized as low (≤10 MVC) and high (>10 MVC). Also, the inflammatory infiltrate density was assessed using an arbitrary scale based on the percentage of tissue area involved by inflammatory cell infiltrate, which is as follows: mild (≤20 % inflammatory cells/HPF, most of which are separated by distinct intervening spaces) and moderate to severe (>20 % inflammatory cells/HPF showing a tendency to confluence). Likewise, interstitial fibrosis was scored and described as mild (≤20 % interstitial fibrosis/HPF) and moderate to severe (>20 % interstitial fibrosis/HPF). In
For immunostaining, paraffin-embedded tissues, sectioned at 4 μm and collected in serial sections on electrostatically charged glass slides (ColorFrost Plus, Fisher Scientific, PA, USA), were initially deparaffinized by leaving in an incubator with gravity convection (Binder APT.Line BD23, Tuttlingen, Germany) at 58 °C overnight. Deparaffinization was completed by successive immersions in xylene, hydration through graded alcohol, and washing with tap water. Endogenous peroxidase activity was blocked with 6 % (v/v) H2O2 in water for 5 min. Antigen retrieval was performed by heating slides immersed in citrate buffer (Target Retrieval Solution, DakoCytomation, CA, USA), pH 6.1, in a steamer for 30 min, and endogenous avidinbiotin activity was quenched with a biotin blocking system (DakoCytomation) for 20 min. The slides were immediately incubated for 1 h at room temperature in a humidified chamber with 100 μL of rabbit anti-human IL-4 polyclonal antibody (ab9622, Abcam, Cambridge, MA, USA) diluted to 1:100. The standard streptavidin-biotin-peroxidase complex method was performed to bind the primary antibody with the use of Universal LSAB™/HRP System Kit (DakoCytomation) following the manufacturer’s instructions. Each of the incubation steps was preceded by a 10-min rinse with phosphate-buffered saline-0.05 % Tween 20 (PBS-T, DakoCytomation), pH 7.0. Reaction products were visualized using 0.3 % diaminobenzidine solution (DakoCytomation) and counterstained with Harris hematoxylin (Merck, Darmstadt, Germany). The specificity of immunohistochemical staining was determined by substituting the primary antibody with an antibody diluent (DakoCytomation). In addition, sections of normal human tonsil tissue were obtained as positive controls for immunostaining. For immunohistochemical analysis, ten HPFs were randomly chosen and assessed in each section by the same observer with the use of the ZEN Pro image analyzer system following previous described methods [32]. The cells with a clearly defined immunostaining when compared with the positive controls (as depicted by a brownish cytoplasmic staining, irrespective of labeling intensity) were considered to be positive. Epithelial cells were excluded from counting in order to prevent false-positive scores. Each HPF was evaluated for the proportion of stained cells and the intensity of overall staining. The proportion of stained cells in each HPF was assessed as follows: 0, absence of immunostained cells; 1, 50 % stained cells. The staining intensity was graded as follows: 0, negative staining; 1, light staining; 2, moderate staining; and 3, intense staining. Finally, a staining intensity distribution
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(SID) score was calculated for each sample as follows: the score of the proportion of stained cells for each HPF was multiplied by the score of the staining intensity in that field to provide a SID score for the field. The average of the 10 HPFs was the SID score for the sample. To increase the confidence level of positive staining, sections with too weak and/or equivocal staining were included in the negatively stained group. Likewise, for the analysis, the mean value of IL-4 SID scores obtained from gingival PG cases with periodontal breakdown was chosen as the cutoff point for dividing the cases into subgroups of 0.7 represented indicated fair to good reproducibility, and a value of >0.9 represented excellent reproducibility. All parameters were tested for normal distribution using the Shapiro-Wilk test for small sample size. Because the data conformed to a normal distribution, the variables were analyzed using parametric methods. Consequently, whereas differences between IL-4 SID score concerning demographic, clinical, and histological parameters were tested by unpaired t test or one-way analysis of variance (ANOVA) when indicated, Pearson’s correlation coefficient analysis was performed to describe the relationship between pairs of significant quantitative variables. Afterwards, for the assessment of periodontal breakdown predictors, statistical analysis was performed on three levels: (a) significant associations between the presence of periodontal breakdown and all investigated variables (15 in total) were explored in bivariate analyses using Pearson’s chisquare test (χ2) or Fisher’s exact test (when frequency was 20 % inflammatory cells/HPF) scores (mean 27.55, range 20.2–41.3). Concerning interstitial fibrosis, it was observed that 71.7 % of biopsies (33 cases) exhibited mild (≤20 % fibrosis/HPF) scores (mean 13.12, range 1.5–19.9) and 28.3 % biopsies (13 cases), moderate-to-severe (>20 % fibrosis/HPF) scores (mean 28.63, range 20.6–46.4). Twenty-four cases (52.2 %) showed histological features of LCH-type and 22 cases (47.8 %) of non-LCH-type PG. Although there were no significant differences between histological subtypes regarding epithelial lining (P=0.053, Fisher’s exact test), mean values of microvessel density and inflammatory infiltrate density were significantly lower in the LCH subtype (P0.05, unpaired t test and ANOVA) were evident in the IL-4 SID scores with respect to age stratum, gender, smoking habit, location of the lesions, macroscopic appearance, predisposing factors, recurrence, epithelial lining, interstitial fibrosis, or histological subtype, mean IL-4 SID values were significantly increased (all P1 cm), presence of periodontal breakdown, high microvessel density (>10 MVC/HPF), and moderate-to-severe inflammatory infiltrate density (>20 % inflammatory cells/HPF). Moreover, Pearson’s correlation analysis showed moderate-to-strong significant positive correlations (P1 cm (P=0.023). It was also noteworthy that the effects of histological/ immunohistochemical predictor variables such as microvessel density, inflammatory infiltrate density, and IL-4 SID score were found to be significant on the proportion of periodontal breakdown (P30 years Male Female Smokers Nonsmokers 1 cm Pedunculated Sessile Local irritants Pregnancy Unknown Present Absent Recurrent Nonrecurrent Ulcerated Atrophic Acanthotic Low (≤10 MVC) High (>10 MVC) Mild (≤20 % inflammatory cells) Moderate to severe (>20 % inflammatory cells) Mild (≤20 % fibrosis) Moderate to severe (>20 % fibrosis) LCH PG Non-LCH PG
23 (50.0) 23 (50.0) 26 (56.5) 20 (43.5) 23 (50.0) 11 (23.9) 12 (26.1) 14 (30.4) 32 (69.6) 11 (23.9) 35 (76.1) 25 (54.3) 7 (15.2) 14 (30.4) 33 (71.7) 13 (28.3) 20 (43.5) 26 (56.5) 33 (71.7) 13 (28.3) 24 (52.2) 22 (47.8)
4.65±2.76 6.67±2.90 5.84±3.24 5.42±2.66 6.28±2.52 4.49±3.46 5.54±3.25 8.04±1.12 4.62±2.95 6.58±2.82 5.37±3.01 5.67±2.98 4.71±3.80 6.11±2.64 5.10±2.96 7.08±2.61 4.41±2.87 6.63±2.74 5.70±3.16 5.57±2.59 5.06±3.02 6.32±2.85
0.056d 0.202d 0.002d 0.810e
0.020d 0.632d 0.261e
1 cm and high microvessel density in the univariate analysis, a confounding effect was evident after adjustment by the other covariables, as
these associations failed to achieve statistical significance. Even so, there was no confounding of the relationship neither between evolution course of ≥12 months, moderate-to-severe inflammatory infiltrate density, nor IL-4 SID score of ≥8.04 regarding periodontal breakdown. In these three cases, each relationship remained associated with the damage status (P1 cm/IL-4 SID score of ≥8.04
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Fig. 1 Scatterplot matrix demonstrating the statistical correlations of IL4 SID score (y-axis) in regard to evolution course, lesion size, microvessel density, and inflammatory infiltrate density of gingival PG (x-axis). Each panel of the matrix is a scatterplot of one variable against another. Solid black dots represent cases with periodontal breakdown, whereas open
black dots represent cases with no periodontal involvement. The regression lines are presented for each plot along Pearson’s rank correlation coefficient (r). All correlations were statistically significant at the 0.01 level of significance
(adjusted OR, 11.2; 95 % CI, 1.98 to 63.47; P=0.006, Wald’s test), lesion size of >1 cm/high microvessel density (adjusted OR, 15.0; 95 % CI, 2.54 to 88.39; P=0.003), high microvessel
density/evolution course of ≥12 months (adjusted OR, 8.3; 95 % CI, 1.37 to 50.46; P=0.021), high microvessel density/ moderate-to-severe inflammatory infiltrate density (adjusted
Fig. 2 Control experiments for IL-4 immunostaining (streptavidinbiotin-peroxidase complex, hematoxylin counterstain, original magnification×400): a Negative control performed by substituting the primary antibody with an antibody diluent. Note complete absence
of staining. b Positive control showing intense cytoplasmic staining of macrophages and B lymphocytes of the germinal center, as well as T lymphocytes of paracortex of human tonsil tissue
Clin Oral Invest Table 2 Bivariate associations between demographic risk variables and periodontal breakdown in gingival pyogenic granuloma Periodontal breakdowna
Risk variables
Gender Age stratum Smoking habit Evolution course
Male Female ≤30 years >30 years Smokers Nonsmokers 1 cm Pedunculated Sessile Local irritants Pregnancy Unknown Recurrent Nonrecurrent
5 (10.9) 7 (15.2) 20 (43.5) 12 (26.1) 17 (37.0) 15 (32.5) 14 (30.4) 8 (17.4) 10 (21.7) 6 (13.0) 26 (56.5)
4 (8.7) 1 (2.2) 3 (6.5) 11 (23.9) 9 (19.6) 5 (10.9) 9 (19.6) 3 (6.5) 2 (4.3) 5 (10.9) 9 (19.6)
Values are given as n (%) of cases
b
Two-sided Fisher’s exact test
c
Two-sided Pearson’s chi-square test (χ2 )
0.199b
0.023b 0.482c 0.378b
0.215c
Clin Oral Invest Table 4 Bivariate associations between histological/immunohistochemical risk variables and periodontal breakdown in gingival pyogenic granuloma Periodontal breakdowna
Risk variables
Absent (n=32) Epithelial lining
Microvessel densityb Inflammatory infiltrate densityb Interstitial fibrosisb Histological subtype IL-4 SID scorec a
P value
Present (n=14)
Ulcerated Atrophic
17 (37.0) 5 (10.9)
8 (17.4) 2 (4.3)
Acanthotic Low (≤10 MVC) High (>10 MVC) Mild (≤20 % inflammatory cells) Moderate-to-severe (>20 % inflammatory cells) Mild (≤20 % fibrosis) Moderate to severe (>20 % fibrosis) LCH PG Non-LCH PG 1 cm) lesions, as well as in those with periodontal breakdown, high microvessel density, and moderate-to-severe inflammatory infiltrate density. Moreover, as such differences were mainly based on the greatest number of immunoreactive inflammatory and endothelial cells, the finding of significant positive correlations between IL-4 SID scores regarding the evolution course, lesion size, microvessel density, and inflammatory infiltrate density of the lesions seems to highlight the proinflammatory role of IL-4 in gingival PG. Although nothing is known about the immunoexpression of IL-4 in regard to gingival PG on which would be established as a basis of comparison, several studies performed on gingival biopsies obtained from periodontitis-affected patients have demonstrated that IL-4 is widely expressed in diseased periodontal tissues, suggesting that a bias towards Th2-type cytokine dominance could be an exacerbating factor [29, 35–37]. In agreement with the former, it has been postulated that IL-4 may serve as an autocrine growth factor responsible for the maintenance and proliferation of inflammatory cells in vivo [38] as well as a potent inducer of angiogenesis [39–41]. Consequently, it is possible that IL-4 is involved in the immunopathogenesis of gingival PG, driving the migration and the maintenance of several cell types such as polymorphonuclear leukocytes, macrophages, subsets of lymphocytes, and microvascular endothelial cells within the gingival tissues.
Clin Oral Invest Table 5 Initial and final model of bivariate binary logistic regression for the association of significant predictor variables with periodontal breakdown in gingival PG Risk variable
Unadjusted OR (95 % CI)a
P valueb
Evolution course ≥12 months Lesion size Microvessel density Inflammatory infiltrate density
7.50 (1.83–30.68)
0.005
IL-4 SID score Lesion size >1 cm Evolution course Microvessel density Inflammatory infiltrate density IL-4 SID score High microvessel density Lesion size Evolution course Inflammatory infiltrate density IL-4 SID score Moderate-to-severe inflammatory infiltrate density Lesion size Evolution course Microvessel density IL-4 SID score IL-4 SID score ≥8.04 Lesion size
6.11 (1.41–26.40)
a
Odds ratio (95 % confidence interval)
b
Wald’s test
5.32 (1.21–23.38) 6.88 (1.59–29.76) 5.02 (1.06–23.60)
0.027 0.010 0.041
6.12 (1.23–30.34)
0.026
4.01 (0.84–19.21) 5.11 (1.31–23.04) 6.26 (1.21–32.23) 2.31 (0.40–13.29)
0.081 0.034 0.028 0.346
3.34 (0.77–14.49) 3.78 (0.82–17.33) 2.39 (0.51–11.07) 2.39 (0.48–11.93)
0.106 0.086 0.265 0.285
19.36 (2.07–180.34) 13.72 (1.50–125.32) 15.14 (1.62–141.28) 18.83 (1.77–200.45)
0.009 0.020 0.017 0.015
8.17 (1.47–45.23)
0.016
10.56 (2.04–54.65) 9.96 (2.08–47.68) 12.49 (2.04–76.15)
0.005 0.004 0.006
0.036
19.00 (2.20–163.57)
Evolution course Microvessel density Inflammatory infiltrate density
P valueb
0.015
4.33 (1.09–17.10)
12.60 (2.77–57.27)
Adjusted OR (95 % CI)a
0.007
0.001
Hosmer and Lemeshow’s goodness-of-fit test >0.208 C-statistics >0.761
On the other hand, in the present study, a significant association of periodontal breakdown with the evolution course of ≥12 months, lesion size of >1 cm, high microvessel density, moderate-to-severe inflammatory infiltrate density, and IL-4 SID score of ≥8.04 in both bivariate and univariate analyses was found. However, despite of these observations, after bivariate logistic regression analysis, both the lesion size >1 cm and the high microvessel density met the criteria to be considered confounders, as the associations weakened considerably when adjusted for the other covariables individually. On the contrary, it is also important to emphasize that although this study confirmed the strong and independent association of evolution course of ≥12 months, moderate-to-severe inflammatory infiltrate density, and IL-4 SID score of ≥8.04 as robust predictors of periodontal damage in gingival PG, synergistic biological interactive effects were also significantly detected regarding the lesion size and the microvessel density. Although there is no available data on a direct relationship between
clinicopathological predictors and the increasing periodontal breakdown in gingival PG, these findings might indicate that while both the lesion size and the high microvessel density are significant factors modifying for the other three significant predictors, the evolution course and inflammatory infiltrate density might promote the periodontal breakdown in gingival PG when IL-4 is overexpressed within the lesional tissue. As IL-4 has been regarded as having angiogenic [39–41] and proinflammatory functions [13, 24–28], it is quite probable that the effects on the vascular and immune systems can act synchronously to promote the bone resorption and tissue destruction observed in gingival PG.
Conclusions and clinical relevance The findings of this study suggest that while evolution course, inflammatory infiltrate density, and overexpression of IL-4 may
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act as predictors of periodontal breakdown in gingival PG, there are mutual confounding and synergistic biological interactive effects with respect to lesion size and microvessel density in the susceptible host that may be also associated with the bone resorption and tissue destruction. Although, the first-line therapy of gingival PG continues to be the surgical excision, this approach poses unwanted complications such as disagreeable mucogingival defects and recurrence. Hence, early diagnosis and detection of these three significant predictor variables as well as timely surgical excision might help prevent the periodontal tissue destruction observed in some of these lesions.
Conflict of interest The authors declare that they have no financial interest in the products in this work. This study has been fully supported by a grant of the Research Development Committee of the University of Antioquia (CODI-Code 8700-1617/2009).
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ORIGINAL ARTICLE
Association study between clinicopathological variables and periodontal breakdown in gingival pyogenic granuloma Leonor V. González-Pérez & Diana M. Isaza-Guzmán & Sergio I. Tobón-Arroyave
Received: 25 May 2013 / Accepted: 17 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Objective The aim was to investigate a possible association between the immunoexpression of interleukin (IL)-4 and clinicopathological parameters with the periodontal breakdown observed in gingival pyogenic granuloma (PG). Materials and methods Paraffin-embedded samples of gingival PG (n=46) were prepared for histological and immunohistochemical assessment. Demographic and clinical parameters were assessed by criteria based on age stratum, gender, smoking habit, evolution course, location, lesion size, macroscopic appearance, predisposing factors, recurrence, and periodontal breakdown. Histological assessment included the appearance of epithelial lining, microvessel density, inflammatory infiltrate density, interstitial fibrosis, and histological arrangement. A staining intensity distribution (SID) score was used to assess IL-4 immunoreactivity. The association between candidate predictor variables and periodontal breakdown was analyzed individually and adjusted for confounding using a bivariate binary logistic regression model. Results Mean IL-4 SID values were significantly increased for long-standing and large lesions, presence of periodontal breakdown, high microvessel density, and moderate-to-severe inflammatory infiltrate density. While bivariate and univariate analyses revealed a positive association of the evolution course ≥12 months, lesion size >1 cm, high microvessel density, moderate-to-severe inflammatory infiltrate density, and IL-4 SID score ≥8.04 with periodontal breakdown, after
bivariate logistic regression analysis, only the evolution course ≥12 months, moderate-to-severe inflammatory infiltrate density, and IL-4 SID score ≥8.04 remained as robust predictors of periodontal damage. Confounding and interaction effects between candidate predictor variables were also noted. Conclusion These findings suggest that while evolution course, inflammatory infiltrate density, and the overexpression of IL-4 may act as predictors of periodontal breakdown in gingival PG, there are mutual confounding and synergistic biological interactive effects with respect to the lesion size and microvessel density in the susceptible host that may be also associated with the bone resorption and tissue destruction. Clinical relevance Although the first-line therapy of gingival PG continues to be the surgical excision, this approach poses unwanted complications such as severe mucogingival defects and recurrence. Hence, early diagnosis and detection of these three significant predictor variables as well as timely surgical excision might help prevent the periodontal tissue destruction observed in some of these lesions.
L. V. González-Pérez : D. M. Isaza-Guzmán : S. I. Tobón-Arroyave (*) POPCAD Research Group, Laboratory of Immunodetection and Bioanalysis, Faculty of Dentistry, University of Antioquia, Calle 64 N° 52-59, Medellín, Colombia e-mail: [email protected]
Pyogenic granuloma (PG) is one of the inflammatory hyperplasias seen in the oral cavity that occurs mainly in the gingiva between two teeth (almost 75 %) and less frequently at other sites like the tongue, lips, palate, and cheeks [1, 2]. However, this term is a misnomer because the condition is not associated with pus production and does not represent a granulomatous inflammation histologically [3, 4]. Notwithstanding, the term continues to be widely used in the literature even though it
S. I. Tobón-Arroyave e-mail: [email protected]
Keywords Clinicopathological features . IL-4 . Immunoregulatory cytokines . Immunopathogenesis . Gingival pyogenic granuloma . Periodontal breakdown
Introduction
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causes confusion. It usually arises in response to various stimuli such as chronic low-grade local irritation, traumatic injury, hormonal factors, or certain kinds of drugs, and reactions to grafts [3]. Clinically, PG occurs as a smooth or lobulated exophytic lesion manifesting as small, red, erythematous papules on a pedunculated or sometimes sessile base, which is usually hemorrhagic [5]. Microscopically, PG has been classified into two subtypes: (a) lobular capillary hemangioma (LCH) type, characterized by proliferating smalland medium-sized blood vessels lined by discrete endothelial cells that are organized in lobular aggregates separated by a fibrous stroma with mild or no inflammation, and (b) nonLCH type, represented by a highly vascular proliferation similar to the granulation tissue which is more infiltrated by chronic inflammatory cells as compared to the LCH type [3, 6, 7]. The inflammatory cell infiltration is composed predominantly of mature lymphocytes admixed with neutrophils, and plasma cells [3, 4, 7]. The epithelial lining may be intact, atrophic, or may show areas of ulceration covered by a thick fibrin membrane or even exhibiting acanthosis [3, 8]. Although over time, some of the lesions may undergo fibrous maturation [7], large and long-standing gingival lesions may lead to cupping erosions and/or interseptal resorption of the underlying bone [9], thus generating a localized periodontal breakdown. It has been recognized that the formation of hyperplasic granulation tissue, which includes the migration of inflammatory cells, migration and proliferation of vascular endothelial cells and fibroblasts, and synthesis of extracellular matrix (ECM), is one of the most distinct histological finding of PG [10, 11]. Hence, it would be possible to assume that such process could be resultant of pro- and antiinflammatory pathways controlled by various kinds of inflammatory angiogenesis-associated factors. In this sense, the authors’ group recently demonstrated that the deregulation between cyclooxygenase (COX)-2 and interleukin (IL)-10 immunoexpression may be an important event contributing to the onset and evolution of oral PG [8]. However, other factors might be modulating different cell-cell and/or cell-matrix interactions within the lesional tissue and could offer an interesting model for immunological evaluation of granulation tissue organization. Among these factors, IL-4, a member of the CD132dependent cytokines [12], has been regarded to possess both anti- and proinflammatory functions [13]. This cytokine is secreted mainly by T-helper 2 (Th2) cells and also by macrophages, monocytes, mast cells, basophils, and nonimmune cells such as fibroblasts and endothelial cells [14]. The antiinflammatory effect of IL-4 results partly from its efficient inhibition of the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), IL-1α, IL-1β, IL-6, and IL-8 by monocytes/ macrophages [15–17]. Additionally, it significantly inhibits
the expression of COX-2 both at mRNA and protein levels [18–21] as well as the production of other proinflammatory mediators such as reactive oxygen species, reactive nitrogen species, and prostaglandins in monocytes/macrophages [22]. On the contrary, IL-4 has also been shown to have other proinflammatory functions, such as the recruitment of inflammatory cells by increasing the expression of vascular cell adhesion molecule (VCAM)-1 on the endothelial surface [23] and increasing neutrophil, eosinophil, macrophage, and B cell chemoattraction [13, 24–26] and antibody production [13, 27, 28]. Because there is evidence suggesting that IL-4 plays an important role in the regulation of the immunoinflammatory process by inducing a Th2-biased local host response [13, 29, 30], it would possible to speculate that the alteration in the mechanisms regulating the synthesis of this inflammatory and angiogenic mediator might have a potential impact on the onset, evolution, and destructive behavior of gingival PG. Therefore, the aim of the present study was to investigate a possible association between the immunohistochemical expression of IL-4 and clinicopathological variables and the periodontal breakdown observed in the cases of gingival PG.
Materials and methods Tissue samples and inclusion/exclusion criteria Tissue samples consisted of cases of gingival PG which were followed up until 1.5 years after surgical excision, so that long-term data were not available. The cases were treated in the Postgraduate Periodontics Clinic and diagnosed histologically in the Laboratory of Immunodetection and Bioanalysis of the Faculty of Dentistry, University of Antioquia between 2006 and 2011. The specimens were obtained during periodontal surgery or biopsy for pathological diagnosis. All cases represented excisional biopsies, fixed in 10 % buffered formalin, and embedded in paraffin wax. Informed consent for use of material in the study was obtained from each patient according to the Helsinki Declaration before the collection of samples. Ethical approval for the study was obtained from the Ethics Committee for Human Studies of the Faculty of Dentistry of the University of Antioquia. All of the cases were assessed and confirmed by an experienced researcher (L.V.G-P.) following previous defined criteria [6]. To obtain a more homogeneous sample material, only gingival PG cases with initial surgical treatment were included. None of the patients had received any treatment with a therapeutic agent prior to this study. Patients with PGs developed at other sites in the oral cavity, as well as selective lesions with clinical resemblance to PGs, but with different histological features such as peripheral giant cell granuloma, peripheral ossifying fibroma, peripheral
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odontogenic fibroma, hemangioma, hyperplastic gingival inflammation, and Kaposi’s sarcoma [3], were excluded.
addition, the cases were stratified as LCH and non-LCH PG subtypes according to histological arrangement [6, 31].
Demographic, clinical, and radiographic data collection
Immunohistochemical staining and evaluation of IL-4-bearing cells
All of the clinical and demographic data were investigated retrospectively from review of preexisting medical records. Consequently, the study included only cases with detailed clinical and radiographic information, which were analyzed and categorized focusing on age stratum (i.e., ≤30 vs. >30 years), gender (male vs. female), smoking habit (i.e., smokers vs. nonsmokers), evolution course (i.e., 1 cm), macroscopic appearance (i.e., pedunculated vs. sessile), predisposing factors (i.e., local irritants, pregnancy, and unknown), recurrence (i.e., recurrent vs. nonrecurrent), and periodontal breakdown demonstrated radiographically by an interseptal resorption of the underlying bone >2 mm from the cemento-enamel junction and/or widening of the periodontal ligament space, along with evidence of periodontal pocket formation, clinical attachment loss, localized destruction of cortical plates, and/or tooth mobility, as described within the intraoperative findings of the operation reports (i.e., present vs. absent). Histological assessment Histological assessment was performed on 4-μm-thick sections mounted on common glass slides and stained with hematoxylin and eosin (H&E) by a routine protocol. Ten high-power nonoverlapping fields (HPFs, ×400 magnification) were observed randomly on each slide by a single rater (L.V.G-P.) using an image analyzer system (ZEN Pro, Carl Zeiss, Oberkochen, Germany) coupled to a Zeiss Axiolab light microscope (Carl Zeiss). Measurements of captured images were calibrated using a microscope stage micrometer (Carl Zeiss) as the reference. Histological evaluation included the record of appearance of epithelial lining which was classified based on the predominant morphological aspect as ulcerated (necrotic), atrophic (thinned), and acanthotic (thickened). In addition, the microvessel density of the lesions was determined based on the average of microvessel count (MVC) per HPF and was categorized as low (≤10 MVC) and high (>10 MVC). Also, the inflammatory infiltrate density was assessed using an arbitrary scale based on the percentage of tissue area involved by inflammatory cell infiltrate, which is as follows: mild (≤20 % inflammatory cells/HPF, most of which are separated by distinct intervening spaces) and moderate to severe (>20 % inflammatory cells/HPF showing a tendency to confluence). Likewise, interstitial fibrosis was scored and described as mild (≤20 % interstitial fibrosis/HPF) and moderate to severe (>20 % interstitial fibrosis/HPF). In
For immunostaining, paraffin-embedded tissues, sectioned at 4 μm and collected in serial sections on electrostatically charged glass slides (ColorFrost Plus, Fisher Scientific, PA, USA), were initially deparaffinized by leaving in an incubator with gravity convection (Binder APT.Line BD23, Tuttlingen, Germany) at 58 °C overnight. Deparaffinization was completed by successive immersions in xylene, hydration through graded alcohol, and washing with tap water. Endogenous peroxidase activity was blocked with 6 % (v/v) H2O2 in water for 5 min. Antigen retrieval was performed by heating slides immersed in citrate buffer (Target Retrieval Solution, DakoCytomation, CA, USA), pH 6.1, in a steamer for 30 min, and endogenous avidinbiotin activity was quenched with a biotin blocking system (DakoCytomation) for 20 min. The slides were immediately incubated for 1 h at room temperature in a humidified chamber with 100 μL of rabbit anti-human IL-4 polyclonal antibody (ab9622, Abcam, Cambridge, MA, USA) diluted to 1:100. The standard streptavidin-biotin-peroxidase complex method was performed to bind the primary antibody with the use of Universal LSAB™/HRP System Kit (DakoCytomation) following the manufacturer’s instructions. Each of the incubation steps was preceded by a 10-min rinse with phosphate-buffered saline-0.05 % Tween 20 (PBS-T, DakoCytomation), pH 7.0. Reaction products were visualized using 0.3 % diaminobenzidine solution (DakoCytomation) and counterstained with Harris hematoxylin (Merck, Darmstadt, Germany). The specificity of immunohistochemical staining was determined by substituting the primary antibody with an antibody diluent (DakoCytomation). In addition, sections of normal human tonsil tissue were obtained as positive controls for immunostaining. For immunohistochemical analysis, ten HPFs were randomly chosen and assessed in each section by the same observer with the use of the ZEN Pro image analyzer system following previous described methods [32]. The cells with a clearly defined immunostaining when compared with the positive controls (as depicted by a brownish cytoplasmic staining, irrespective of labeling intensity) were considered to be positive. Epithelial cells were excluded from counting in order to prevent false-positive scores. Each HPF was evaluated for the proportion of stained cells and the intensity of overall staining. The proportion of stained cells in each HPF was assessed as follows: 0, absence of immunostained cells; 1, 50 % stained cells. The staining intensity was graded as follows: 0, negative staining; 1, light staining; 2, moderate staining; and 3, intense staining. Finally, a staining intensity distribution
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(SID) score was calculated for each sample as follows: the score of the proportion of stained cells for each HPF was multiplied by the score of the staining intensity in that field to provide a SID score for the field. The average of the 10 HPFs was the SID score for the sample. To increase the confidence level of positive staining, sections with too weak and/or equivocal staining were included in the negatively stained group. Likewise, for the analysis, the mean value of IL-4 SID scores obtained from gingival PG cases with periodontal breakdown was chosen as the cutoff point for dividing the cases into subgroups of 0.7 represented indicated fair to good reproducibility, and a value of >0.9 represented excellent reproducibility. All parameters were tested for normal distribution using the Shapiro-Wilk test for small sample size. Because the data conformed to a normal distribution, the variables were analyzed using parametric methods. Consequently, whereas differences between IL-4 SID score concerning demographic, clinical, and histological parameters were tested by unpaired t test or one-way analysis of variance (ANOVA) when indicated, Pearson’s correlation coefficient analysis was performed to describe the relationship between pairs of significant quantitative variables. Afterwards, for the assessment of periodontal breakdown predictors, statistical analysis was performed on three levels: (a) significant associations between the presence of periodontal breakdown and all investigated variables (15 in total) were explored in bivariate analyses using Pearson’s chisquare test (χ2) or Fisher’s exact test (when frequency was 20 % inflammatory cells/HPF) scores (mean 27.55, range 20.2–41.3). Concerning interstitial fibrosis, it was observed that 71.7 % of biopsies (33 cases) exhibited mild (≤20 % fibrosis/HPF) scores (mean 13.12, range 1.5–19.9) and 28.3 % biopsies (13 cases), moderate-to-severe (>20 % fibrosis/HPF) scores (mean 28.63, range 20.6–46.4). Twenty-four cases (52.2 %) showed histological features of LCH-type and 22 cases (47.8 %) of non-LCH-type PG. Although there were no significant differences between histological subtypes regarding epithelial lining (P=0.053, Fisher’s exact test), mean values of microvessel density and inflammatory infiltrate density were significantly lower in the LCH subtype (P0.05, unpaired t test and ANOVA) were evident in the IL-4 SID scores with respect to age stratum, gender, smoking habit, location of the lesions, macroscopic appearance, predisposing factors, recurrence, epithelial lining, interstitial fibrosis, or histological subtype, mean IL-4 SID values were significantly increased (all P1 cm), presence of periodontal breakdown, high microvessel density (>10 MVC/HPF), and moderate-to-severe inflammatory infiltrate density (>20 % inflammatory cells/HPF). Moreover, Pearson’s correlation analysis showed moderate-to-strong significant positive correlations (P1 cm (P=0.023). It was also noteworthy that the effects of histological/ immunohistochemical predictor variables such as microvessel density, inflammatory infiltrate density, and IL-4 SID score were found to be significant on the proportion of periodontal breakdown (P30 years Male Female Smokers Nonsmokers 1 cm Pedunculated Sessile Local irritants Pregnancy Unknown Present Absent Recurrent Nonrecurrent Ulcerated Atrophic Acanthotic Low (≤10 MVC) High (>10 MVC) Mild (≤20 % inflammatory cells) Moderate to severe (>20 % inflammatory cells) Mild (≤20 % fibrosis) Moderate to severe (>20 % fibrosis) LCH PG Non-LCH PG
23 (50.0) 23 (50.0) 26 (56.5) 20 (43.5) 23 (50.0) 11 (23.9) 12 (26.1) 14 (30.4) 32 (69.6) 11 (23.9) 35 (76.1) 25 (54.3) 7 (15.2) 14 (30.4) 33 (71.7) 13 (28.3) 20 (43.5) 26 (56.5) 33 (71.7) 13 (28.3) 24 (52.2) 22 (47.8)
4.65±2.76 6.67±2.90 5.84±3.24 5.42±2.66 6.28±2.52 4.49±3.46 5.54±3.25 8.04±1.12 4.62±2.95 6.58±2.82 5.37±3.01 5.67±2.98 4.71±3.80 6.11±2.64 5.10±2.96 7.08±2.61 4.41±2.87 6.63±2.74 5.70±3.16 5.57±2.59 5.06±3.02 6.32±2.85
0.056d 0.202d 0.002d 0.810e
0.020d 0.632d 0.261e
1 cm and high microvessel density in the univariate analysis, a confounding effect was evident after adjustment by the other covariables, as
these associations failed to achieve statistical significance. Even so, there was no confounding of the relationship neither between evolution course of ≥12 months, moderate-to-severe inflammatory infiltrate density, nor IL-4 SID score of ≥8.04 regarding periodontal breakdown. In these three cases, each relationship remained associated with the damage status (P1 cm/IL-4 SID score of ≥8.04
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Fig. 1 Scatterplot matrix demonstrating the statistical correlations of IL4 SID score (y-axis) in regard to evolution course, lesion size, microvessel density, and inflammatory infiltrate density of gingival PG (x-axis). Each panel of the matrix is a scatterplot of one variable against another. Solid black dots represent cases with periodontal breakdown, whereas open
black dots represent cases with no periodontal involvement. The regression lines are presented for each plot along Pearson’s rank correlation coefficient (r). All correlations were statistically significant at the 0.01 level of significance
(adjusted OR, 11.2; 95 % CI, 1.98 to 63.47; P=0.006, Wald’s test), lesion size of >1 cm/high microvessel density (adjusted OR, 15.0; 95 % CI, 2.54 to 88.39; P=0.003), high microvessel
density/evolution course of ≥12 months (adjusted OR, 8.3; 95 % CI, 1.37 to 50.46; P=0.021), high microvessel density/ moderate-to-severe inflammatory infiltrate density (adjusted
Fig. 2 Control experiments for IL-4 immunostaining (streptavidinbiotin-peroxidase complex, hematoxylin counterstain, original magnification×400): a Negative control performed by substituting the primary antibody with an antibody diluent. Note complete absence
of staining. b Positive control showing intense cytoplasmic staining of macrophages and B lymphocytes of the germinal center, as well as T lymphocytes of paracortex of human tonsil tissue
Clin Oral Invest Table 2 Bivariate associations between demographic risk variables and periodontal breakdown in gingival pyogenic granuloma Periodontal breakdowna
Risk variables
Gender Age stratum Smoking habit Evolution course
Male Female ≤30 years >30 years Smokers Nonsmokers 1 cm Pedunculated Sessile Local irritants Pregnancy Unknown Recurrent Nonrecurrent
5 (10.9) 7 (15.2) 20 (43.5) 12 (26.1) 17 (37.0) 15 (32.5) 14 (30.4) 8 (17.4) 10 (21.7) 6 (13.0) 26 (56.5)
4 (8.7) 1 (2.2) 3 (6.5) 11 (23.9) 9 (19.6) 5 (10.9) 9 (19.6) 3 (6.5) 2 (4.3) 5 (10.9) 9 (19.6)
Values are given as n (%) of cases
b
Two-sided Fisher’s exact test
c
Two-sided Pearson’s chi-square test (χ2 )
0.199b
0.023b 0.482c 0.378b
0.215c
Clin Oral Invest Table 4 Bivariate associations between histological/immunohistochemical risk variables and periodontal breakdown in gingival pyogenic granuloma Periodontal breakdowna
Risk variables
Absent (n=32) Epithelial lining
Microvessel densityb Inflammatory infiltrate densityb Interstitial fibrosisb Histological subtype IL-4 SID scorec a
P value
Present (n=14)
Ulcerated Atrophic
17 (37.0) 5 (10.9)
8 (17.4) 2 (4.3)
Acanthotic Low (≤10 MVC) High (>10 MVC) Mild (≤20 % inflammatory cells) Moderate-to-severe (>20 % inflammatory cells) Mild (≤20 % fibrosis) Moderate to severe (>20 % fibrosis) LCH PG Non-LCH PG 1 cm) lesions, as well as in those with periodontal breakdown, high microvessel density, and moderate-to-severe inflammatory infiltrate density. Moreover, as such differences were mainly based on the greatest number of immunoreactive inflammatory and endothelial cells, the finding of significant positive correlations between IL-4 SID scores regarding the evolution course, lesion size, microvessel density, and inflammatory infiltrate density of the lesions seems to highlight the proinflammatory role of IL-4 in gingival PG. Although nothing is known about the immunoexpression of IL-4 in regard to gingival PG on which would be established as a basis of comparison, several studies performed on gingival biopsies obtained from periodontitis-affected patients have demonstrated that IL-4 is widely expressed in diseased periodontal tissues, suggesting that a bias towards Th2-type cytokine dominance could be an exacerbating factor [29, 35–37]. In agreement with the former, it has been postulated that IL-4 may serve as an autocrine growth factor responsible for the maintenance and proliferation of inflammatory cells in vivo [38] as well as a potent inducer of angiogenesis [39–41]. Consequently, it is possible that IL-4 is involved in the immunopathogenesis of gingival PG, driving the migration and the maintenance of several cell types such as polymorphonuclear leukocytes, macrophages, subsets of lymphocytes, and microvascular endothelial cells within the gingival tissues.
Clin Oral Invest Table 5 Initial and final model of bivariate binary logistic regression for the association of significant predictor variables with periodontal breakdown in gingival PG Risk variable
Unadjusted OR (95 % CI)a
P valueb
Evolution course ≥12 months Lesion size Microvessel density Inflammatory infiltrate density
7.50 (1.83–30.68)
0.005
IL-4 SID score Lesion size >1 cm Evolution course Microvessel density Inflammatory infiltrate density IL-4 SID score High microvessel density Lesion size Evolution course Inflammatory infiltrate density IL-4 SID score Moderate-to-severe inflammatory infiltrate density Lesion size Evolution course Microvessel density IL-4 SID score IL-4 SID score ≥8.04 Lesion size
6.11 (1.41–26.40)
a
Odds ratio (95 % confidence interval)
b
Wald’s test
5.32 (1.21–23.38) 6.88 (1.59–29.76) 5.02 (1.06–23.60)
0.027 0.010 0.041
6.12 (1.23–30.34)
0.026
4.01 (0.84–19.21) 5.11 (1.31–23.04) 6.26 (1.21–32.23) 2.31 (0.40–13.29)
0.081 0.034 0.028 0.346
3.34 (0.77–14.49) 3.78 (0.82–17.33) 2.39 (0.51–11.07) 2.39 (0.48–11.93)
0.106 0.086 0.265 0.285
19.36 (2.07–180.34) 13.72 (1.50–125.32) 15.14 (1.62–141.28) 18.83 (1.77–200.45)
0.009 0.020 0.017 0.015
8.17 (1.47–45.23)
0.016
10.56 (2.04–54.65) 9.96 (2.08–47.68) 12.49 (2.04–76.15)
0.005 0.004 0.006
0.036
19.00 (2.20–163.57)
Evolution course Microvessel density Inflammatory infiltrate density
P valueb
0.015
4.33 (1.09–17.10)
12.60 (2.77–57.27)
Adjusted OR (95 % CI)a
0.007
0.001
Hosmer and Lemeshow’s goodness-of-fit test >0.208 C-statistics >0.761
On the other hand, in the present study, a significant association of periodontal breakdown with the evolution course of ≥12 months, lesion size of >1 cm, high microvessel density, moderate-to-severe inflammatory infiltrate density, and IL-4 SID score of ≥8.04 in both bivariate and univariate analyses was found. However, despite of these observations, after bivariate logistic regression analysis, both the lesion size >1 cm and the high microvessel density met the criteria to be considered confounders, as the associations weakened considerably when adjusted for the other covariables individually. On the contrary, it is also important to emphasize that although this study confirmed the strong and independent association of evolution course of ≥12 months, moderate-to-severe inflammatory infiltrate density, and IL-4 SID score of ≥8.04 as robust predictors of periodontal damage in gingival PG, synergistic biological interactive effects were also significantly detected regarding the lesion size and the microvessel density. Although there is no available data on a direct relationship between
clinicopathological predictors and the increasing periodontal breakdown in gingival PG, these findings might indicate that while both the lesion size and the high microvessel density are significant factors modifying for the other three significant predictors, the evolution course and inflammatory infiltrate density might promote the periodontal breakdown in gingival PG when IL-4 is overexpressed within the lesional tissue. As IL-4 has been regarded as having angiogenic [39–41] and proinflammatory functions [13, 24–28], it is quite probable that the effects on the vascular and immune systems can act synchronously to promote the bone resorption and tissue destruction observed in gingival PG.
Conclusions and clinical relevance The findings of this study suggest that while evolution course, inflammatory infiltrate density, and overexpression of IL-4 may
Clin Oral Invest
act as predictors of periodontal breakdown in gingival PG, there are mutual confounding and synergistic biological interactive effects with respect to lesion size and microvessel density in the susceptible host that may be also associated with the bone resorption and tissue destruction. Although, the first-line therapy of gingival PG continues to be the surgical excision, this approach poses unwanted complications such as disagreeable mucogingival defects and recurrence. Hence, early diagnosis and detection of these three significant predictor variables as well as timely surgical excision might help prevent the periodontal tissue destruction observed in some of these lesions.
Conflict of interest The authors declare that they have no financial interest in the products in this work. This study has been fully supported by a grant of the Research Development Committee of the University of Antioquia (CODI-Code 8700-1617/2009).
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