“Efficacy of cytotoxic effect of green tea catechins on the human periodontal fibroblasts and human dental pulp fibroblasts -An in vitro study”

Abstract

Background:

Inflammation of tooth-supporting tissue and the pulp tissue is followed by wound healing and regeneration process that involves the specific type of connective tissue cells, the fibroblasts. During periodontitis and pulpitis, the inflammation of the tissue causes damage to the fibroblasts. These fibroblasts secrete collagen proteins and maintain the structural framework; along with this the inflammatory process moves toward healing where in the specific cells such as the fibroblast cells play important roles. Green tea catechins epigallocatechin-3-gallate (EGCG) being one of the major catechins is known to have multiple beneficial effects on human fibroblasts.

Objective:

To assess the in vitro cytotoxicity of green tea catechins on the human periodontal ligament (PDL) fibroblasts and human dental pulp fibroblasts.

Materials and Methods:

Human PDL fibroblasts (hPDLFs) and human dental pulp fibroblasts were isolated from the two extracted premolar teeth that were indicated for orthodontic treatment. The fibroblasts were then seeded in 96 well tissue culture plate for cell viability study. EGCG was used at different concentration to treat the cells. After 48 h; (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) assay was performed to determine the cell viability.

Results:

The vitality of hPDLFs and human dental pulp fibroblasts was found to be inversely proportional to EGCG concentrations.

Conclusions:

hPDLFs have shown 37% proliferation at lowest concentration of EGCG used and human dental pulp fibroblasts have shown 99% viability at lowest concentration of EGCG used.

INTRODUCTION

Fibroblast cells play an important role during the healing process. During injury to the tissue, the fibroblast migrates at the site of injury and they by facilitate the process of healing by depositing collagen and extracellular matrix. The fibroblast cells serve a role in inflammation and immune cell recruitment to the sites of tissue injury. The underlying cells, such as the fibroblasts and undifferentiated mesenchymal cells in the cell-rich zone of the pulp tissue get stimulated and actively participate in the host response, thereby commencing reparative dentin formation in the dental pulp tissue.[1-3] Similarly, in the human periodontal ligament (PDL), the connective tissue contains numerous cells, these cells including the fibroblast play as promoters of periodontal inflammation in response to the inflammation. Green tea is one of the teas that has been consumed widely and is quite a popular beverage. The catechins present in green tea are more than the other teas. Green tea contains four main catechins namely (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-EC-3-gallate, and (-)-EGC -3-gallate (EGCG). EGCG is the prevalent catechin (~60%) found in green tea. EGCG’s anti-inflammatory and antioxidant properties protect cells from oxidative stress-related damage and inhibit the production of pro-inflammatory molecules, which is why it has such a wide range of disease-prevention applications. Studies have shown the effect of green tea catechins on gingival and periodontal fibroblast. EGCG has an anti-inflammatory effect on human gingival fibroblasts.[4-6] Thereby, it is effective in preventing gingival and periodontal inflammation. Healing occurs in the pulp during the inflammation phase in pulpitis, where transforming growth factor-1 and fibroblast growth factor drive fibroblast activity and proliferation.[7,8] When there is any mechanical trauma to a tooth, the choice of treatment of such teeth would be direct pulp capping (DPC) treatment[9] where the material of choice is calcium hydroxide that has been considered as standard material, but due to some disadvantages of calcium hydroxide, newer material’s that are biocompatible should be considered. EGCG that is present in green tea seems to have more benefits on pulp healing, thereby enhancing and increasing the number of fibroblast cells as these cells play a role in wound healing. A study by Widjiastuti et al. in their study concluded that the different hydrogel EGCG concentration was effective in increasing the number of fibroblast cell proliferation when a concentration of 90 ppm was used.[10] Periodontitis is one of the most common infectious disorders. An in vitro study showed that the infected human PDL fibroblasts (hPDLFs) and human PDL stem cells (hPDLSCs) infected by a periodontal pathogen (porphyromonas gingivalis) EGCG showed anti-inflammatory effects on such fibroblast cells and found that EGCG could reduce inflammation in hPDLFs and hPDLSCs in response to P. gingivalis Lipopolysaccharide by Jung et al.[11] Based on the evidence that catechins have a beneficial effect on PDL, gingival, and pulpal cells, green tea extract application as local delivery systems in form of strips, mouthwashes, chewing gums, chips, and fibres, as regenerative materials can be used to improve pulp and periodontal tissue regeneration should be focused.

MATERIALS AND METHODS

This study was conducted in our institute.

The preparation of EGCG was done by using the purest form of Green Tea Polyphenols EGCG was purchased from Ambe NS Agro products private limited, Vasundhara Ghaziabad, Utter Pradesh. EGCG stocks were prepared by dissolving in dimethyl sulfoxide (DMSO) to concentrations of 100 mM. Phosphate-buffered saline ( PBS) was further used to dilute EGCG in to different concentration of 100 mg/ml, 75 mg/ml, 50 mg/ml, 25 mg/ml, and 10 mg/ml before using it on the fibroblast cells.[12]

hPDLFs culture was done using human PDL (HPDL) cells that was obtained from freshly extracted 3 premolar teeth that had no caries, periodontal disease or restoration from two healthy patients who were undergoing orthodontic treatment after getting informed written permission. The extracted teeth were immediately brought to the laboratory for fibroblast culture in Dulbecco’s modified eagle’s medium (DMEM) (gibco, USA).

These teeth were then carefully placed on sterile dishes to this then 10% foetal bovine serum (FBS) was supplemented along with ×100 antibiotic antimycotic to prevent any kind of bacterial and fungal contamination to the fibroblast cells. The PDL cells were then removed aseptically and carefully from two-thirds of the root surface with a scalpel blade. The tissue obtained was then transferred to a polypropylene tube containing 70% DMEM and 30% enzymes (3 mg/ml collagenase and 4 mg/ml dispase). The tube was then incubated at the room temperature for 45 min. After 45 min, it was centrifuged at 2000 rpm for 3 min, the supernatant was discarded and pellet obtained was washed with PBS (phosphate-buffered saline) and then transferred to 24 well micro titer plate containing DMEM supplemented with 10% FBS gibco, UK and 1 ml of 100x antibiotic antimycotic (Hi Media Laboratories, Mumbai, Maharashtra, India) solution. The plate was then incubated at 37°C, 5% CO₂ with 98% humidity (Thermo Scientific, Massachusetts, USA) for 48 h for fibroblast culture. After obtaining 90% confluence [Figure 1], the cells were trypsinized and seeded in 96 well plate for further study. Fourth generation cells were used for the cell viability study.

Figure 1.

Ninety percent confluence of fibroblasts

The human dental pulp fibroblast culture was done on the same teeth. After scraping off the PDL cells, the cells were transferred back to DMEM. The teeth were then subjected to access opening to obtain the pulp tissue which was transferred to DMEM. The obtained tissue was then subjected to enzymatic dissociation as above, so as to achieve fibroblast culture up to 90% confluence. Cells were then seeded in 96 well tissue culture plate [Figure 2] for cell viability study by MTT assay. These plates were incubated for 24–48 h to facilitate the attachment of the cells.

Figure 2.

Cells seeded in 96 well tissue culture plate

The cell viability was studied by using the commercially available EGCG powder which was procured from Ambe NS Agro products Ghaziabad. Ten milligrams of EGCG powder was diluted in 1 ml of DMEM and further 9 serial dilutions were prepared. In triplicates, 100 µl of each dilution was applied to a 96 well microtiter plate containing 1 × 104 cells per well. Untreated wells were used as negative control. The plates were incubated for 48 h and then subjected to MTT assay.

The MTT assay was performed using the stock solution of MTT (HiMedia^©) that was prepared by adding 5 mg of MTT powder to 1 ml of PBS (phosphate-buffered solution). The MTT conversion assessment was done using the colorimetric or spectrophotometric method to assess the test compound’s in vitro cell viability and proliferation effect. Stock solution of MTT was added to each well (20 µl, 5 mg per ml in sterile PBS) and kept for 4 h incubation. The supernatant was aspirated and the precipitated crystals of “Formazan blue” were solubilised using DMSO (100 ml) solution. The optical density was measured at 595 nm wavelength by using LISA plus. The results obtained represent the mean of three readings. Cell viability was calculated using the following formula

Formula:

RESULTS

MTT assay was done to know the effect of EGCG extract on both types of fibroblasts. The hPDLFs and human dental pulp fibroblasts when treated with EGCG extract for 48 h, it was observed that at the highest concentration (10 mg/ml) of EGCG extract the cell inhibition was 29% whereas the cell inhibition of EGCG extract at lowest concentration (0.0195) was 1%. Thus, the results showed viability of hPDLFs cells and human dental pulp fibroblasts was inversely proportional to the concentrations of EGCG, as shown in Tables 1, 2 and Figures 3, 4.

Table 1.

Cell viability and cell inhibition of human dental pulp fibroblasts after 48 h of treatment with 10 concentrations of epigallocatechin-3-gallate extract

S. No.	Concentrations (mg/mL)	EGCG extract
		Absorbance at 490 nm wavelength	Cell viability (%)	Cell inhibition (%)
1	10	0.319	71	29
2	5	0.340	76	24
3	2.5	0.367	81	19
4	1.25	0.370	82	18
5	0.625	0.372	83	17
6	0.312	0.380	84	16
7	0.156	0.389	86	14
8	0.078	0.399	88	12
9	0.039	0.409	91	11
10	0.0195	0.449	99	1
Negative control	0	0.450	100	0

S.No. – Serial number; mg/ml – Milligram/milliliter; nm – Nanometer; EGCG – Epigallocatechin-3-gallate

Table 2.

Cell viability and cell inhibition of human periodontal ligament fibroblasts after 48 h of treatment with 10 concentrations of epigallocatechin-3-gallate extract

S. No.	Concentrations (mg/mL)	EGCG extract
		Mean absorbance at 490 nm wavelength	Cell viability (%)	Cell inhibition (%)
1	10	0.091	78	22
2	5	0.092	79	23
3	2.5	0.096	83	17
4	1.25	0.099	85	15
5	0.625	0.100	86	14
6	0.312	0.104	90	10
7	0.156	0.107	92	8
8	0.078	0.122	105	−5
9	0.039	0.137	118	−18
10	0.0195	0.160	137	−37
Negative control	0	0.116	100	0

S.No. – Serial number; mg/ml – Milligram/milliliter; nm – Nanometer; EGCG – Epigallocatechin-3-gallate

Figure 3.

Line diagram depicting cell viability and cell inhibition of human dental pulp fibroblasts after 48 h of treatment with ten concentrations of EGCG extract. EGCG – Epigallocatechin-3-gallate

Figure 4.

Line diagram depicting cell viability and cell inhibition of human PDL fibroblasts after 48 h of treatment with ten concentrations of EGCG extract. EGCG – Epigallocatechin-3-gallate, PDL – Periodontal Ligament

DISCUSSION

The primary function of a fibroblast is to maintain the structural integrity within the connective tissue. Because fibroblasts can be easily isolated from many body regions, their application in the medical research allows researchers to better understand the etiology of a variety of disorders. The PDL fibroblast constitutes as major cell in the PDL, during inflammatory process of PDL there will be destruction of the fibroblast due to release of inflammatory cytokines. Yamamoto et al. study reported that the production of these inflammatory cytokines during a periodontal disease on the HPDL cells could play an important role.[13] Since the cells of PDL have the capability to proliferate and form cementum and PDL like tissues their therapeutic potential has been demonstrated in the restoration of a damaged or diseased periodontal tissue.[14] Studies have reported that EGCG improves the periodontal condition, thereby helping us in understanding the anti-inflammatory effect of EGCG.

Pulpitis is another inflammatory condition of dental pulp that can occur due to caries or because of deep cavity preparation where there can be a perforation. To protect the underlying pulp tissue, that has been damaged due to a trauma would require the use of a barrier material like calcium hydroxide which is placed directly over the damaged pulp tissue and this procedure is called as DPC where calcium hydroxide is considered as the gold standard pulp capping material for the perforated teeth. Although there have been studies that have found weaknesses with the use of calcium hydroxide.[15] In vivo, EGCG has shown to exert inhibitory effects on the pulp tissue that is inflamed and has significantly inhibited the inflammatory response of hDPSCs and apoptosis of hDPSCs caused by hypoxia injury.[16] Thus, the anti-inflammatory property of EGCG can help patients with periodontitis and periapical lesions.[17,18] in controlling the spread of inflammation.

EGCG is known to have effect in raising various cells of oral the epithelium. In a study EGCGs was found to be beneficial in raising the number of fibroblast cell proliferation in a perforated dental pulp of Wistar rats in vitro and showed its influence on the inflamed pulp tissue in rats in vivo, according to a study.[16] The potential effects of EGCG have been explored in the dental field and medical fields.[17-29] EGCG has shown to increase the lifespan of dental cells and tissues and also keep the cells stable.[24,25] One study done by Vaillancourt. et al. investigated the ability of green tea extract and EGCG on oral epithelial cells and oral keratinocytes against the effects of chemotherapeutic agent, the study concluded that the green tea extract and EGCG protect the oral mucosa against the deleterious effects of the chemotherapeutic agent irinotecan though anti-cytotoxic, anti-inflammatory, and anti-oxidative property of green tea catechins.[30]

Hence, with the above understanding properties of EGCG effects, we evaluated the EGCG effects on the proliferation of the hPDLFs cells and human dental pulp fibroblasts cells at different concentrations. When hPDLFs were treated with EGCG extract, it was observed that, at the highest concentration of EGCG, the cell viability was 78%, but at the concentration of 0.078 mg/ml, there was cell proliferation by 5% and at the lowest concentration used, the proliferation was by 37%. On the other hand, the highest concentration of EGCG used has shown the cell viability of 71% and the lowest concentration of EGCG used has shown the cell viability of 99% in the case of human dental pulp fibroblasts. There by showing more benefits in increasing the number of fibroblasts proliferation. Thus, with these results, the use of EGCG on the fibroblasts of PDL and pulp fibroblasts can be of great therapeutic strategy when restoration of damaged periodontal tissue and pulp tissue is the ultimate purpose of any therapy.

CONCLUSIONS

The current study demonstrated that EGCG showed potent proliferation of hPDLFs cells and human dental pulp fibroblasts cells in a dose-depended manner, suggesting a potential therapeutic of EGCG as a beneficial supplement in treating patients with periodontitis and pulpal inflammation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.