| Abstract|| |
Objective: To isolate, culture and characterize fibroblasts from the buccal mucosa of areca nut chewers with and without oral submucous fibrosis (OSF).
Materials and Methods: Primary fibroblast cultures were established by the collagenase disaggregation technique and the phenotypic and growth characteristics were studied.
Results: Cells cultured from OSF showed a statistically significant increase in both the post-mitotic fibroblast subpopulation and the population doubling time when compared with controls.
Conclusion: There was a significant increase in the pro-fibrotic, post-mitotic subpopulation of fibroblasts in areca nut chewers with OSF.
Keywords: Areca nut, chewers mucosa, culture, fibroblasts, oral submucous fibrosis
|How to cite this article:|
Mathew DG, Skariah K S, Ranganathan K. Proliferative and morphologic characterization of buccal mucosal fibroblasts in areca nut chewers: A cell culture study. Indian J Dent Res 2011;22:879
Oral submucous fibrosis (OSF) is a chronic, progressive and potentially malignant condition characterized by submucosal fibrosis and epithelial atrophy.  There is a strong association between areca nut chewing and OSF.  The fibrosis is considered to be due to a combination of factors involving mutation and selection of fibroblast subpopulations that produce altered collagen in a tissue environment conditioned by fibrogenic-cytokines, induced by exposure to areca nut alkaloids.  In addition to genetic, racial and ethnic factors, fibroblasts in OSF can be altered due to the various sources of areca nut, as the contents including alkaloids vary depending upon the geographical area of origin. ,, Studies on primary cultures are few and, to our knowledge, no published data exists for cases from the Indian subcontinent that has a high burden of OSF. This study was conducted to address this lacunae and it is the first of its kind from India. It is the first time to our knowledge that culture characteristics of cells from areca nut chewers with no OSF were studied.
|How to cite this URL:|
Mathew DG, Skariah K S, Ranganathan K. Proliferative and morphologic characterization of buccal mucosal fibroblasts in areca nut chewers: A cell culture study. Indian J Dent Res [serial online] 2011 [cited 2017 Nov 18];22:879. Available from: http://www.ijdr.in/text.asp?2011/22/6/879/94693
| Materials and Methods|| |
The study group comprised of areca nut chewers with clinically normal mucosa (Group A; n=3) and areca nut chewers with clinical and histopathologic features  of OSF (Group B; n=3). Subjects in both these groups had a chewing habit for a minimum of 1 year. The control group (Group C; n=6) comprised of subjects without areca nut chewing habits and with clinically normal oral mucosa. Buccal mucosal biopsies for fibroblast cultures were obtained from the patients who reported at the Ragas Dental College and Hospital, Chennai, India. In Groups A and C, tissues were obtained from the non-inflamed posterior buccal mucosa during surgical extraction of the 3 rd molar and in Group B, from the fibrotic areas in the buccal mucosa. The study was approved by an Institutional Review Board. Informed consent was obtained from all subjects who participated in the study.
Tissues were obtained by incisional biopsy under local anesthesia and minced into 1 mm × 1 mm × 1 mm pieces and washed in Dulbecco's phosphated buffer saline (DPBS). Tissue pieces were incubated in working media [Dulbecco's Modified Eagle Media (Invitrogen TM : Grand Island, New York state, USA) supplemented with 10% Fetal bovine serum (Invitrogen TM : Grand Island, New York state, USA), 100 μg/ml of penicillin, 100 μg/ml of streptomycin and 1 μg/ml of amphotercin B] containing crude collagenase (Sigma TM : St Louis, Missouri, USA) for 18 h following which they were centrifuged at 2000 rpm for 5 min. The sediment was plated on a 60-mm tissue culture plate (Tarson TM : India) containing working media for 48 h to facilitate cell attachment. Media change was performed every 3 rd day. Confluency was reached in 2-3 weeks, after which the cells were subcultured. The cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . The fourth passage cells were used for the study. Morphologic characteristics and vimentin positivity by immunocytochemistry were used to confirm the fibroblast lineage of the cultured cells. 
Estimation of fibroblast subpopulations based on morphology
Cell lines from the fourth passage were plated on three 60-mm tissue culture plates at a concentration of 0.5 × 10 4 cells/ml. Using a phase contrast microscope (Olympus CKX41 TM : Japan), cells were observed at ×200 magnification and counted daily for 8 consecutive days. Fibroblast subpopulations were classified using two models based on their morphology; the conventional rat skin model F 1 , F 2 , F 3 (F1 are spindle shaped, F2 are epitheloid shaped and F3 are stellate-shaped cells) that has been widely used , and the human skin model; Mitotic: f1, f2, f3 and post-mitotic fibroblasts: f4, f5, f6, f7 [f1 are small spindle-shaped cells, f2 cells are small and epitheloid, f3 cells are large pleomorphic and epitheloid, f4 cells are large spindle shaped, f5 cells are larger epitheloid, larger than f3, f6 cells are the largest epitheloid and f7 cells are the degenerating fibroblasts] [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11] and [Figure 12].  The percentages of subpopulation in the log phase (day 1-6) and stationary phase (day 7-8) were calculated.
|Figure 9: Comparative growth curve of cell linets. CM- Chewers mucosa cell line, SM- OSF cell line, NB- Control buccal mucosa cell line|
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Cells from the fourth passage were inoculated at a concentration of 1.2 × 10 4 cells/ml/well in 24-welled plates and allowed to attach overnight. The following day, cells from three randomly selected wells were trypsinized and counted using a hemocytometer. Counting was repeated every 24 h for 8 days. Cells from each well were counted thrice and the average was used to plot the growth curve. The seeding efficiency was estimated using the following equation,
The population doubling time (PDT) was calculated from the slope of the growth curve at its log phase (days 1-6). ,
All data were entered and analysed using SPSS (Statistical package for social science) v11.5. Regression line was derived from the slope of the growth curve (from day 1 to 6) and the PDT was calculated.  Independent sample Student's t test was used to compare between the control and study groups for the percentage of cells in log phase (day 1-6) and stationary phase (day 7-8) derived from the subpopulation studies, the seeding efficiency and PDT of growth curves. A P value <0.05 was considered to be statistically significant.
| Results|| |
Limited cell lines were established from patients with areca nut chewing habit but no clinical signs of OSF (Group A; n=3), areca nut chewers with OSF (Group B; n=3) and controls (Group C; n=6). The average duration of exposure to areca nut products in the study population was 9 years (range 1-35 years). The average age of the subjects in this study was 34 years (range 17-60 years) [Table 1].
Fibroblast subpopulation studies
Rat skin model
There were no statistically significant differences in the percentage of cell subpopulations between controls and study groups [Table 2].
|Table 2: Mean difference of rat fibroblast subpopulation percentages in its various growth phases between control group (Group C; n=6) and study groups-chewers with no OSF (Group A; n=3), chewers with OSF (Group B; n=3)|
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Human skin model
When the fibroblasts were classified as mitotic and post-mitotic, there was a statistically significant reduction of mitotic cells and increase in the post-mitotic cells during the log phase of growth for chewers with OSF when compared with controls (P=0.043). In chewers without OSF, alterations in the mitotic and post-mitotic subpopulations, although present, were not significant when compared with controls [Table 3].
|Table 3: Mean difference of human fibroblast subpopulation percentages in its various growth phases between control group (Group C; n=6) and study groups-chewers with no OSF (Group A; n=3), chewers with OSF (Group B; n=3)|
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Growth curve and its derivatives
Growth curves of limited cell lines showed an initial lag phase, an exponential log phase followed by a stationary phase. After the initial lag phase, there was a steady increase in the slope of the growth curves from day 1 to day 6 in Groups A and C. This steady increase in the cell concentration was not found in the OSF cell lines [Figure 9], [Table 4] and [Table 5]. By the 6 th day, cell lines from Groups A and C reached the stationary phase due to confluency. At the 8 th day of observation, this stationary phase was not observed in OSF cell lines. The OSF group had PDT three times greater than that in Groups A and C (P=0.022) [Figure 9], [Table 4] and [Table 5]. There was a statistically significant decrease in the seeding efficiency of OSF cell lines when compared with controls (P=0.018) [Table 4] and [Table 5].
|Table 5: Mean difference of seeding efficiency and population doubling time between control group (Group C; n=6) and study groups-chewers with no OSF (Group A; n=3), chewers with OSF (Group B; n=3)|
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| Discussion|| |
Seven subpopulations (f1-f7) of fibroblasts were identified in the study groups. Fibroblast cellular characteristics were studied for the first time in areca nut chewers with clinically normal buccal mucosa. The senescent phenotypes (f4-f7) were more prominent in the cell lines from chewers with OSF.
When the rat model was used in studies in OSF, a significant increase in the senescent F3 subpopulation was reported.  However, in this study, we could not find any significant difference in the F1, F2 and F3 subpopulations between areca nut chewers and controls [Table 2]. Consequently, we used the human model to study the subpopulation shift during the 8- day observation period as (i) unlike human fibroblasts, rat fibroblasts undergo a lesser degree of senescence in vitro, , (ii) in the rat model, F3 is considered as a senescent post-mitotic subpopulation; however, our earlier observations (data not published) showed that F3 was not a homogenous population but a mixture of proliferating mitotic and senescent post-mitotic stellate-shaped fibroblasts. When the fibroblasts were subclassified using a human model, subjects with OSF showed a statistically significant decrease in the mitotic phenotypes and increase in the post-mitotic phenotypes during the log phase of growth (day 1-6) compared with the controls [Table 3]. The increase in the post-mitotic phenotypes in OSF patients is the result of the effect of areca nut alkaloids on the fibroblasts, resulting in a shift in the fibroblast subpopulations to a more-differentiated, less-proliferative and high collagen-producing phenotypes, as described by De Waal et al. in1997.  This change was significant only in chewers with OSF. The changes in the sub-populations were more obvious during the log phase of the cell growth than in its stationary phase as cell division facilitates sub-population shifts with greater differentiation.
Changes described in the study groups were apparent when fibroblasts were classified into the two broad groups based on the human fibroblast model  rather than with the rat fibroblast model,  which has been widely used to study the fibroblast subpopulation shifts in earlier studies. , As rat fibroblasts undergo senescence to a lesser degree than human fibroblasts in culture conditions, they have a lesser proportion of senescent phenotypes. , Also, the rat model has the flaw of grouping both mitotic and post-mitotic cells into the same group. Stellate-shaped mitotic f3, post-mitotic cells f5 and f6 of the human model correspond to the senescent F3 subpopulation of the rat model and among the spindle-shaped cells, mitotic f1 and post-mitotic f4 of the human model corresponds to the proliferative subpopulation F1 of the rat model. Based on these findings, we propose that future studies on the fibroblast subpopulation analysis should use a human model rather than the conventionally used rat model.
The PDT of OSF cell lines was three times longer than that of the cell lines derived from areca nut chewers with no OSF and controls, reflecting the increase in the post-mitotic fibroblast subpopulations in OSF [Figure 9], [Table 3], [Table 4] and [Table 5]. This finding is in contrast to previous studies on the proliferation rates of OSF fibroblasts. In 1995,Van Wyk et al. found no statistically significant difference between the growth rates of normal and OSF fibroblasts  and in 1995, Ma et al. reported that OSF fibroblasts have a shorter PDT of 3.2 days when compared with normal controls, with a PDT of 3.5 days.  This difference between our study and the previous studies may probably be due to variation in the alkaloid content in the areca nut grown in the Indian environment  and the differences in the fibroblast cellular characteristics due to racial origins of the subject. , However, in in vitro studies, where cultured oral fibroblasts were exposed to areca nut extracts at concentrations of 50-150 μg/ml, there was growth inhibition in a dose-dependent manner. 
Cell lines derived from areca nut chewers without OSF exhibited an increase in the post-mitotic phenotypes during the log phase and longer PDT similar to the OSF cell lines. But, these changes were not statistically significant from controls, probably indicating early pathologic changes prior to the clinical manifestation of disease.
In the present study, we have standardized the culture technique of fibroblasts from the buccal mucosa of areca nut chewers and controls. We have also shown that grouping the fibroblasts as mitotic and post-mitotic based on the human fibroblast model is a better indicator of the phenotypic alterations in areca nut chewers than in the rat model. PDT and phenotypic changes occur in chewers with no OSF and become significant in chewers with clinically established OSF. Identifying changes before the clinical onset of OSF will give us an opportunity to better understand the pathologic process and also prevent or reverse the disease progress.
| Acknowledgment|| |
The authors would like to acknowledge Drs. S. Ramachandran, Umadevi K. Rao, E. Joshua, T. Rooban, W. Kavitha and S. Deepa of Ragas Dental College, T. R. Saraswathi of Vishnu Dental College and R. Gunaseelan and B. Praveen of CDRF for the invaluable help rendered in this study.
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Deepu George Mathew
Department of Oral and Maxillofacial Pathology, Ragas Dental College and Hospital, Chennai
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]