Phenotypic Expression of Oral Fibroblasts Derived from Oral Submucous Fibrosis: An Assay through Cell Culture.

OBJECTIVE: The present study aimed to establish cell lines of fibroblast from human OSF tissues and their response to varying concentrations of arecoline. The various morphological forms of fibroblasts were identified to establish phenotypic change. MATERIALS AND
METHOD: Fibroblast cell lines were obtained from control samples as well as from OSF cases. The cell lines were treated with 50/100/150/300/500 ug/ml of arecoline and morphology were determined.
RESULTS: Three morphological forms were detected; F1 spindle, F2 epitheloid and the F3 stellate. The F3 to F1 ratio was higher in OSF. Arecoline at 50ug/ml was stimulatory and at 150ug/ml cytotoxic to the cell lines.
CONCLUSION: Arecoline seems to enhance proliferation of the fibroblast at lower concentrations but cytotoxic at higher levels. This is probably due to the generation of new cell lines and response of the arecoline receptors indicating phenotypic change. Copyright:

INTRODUCTION

Oral submucous fibrosis (OSF) is characterized by excessive deposition of collagen in the subepithelial tissue by the fibroblasts. Previous studies on the pathogenesis of OSF have suggested that the occurrence is due to stimulation of fibroblasts proliferation and collagen synthesis due to areca nut. It was also found that there was a decreased secretion of collagenase, deficiency of collagen phagocytosis by OSF fibroblasts.[12] In vivo techniques like cell culture are more precise mechanisms for assessing the cellular behavior of the lesion.

Cell cultures of OSF fibroblasts indicate multiple morphological patterns of the cells. The various morphological phenotypes reported of fibroblasts in cell cultures are of three types, i.e., F1, F2, and F3. The F1 fibroblast is spindle shaped, highly proliferative, and secretes low levels of type 1 and type 3 collagen. F2 is more epithelioid, less proliferative, and synthesizes relatively more collagen, while F3, a large stellate cell and the least proliferative, produces large quantities of collagen type 1 and 3. It is known that this population of cells arises in a hierarchical pattern, i.e., F1 gives rise to F2, F2 gives rise to F3, and this process seems to be irreversible. The shape of the fibroblast is determined by the pseudopods and the movement of the cells that involve the pseudopods. In cases of fibrosis, it was found that F3 fibroblasts predominate and similar observation has been reported in OSF.[3]

Arecoline is the major alkaloid in betel quid and plays an important role in pathogenesis. The response of the fibroblasts to arecoline is concentration dependent.[456] There are previous studies done to study the morphology of fibroblast as well as effects of arecoline, but there were no studies done to compare the characteristic and time bound response of normal and OSF fibroblasts from humans on application of arecoline.

MATERIALS AND METHODS

The study group comprised 13 cases of OSF from the department of oral and maxillofacial pathology. The clinical grading was done according to Lai DR et al. classification (1995) based on mouth opening. The incisional biopsy samples of 0.5 cm × 0.5 cm × 0.5 cm were obtained from the fibrotic areas of the buccal mucosa and were divided into two portions. One portion of the tissue was used for histopathological assessment and the other was transported from the clinic to the cell culture laboratory in the cell culture media which also acts as the transport media (DMEM-Dulbecco Modified Eagles Media- Sigma Aldrich). During the initial assessment of the cell culture wells, it was noticed four wells got contaminated by adipocytes due to deeper biopsy and were discarded.

The control group comprised subjects without areca nut chewing habits with clinically normal mucosa. A total of 12 biopsy samples were obtained, but due to contamination, two samples had to be discarded. The control group biopsies were obtained from the noninflamed posterior buccal mucosa during surgical extraction of the third molar.

The biopsy vials were sealed with paraffin-coated tape and were transferred from the clinic to the laminar hood in the cell culture laboratory.

Cell culture

The tissue is retrieved from the transport media and is minced into 1 mm × 1 mm × 1 mm pieces with the help of sterile BP blade no. 15 on the Petri dish containing the culture media (DMEM). Then, it is subsequently washed in Dulbecco phosphate-buffered saline (Sigma-Aldrich) supplemented with 100 u/ml gentamycin and 2.5 u/ml fungicide.

Minced tissues were further incubated into 35 wells of 60 mm size and allowed to adhere and maintain in 5 ml of DMEM supplemented with 10% bovine calf serum and gentamycin. These Petri dishes were incubated at 37° centigrade in a humidifying atmosphere of 5% carbon dioxide for 24 h in water-jacketed carbon dioxide incubator (Thermo Electron Corporation). This was followed by centrifugation at 2000 rpm for 5 min. The sediment was then plated using pipettes onto 30 mm tissue culture wells containing the working media for 48 h. This ensures the attachment of the cells to the culture plate.

The cells were later observed every alternate day under inverted microscopy during culture. In addition, the fibroblastic nature of the cells in the early passage (third passage) is confirmed by the determination of the morphological phenotypes. The primary fibroblasts were treated with 0.05% trypsin (Sigma-Aldrich) in 0.1% ethylenediaminetetraacetic acid (Nice Chemicals) containing phosphate buffer of PH 7.4; the process of trypsinization leads to the separation of the coalesced cells and helps in the passaging of the cells.

Cell counting

For the determination of growth rate and the cumulative population doubling level of the cell passage, the cells were counted in manual cell-counting chamber after staining with 0.4% trypan blue.

During these observations, the media was changed every 3rd day till 1 week, till the cells were subcultured for obtaining the next passage of cell lines to reach the confluence. These cultures were maintained at 37°C in a humidifying atmosphere of 95% air and 5% carbon dioxide in water-jacketed carbon dioxide incubator.

Passages or subculturing

The third passage was taken into consideration for the morphological assessment of the fibroblasts and the effects of arecoline (Sigma-Aldrich) and collagenase (HiMedia) on a concentration in a dose-dependent manner.

Assessment of the fibroblast subpopulation based on morphology

Cell lines from the third passage were seeded on three 30 mm tissue culture plates at a concentration of 104 and maintained for 4 days, during which they expressed their specific cell type morphology. The assessment of the morphological phenotypes was done 24 h after the revival of the cell culture plates from the ultra-freeze (New Brunswick Scientific) for both the cases and controls. An assessment of the correlation of the varied morphology of the fibroblasts in respect to the histopathological grading was done.

Morphological phenotypes of fibroblast cells

Using inverted microscope, the cells were observed under ×20 magnification and counted daily for 8 days. Fibroblast subpopulations were classified using two models. The morphology identification was based on the rat skin model where F1, F2, and F3 (F1 are spindle-shaped, F2 are epithelioid, and F3 are stellate-shaped cells) were identified as fibroblast morphological phenotypes.

Assessment of the cell count after treatment with arecoline

At various concentrations of 50 ug, 100 ug, 150 ug, 300 ug, and 500 ug of arecoline, the cell population was assessed for 8 days after obtaining the third passage of subcultured fibroblasts of cases and control tissues.

Assessment of cell count after treatment with collagenase type

Two groups of fibroblast cell lines, i.e., the controls and cases which were treated with 50 ug, 100 ug, and 150 ug of arecoline as well as the untreated groups, were subjected to 50 ul of 0.1% type 2 collagenase and were evaluated over a period of 8 days to observe the morphological forms of fibroblasts and the variation in the cell count.

RESULTS

The present study involves culturing of the human oral fibroblasts obtained from the normal buccal mucosa and OSF tissues and to know the various morphological phenotypes of the fibroblasts in those tissues and assess the response of those cultured fibroblasts to various concentrations of arecoline. The OSF tissues were histopathologically assessed and the other portion was used for culturing the oral fibroblasts.

Assessment of fibroblast cell culture lines in the study

The assessments of the response of the fibroblasts were based on the cell counts from each culture well which confirmed viable cells assayed through trypan blue staining [Figure 1a]. The cells were visualized by inverted microscope. The cell counts were determined by cell-counting chamber. The cells were subcultured from the first passage cell lines showing viable cells and it was continued till the third passage. The third passage yielded more pure cell lines of fibroblasts which were needed for assessment. The cell counts were noted after the third passage over a period of 8 consecutive days.

Figure 1

(a) Trypan blue assessment to ascertain the viability of the cell lines in the culture well. The nonviable cells uptake the stain. The lesser the stained cells, the more viable are the cell lines. (b) Spindle form of fibroblasts (F1), the most proliferative population of fibroblasts and seen in the early stages of oral submucous fibrosis. (c) Epithelioid form of fibroblasts (F2), more committed toward production of type I and III collagen. (d) Stellate form of fibroblast, least proliferative among all but produces more collagen and seen mostly in the advanced stages of oral submucous fibrosis

Morphological assessment of fibroblasts

There are three different morphological phenotypes of fibroblasts noticed in the normal buccal mucosa and the OSF tissues. It was noticed that in both the groups, the epithelioid form [Figure 1c] of the fibroblasts predominated followed by spindle-shaped fibroblasts [Figure 1b]. Stellate varieties [Figure 1d] of fibroblasts were very scanty in the cell culture wells. The F3-to-F1 ratio in OSF cell lines was 0.27, which was higher than the normal buccal mucosa fibroblast cell lines based on the mean cell count of each fibroblast form [Table 1].

Table 1

Mean cell counts of the morphological forms of fibroblasts in cases and controls

Cell form	Control*	Case*
F1	2.7	1.1
F2	39	40.6
F3	0.6	0.3

*All cell counts=n104; (n: Actual cell count). F1: Spindle-shaped cells, F2: Epithelioid cells, F3: Stellate-shaped cells

Mode of assessment of the fibroblasts response (cell count)

The response of the fibroblasts with or without the addition of arecoline and collagenase was analyzed based on the cell counts over a period of 8 days. The cells were counted based on five different high power fields (×100) and multiplied to 104.

The assessment based on varying concentrations of arecoline

Untreated fibroblasts – It was found that over a period of time, i.e., from the 1st day to the 8th day, there was an increase in the cell count in both the controls (normal buccal mucosa) and in the cases. The fibroblasts showed a progressive proliferation over a period of time.

50 ug arecoline – When this concentration of arecoline was added, there was a steep fall in the cell count levels compared to the untreated ones on the 1st day. There was a decrease in cell counts observed progressively over a period of 8 days both in the controls and cases [Figure 2]

Figure 2

Graph showing the overall assessment of the response of the fibroblasts at different concentrations of arecoline over a period of 8 days in oral submucous fibrosis sample (case)

100 ug arecoline – There was a progressive decrease in the cell counts observed over 8 days in both the groups of treated fibroblasts. The cases showed a decreased fibroblast count compared to the controls in all the days. The control cell counts showed a plateau on the 2nd and 3rd days, and there was a decrease in the cell counts which was noticed [Figure 2]

150 ug arecoline – There was a higher cell count in the control cell culture plates compared to the cases in all the days, but it was noticed that there was a progressive decrease in the cell counts in the controls and cases [Figure 2]

300 ug arecoline – There was a progressive decrease in the cell counts observed over 8 days in both the groups of treated fibroblasts. There was a slight proliferation of fibroblasts seen at all days in cases. There was a sharp inhibition of cell proliferation which was noticed from the 6th day to the 7th day and thereafter progressive decrease.

500 ug arecoline – There was a uniform inhibition of cell growth over the progressive days in the controls and the cases. The cell count was always seen higher in the controls on all the days.

The mean cell counts in the cell lines were found to be statistically significant. (Greenhouse-Geisser Test) on the days of evaluation where (P < 0.001) [Table 2] (mentioned in the result section about statistical analysis).

Table 2

The level of changes in the mean cell counts of the individual group of cell lines over a period of 8 days as per concentration gradient

Concentration (ug/ml)	Mean value		F
	Control	Cases	Control	Cases
Untreated	766.37	477.07	18.51**	81.18**
50	1027.84	833.35	34.05**	91.017**
100	1174.87	982.37	18.09**	71.52**
150	1592.75	1279.24	23.55**	58.36**
300	2536.54	2217.49	19.79**	53.91**
500	2698.08	2118.37	18.89**	41.40**

**P<0.001 (highly significant). All cell counts=n104; (n: Actual cell count)

DISCUSSION

The pathogenesis concept was more strengthened by documenting the dose–response relationship of arecoline, the major alkaloid responsible for development of OSF. They undergo nitrosation and give rise to N-nitrosamines, which have a cytotoxic effect on the cells. Arecoline, the most abundant alkaloid, has been shown to influence the process of fibrosis and has been implicated in the pathogenesis of OSF.[789101112]

In our study, the maximum count of fibroblasts was of epithelioid form (F2) followed by spindle forms (F1) of fibroblasts in both the controls and OSF cases. This could be because of the fact that most of the cases belonged to early OSF and moderately advanced stages; the fibroblasts were still in the proliferative stage where most of the spindle cells gave rise to F2 population of cells. F3 are the terminal cells which are responsible for synthesizing high amounts of collagen were very scanty because of the consideration of the third passage of cell lines where the spindle form of fibroblasts probably has transformed to epithelioid variety over progressive subculturing.

The F3-to-F1 ratio in OSF cell lines was 0.27, which was higher than the normal buccal mucosa fibroblast cell lines, which was found to be consistent with previous studies where F3 population was found to higher in OSF than in F1 which signified the prevalence of senescent fibroblasts in OSF. The cell populations were evaluated during initial days of cell culture of third passage but an evaluation of morphological phenotype was not done over a period of 8 days.[3131415]

Fibroblast population derived from the normal buccal mucosa and OSF cases exhibited similar response on getting exposed to 10 ug/ml of arecoline. The population doubling time was also almost similar in the cell culture wells.[1314151718] We also saw a similar response in the fibroblasts of the controls as well as the OSF cases when they were exposed to different concentrations of arecoline. In the untreated groups of fibroblasts, the cell count of the normal buccal mucosa was found to be slightly higher than the OSF cases.[19202122] This is probably due to decreased sensitivity of the OSF fibroblasts that have already got exposed to different components of areca nut.

The salivary concentrations of arecoline were found to be ranging from 5.66 ug/ml to 97.39 ug/ml while chewing areca nut.[19] However, the concentration drastically gets reduced upon reaching the tissue and that concentration actually initiates the response in the fibroblasts. We have considered the same principle of bioavailability and used different concentrations of arecoline ranging from 50 to 500 ug/ml. The concentrations of arecoline, i.e., from 50 ug/ml to 500 ug/ml, were taken into consideration for our study on noting the salivary concentrations of arecoline stated in the previous studies.[1920] We tried to hypothesize the situation of the OSF condition where arecanut was chewed over years and over a period of time the tissue level concentrations of arecanut increased.

In similar studies which used cultured human buccal mucosa fibroblasts and further treated with 50 ug/ml, 100 ug/ml, 150 ug/ml, 300 ug/ml, and 500 ug/ml of arecoline, it was found that the response of the cultured fibroblasts was dose dependent. The concentrations 50–100 ug/ml showed inhibited cell growth followed by cytotoxicity and cell death.[2122] It is interesting to note this response of the fibroblasts to varying concentrations of arecoline; two-phased response seems to be evident. In the initial phase, there is inhibition of cell proliferation as noted by a progressive but limited decrease in cell count. At higher concentrations of 150 ug/ml of arecoline, the decrease was rapid; this is the phase of cytotoxicity. The clinical behavior of the disorder is consistent with the cellular changes.

The fibroblasts derived from kidney and buccal mucosa showed different responses on a particular concentration of arecoline.[2324] Hence, it can be concluded that the response of the fibroblasts varies depending upon the origin or site. At 100 ug/ml concentrations of arecoline, there was an increased cell count noticed at all days followed by a fall in cell count. This phenomenon can take place when the population of fibroblasts tries to overcome the toxic effects of arecoline and shows proliferation to adapt to the condition or arecoline shows slight stimulation of fibroblasts till it is treated with 100 ug/ml concentration of arecoline.

There was an interesting fact that we can derive from our observation that the fibroblast cell lines which were treated with higher concentrations of arecoline simulate the condition when the patient chews areca nut over the years and the tissue level arecoline increases. Hence, cultured fibroblasts were also exposed to higher concentrations of arecoline, i.e., 150 ug/ml, 300 ug/ml, and 500 ug/ml of arecoline, and it was found that these concentrations caused cytotoxicity and progressive cell death. Cytotoxicity creates a self-limiting environment which leads to perpetuation and stabilization of fibrosis condition. Inhibition of cell proliferation and cellular toxicity may also lead to reduced remodeling of collagen fibers and help in the progression of fibrosis condition.[25]

Obtaining primary cell lines of fibroblasts from the OSF tissues was very challenging as they were susceptible to contaminations of the media as well as from environment. Hence, with the cell lines, we could obtain in our study we conclude that there exist different morphological forms of fibroblasts and it is tissue specific. There was a dose-dependent response of arecoline seen on the cultured fibroblasts over a period of 8 days.

CONCLUSION

The experiment was carried out only after obtaining the viable population of cells in the normal buccal mucosa fibroblast cell lines as well as the OSF fibroblast cell lines. The response of the fibroblasts of those two groups of cell lines differed at various concentrations of arecoline ranged from 50 ug/ml to 500 ug/ml. The untreated cell lines of the control tissues showed greater proliferation compared to the OSF cases over a period of 8 days. The cells when treated with various concentrations of arecoline showed a decrease in the cell count over the period which was dose dependent, but the cell lines of the OSF cases showed a slight peak of cell proliferation at 300 ug/ml of arecoline. This could be because of the variability and the sensitivity of the fibroblasts or the development of arecoline receptors in the fibroblasts in the cell lines from OSF samples to the different concentrations of arecoline. The rate of decrease in the cell colony count seems to indicate the action of arecoline concentrations which were initially inhibitory then frankly cytotoxic. Based on the reaction of the controls and OSF cell lines to different concentrations of arecoline, it is safe to assume that differential cell lines of fibroblasts are produced in OSF which react in a different manner to arecoline as compared to controls.

There appears to be incontrovertible proof that arecoline is a potent stimulator of fibroblasts and primary compound in the development of OSF. Attempts to observe these pathways would be one of the primary steps in the targeted therapy of this disorder.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.