Effect of a volatile smoke component (acrolein) on human gingival fibroblasts: An in vitro study.

AIM: Tobacco and some of its volatile and non-volatile components have been found to affect many types of cells including human gingival fibroblasts. The aim of this present study was to estimate the effect of acrolein, a volatile fraction of cigarette smoke on the attachment, proliferation and ultra structure of human gingival fibroblasts in culture.
MATERIALS AND METHODS: Human gingival fibroblasts strains obtained from healthy subjects aged 20-30 years, were grown to confluency and utilized between 3(rd) -6(th) passages. The cell cultures seeded in 96 well microtitration plates at a density of 45,000 cells/well were incubated with acrolein at concentrations of 10(-4), 3×10(-5) and 10(-5) . Attachment ability was evaluated after three hours using Neubauer hemocytometer. For the proliferation assay cell cultures seeded at a density of 10,000 cells/well were incubated at concentrations of 10(-4), 3×10(-5), 10(-5), 3×10(-6), 10(-6) and cell count determined after 5 days using a hemocytometer. Cell morphology was examined under phase contrast microscope.
RESULTS: Acrolein produced a dose-dependent cytotoxic effect on human gingival fibroblasts with complete inhibition of attachment and proliferation at higher concentrations.
CONCLUSION: This supports the hypothesis that cigarette smoke is a great risk factor in the development and progression of periodontal disease.

INTRODUCTION

The risk revolution is one of the most significant advances that have occurred in medicine over the past decades. In the current state of incomplete knowledge of most human diseases, the probabilistic approach allows us to make accurate predictions for a group of individuals’ presenting with specific attributes or exposures. Risk assessment has become increasingly important in the prevention of chronic diseases and has recently been applied to oral diseases due to the realization that severe forms of the two most prevalent dental diseases, i.e., caries and periodontal disease are clustered in a minority of the population who are hypothesized at being at higher risk.[1] Consequently, a new paradigm emerging to control dental disease involves identification and targeting high risk individuals and intercepting the disease process. A targeted prevention based approach is proposed to replace more conventional treatment based approaches.

Cigarette smoking has long been associated with a variety of oral conditions including periodontal diseases. Experimental evidence accumulated over the last two decades has indicated that cigarette smoking is a true risk factor for periodontitis. Smokers have both increased prevalence and more severe extent of periodontal disease compared to non-smokers.[2] Tobacco and some of its volatile and non-volatile components have been found to affect human gingival fibroblasts. Among the multiple volatile components, some reactive aldehydes are thought to have a prominent cytopathic effect, resulting in a dose dependent human gingival fibroblast inhibition of cell adhesion related to morphological alteration of cytoskeletal structure.[3] Acrolein is one subtype of several aldehydes present in cigarette smoke.

Acrolein, a very reactive compound is a volatile flammable liquid with a pungent, choking disagreeable odor. In cigarettes, it has been identified at a mean level of 0.11 mg/cigarette.[4] Experimental data suggests that acrolein is detrimental to Human gingival fibroblasts (HGF) survival and consequently to the oral connective tissue.[5] They are well recognized to have a high cytotoxic and genotoxic effect on cultured human bronchial epithelial cells and fibroblasts, as well as on human skin fibroblasts.[6] This in vitro study is focused on the toxic nature of a volatile smoke component-Acrolein on the proliferation and attachment of human gingival fibroblasts.

MATERIALS AND METHODS

Human gingival fibroblasts (HGF) strains from healthy subjects with non-inflamed gingiva which did not bleed on probing were utilized. Individuals who were never exposed to tobacco were included in the study. Informed consent was obtained prior to the biopsy procedure.

Surgical procedure

A split thickness flap was raised at the donor site using Bard Parker no-15 and careful dissection was carried out [Figure 1a]. A conventional method was preferred over punch biopsy to delineate the epithelium from the underlying connective tissue. The biopsy tissue was collected in the sterile test tube containing Dulbecco's modified Eagle's medium (DMEM) and transported to the laboratory.

Figure 1

(a) Procuring gingival connective tissue; (b) Human gingival fibroblast in a mono layer; (c) Human gingival fibroblasts - acrolein treated (1:100000 molar); (d) Human gingival fibroblasts - acrolein treated(1:10000 molar)

Fibroblast culture

After 3 washes in DMEM supplemented with Gentamycin, the tissues were minced into small pieces plated in Petri dishes. Trypsin-Versene-Glucose (TVG) solution containing 0.25% trypsin was added and incubated at 37°C for 2 hours. The TVG was discarded, and the cells resuspended in DMEM containing 10% fetal calf serum (FCS), and were then seeded into 25 cm2 tissue culture flask and incubated at 37°C in an atmosphere of 5% CO2. The confluent layer of cells obtained was designated as “first passage cells”. For the experiments cells were utilized between the third and the sixth passage.

Attachment assay

Human gingival fibroblasts were seeded in 96 well microtitration plate at a density of 45,000 cells/well in DMEM containing 10% FCS. About 24 wells seeded with 45,000 cells per well formed the sample group wherein three groups of 8 wells were treated with 10-5, 3×10-5 and 10-4 M acrolein respectively in an atmosphere of 5% CO2 and at a temperature of 37°C. Untreated cell cultures, obtained from the same biopsies as the test fibroblast cells, were utilized as the control group and the acrolein treated cells were considered as the study group. Attachment ability was evaluated after 3 hours. The attached cells at each of the different concentrations of acrolein were removed with 0.25% trypsin and evaluated by a Neubauer Hemocytometer.

Proliferation assay

Human gingival fibroblasts were seeded in 96 well microtitration plates at a density of 10,000 cells/dish in DMEM with 10% FCS. Cells were incubated for 24 hours at a temperature of 37°C in an atmosphere of 5% CO2 to allow attachment to the plate. Culture wells were rinsed once to remove unattached cells and the culture medium was changed to fresh DMEM with 10% FCS at 37°C. Cell cultures were incubated in the presence of 10-6 3×10-6, 10-5, 3×10-5 and 10-4 concentrations of acrolein. The proliferation rate was carried out over a 5-day period. The wells were trypsinized (0.25%) to remove the cells and then counted using a Hemocytometer.

Cell counting by hemocytometer

The monolayer formed was trypsinized and resuspended in the medium. The suspension was mixed thoroughly to disperse the cells and a small sample (20 μl) was collected into the tip of a Pasteur pipette and transferred to the edge of the hemocytometer chamber. The surplus fluid was blotted and the slide transferred to the microscope stage. A 10x objective was selected and focused on the grid lines in the chamber. The central area of the grid bounded by three parallel lines was focused. The cells lying within this 1 mm2 area were counted and analyses was made using the formula c=n/v where c is the cell concentration (cells/ml), n is the number of cells counted and v is the volume counted (ml).

A total of 5 squares were counted using a slide of depth 0.1 mm and an area of 1 mm2 and values obtained using the equation c=n×104/5. The cell cultures were studied for morphology before counting, using an inverted phase contrast microscope (Leica, W. Germany) and photomicrographed. Human gingival fibroblasts between 3rd to 6th passages were grown to confluency. For the attachment assay, the cell cultures seeded in a 96 well microtitration plates at a density of 45,000 cells/well. They were then incubated at concentrations of 10-4 (C1), 3×10-5 (C2), 10-5 (C3) acrolein in an atmosphere of 5% C02 and at a temperature of 37°C. Untreated cell cultures were used as controls. Attachment ability was evaluated after 3 hours using a Neubauer hemocytometer. The proliferation assay that was carried over a 5-day period incubated cells at concentrations of 10-4 (C1), 3×10-5 (C2), 10-5 (C3), 3×10-6 (C4), 10-6 (C5) acrolein in an atmosphere of 5% Co2 and at a temperature of 37°c. Proliferation ability was evaluated after 5 days using the hemocytometer. A total of 5 squares were counted and the concentration of human gingival fibroblast was evaluated using the formula c=n×104/5 for 1 ml of suspension. In the present study, geometric mean and standard deviation were estimated for each study group after log transformation. The mean values were compared by One-way Analysis of Variance (ANOVA) and further multiple range tests by Tukey's- Honestly Significant Difference (HSD) were employed to identify the significant groups at 5% level. P<0.05 was considered significant.

RESULTS

The human gingival fibroblast attachment evaluated at three hours indicated that acrolein caused a dose dependent inhibition of cell attachment [Table 1]. At low concentration of acrolein (10-5), the cells maintained the attachment capacity [Figure 2b]. As the concentration of acrolein increased (10-4), the cells rapidly lost their attachment capacity [Table 2]. The number of cells attached/well decreased from a mean value of 29,930 in C3 group (10-5) to 20,505 in C1group (10-4) also the mean values in C3 (29,930) and C2 (27,441) were significantly higher compared to C1 (20,505) [Table 3]. In the present study, the human gingival fibroblast proliferation evaluated at five days indicated that the untreated cell cultures (control group) proliferated from 10,000 cells/well to an average of 29,697 cells/well [Table 4, Figure 2b]. In the acrolein treated cell cultures (study group), the proliferation ability decreases from 21,184 cells/well in the C5 (10-6) group to 10,154 in the C1 (10-4) group [Table 5]. Further statistical evaluation indicates that:

Table 1

Human gingival fibroblast attachment in acrole in treated cell cultures (study group)

Figure 2

(a) Dose-related effects of acrolein on human gingival fibroblast attachment; (b) Dose-related effects of acrolein on human gingival fibroblast proliferation

Table 2

Mean, standard deviation and test of significance of mean values between different study groups for attachment assay (one - way ANOVA)

Table 3

Mean, standard deviation and test of significance of mean values between different groups for attachment assay (Tukey- HSD)

Table 4

Human gingival fibroblast proliferation in acrolein treated cell culture

Table 5

Mean, standard deviation and test of significance of mean values between different groups for proliferation assay (one - way ANOVA)

The mean value in the control group (29,697) is significantly higher than the mean values in the study group.

The mean values of C5 (21,184) and C4 (20,068) are significantly higher than the mean values in C3 (16,691), C2 (16,244) and C1 (10,154).

Further the mean values of C3 and C2 are significantly higher than C1 [Table 6]. Thus a dose-dependent inhibition of proliferation is seen and at very high concentration (10-4) of acrolein a complete inhibition of proliferation is noted.

Table 6

Mean, standard deviation and test of significance of mean values between different groups for proliferation assay

Human gingival fibroblasts seen in the control group are spindle shaped, oriented in a parallel manner forming a cohesive layer [Figure 1b]. In contrast, the test group shows disruption of the fibroblast cytoskeleton leading to loss of shape and orientation. There is a dose dependent increase in vacuolisation and degeneration of the nucleus observed [Figure 1c and d].

DISCUSSION

Periodontal diseases are opportunistic infections caused by specific periopathogenic microorganisms and their metabolic products. The onset, progress, and severity are determined by the individual host response modulated by various factors. Advances in research have led to a fundamental change in the periodontal disease model. The current complex model maintains a bacterial etiology taking into consideration individual susceptibility as well as environmental and host response influences. The current list of risk indicators and putative risk factors is being shaped by ongoing research efforts, and multiple attributes and exposures have been associated with increased odds of periodontitis. This has led to the current understanding that periodontitis is a multifactorial disease whose clinical manifestations are the result of specific interactions between particular genetic susceptibility traits and environmental factors. Experimental evidence accumulated over the last two decades has indicated that cigarette smoking is a true risk factor for periodontitis. Tonetti[7] has indicated that this environmental exposure has been associated with 2-3 fold increase in the odds of developing clinically detectable periodontitis. Regression analyses done by Zambon et al.,[8] have shown that the relative risk of attachment loss among 25-74 year subjects with a history of moderate smoking was 2.77 and 4.75 for heavy smokers. Periodontal disease is the result of an imbalance between tissue destruction and repair. In health, fibroblasts are responsible for the production and maintenance of the connective tissue matrix. They are the predominant cells of the periodontal ligament and have an important role in the development, function and regeneration of the tooth support. In consideration of the important role played by fibroblasts in the periodontal ligament homeostasis, they have been described by Tencate[9] as “the architect, builder, and caretaker” of the periodontal ligament.

In recent years, many clinical and epidemiological studies have indicated that cigarette smoking significantly increases the risk factor for periodontal disease, especially in younger people. Haber et al.,[10] have demonstrated that the noxious effect of cigarette smoking, rather than poor oral hygiene and increased plaque, act directly on periodontal tissues. Evidence indicates that tobacco and some of its volatile and non-volatile components have been found to affect many types of cells including gingival fibroblasts. Among the volatile components of cigarette smoke some reactive aldehydes have a prominent cytopathic effect. Grafstrom et al.,[11] have recognized them to have a high cytotoxic and genotoxic effect on cultured human bronchial epithelial cells and fibroblasts, and inhibit both fibroblast-mediated gel contraction and fibronectin production.

Acrolein, one subtype of several aldehydes present in cigarette smoke is a very reactive volatile compound. In cigarettes, it has been identified at a mean level of 0.11 mg/cigarette. Acrolein being a cytotoxic agent induces microtubule depolymerisation, and triggers feedback inhibition of new tubulin synthesis affecting cell adhesion and proliferation. The adhesion capacity and organization of the cytoskeleton microtubules and vimentin associated filaments in cultured human gingival fibroblasts are affected by acrolein in a dose-dependent manner. In the present study, we examined the attachment, proliferation and morphology of human gingival fibroblasts in culture after exposure to acrolein. For this study, acrolein was selected because Newsome et al.,[12] recognized that it is present in small, but significant or toxic quantities in cigarette smoke. Human gingival fibroblast strains’ from healthy subjects with non-inflamed gingiva who were never exposed to tobacco were included in the study. Human gingival fibroblasts were obtained from periodontally healthy subjects aged 20-30 years. The criteria for selecting the particular age group was based on studies by Haber[10] and Monterio da Silva[13] which have indicated that cigarette smoking significantly increases the risk factor for periodontal disease especially in younger people. Concentration ranges of acrolein used refer to the previous experiments by Grafstrom et al.,[11] Nakamura et al.,[14] and Carnevali et al.[15] Human gingival fibroblasts between 3rd to 6th passages were utilized for the experiment. These results are in agreement with the study done by Rota[16] who stated that acrolein caused a dose-dependent inhibition of human gingival fibroblast adhesion strictly related to the morphological alterations of cytoskeletal structures, and by Cattaneo[17] who stated that acrolein caused a dose dependent inhibition of human gingival fibroblast attachment and proliferation when subjected to different concentrations of acrolein ranging from 10-6 to 10-4. Further evidence is obtained from a study by Poggi,[18] which indicated that acrolein produced a dose-dependent decrease in cell replication and adhesion by causing a disruption of microtubules, vimentin intermediate filaments and actin filaments. Thus, Maurizio S. Tonetti[7] stated that although no direct causative role of cigarette smoke on periodontal diseases has been proven, a connection is considered to be plausible.

CONCLUSION

Volatile fractions of cigarette smoke have a direct cytotoxic effect on human gingival fibroblasts. This study confirms the hypothesis that cigarette smoke is a significant risk factor in the development and progression of periodontal disease and impairs the ability of gingival fibroblasts to maintain the integrity of the periodontal connective tissue.