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Journal of the Formosan Medical Association (2015) xx, 1e8
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.jfma-online.com
ORIGINAL ARTICLE
Citrus polyphenol for oral wound healing in oral ulcers and periodontal diseases Hsiao-Cheng Tsai a,b, Yi-Chen Li a, Tai-Horng Young a,*, Min-Huey Chen b,** a Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan, ROC b Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, Taipei, Taiwan, ROC
Received 16 September 2014; received in revised form 8 November 2014; accepted 5 January 2015
KEYWORDS citrus polyphenol; proliferation; wound healing
Background/purpose: Various polyphenolic compounds from plants have been confirmed to have different pharmaceutical functions. The purpose of this study was to evaluate citrus polyphenol (CP) for dental applications. A medium with CP was developed to improve oral wound healing. The CP could be used as a supplemental compound in mouthwash for periodontal diseases. Method: In this study, the metabolic activity and cell toxicity of CP (1%, 0.1%, and 0.01%) for fibroblasts were investigated by MTT and lactate dehydrogenase assays (n Z 6). The effect of CP on motility of fibroblast was also evaluated via a wound healing model. Results: The growth of Hs68 cells on TCPS was greatly increased in the presence of 0.01% CP. In addition, the significant difference (p < 0.01) of cell toxicity of fibroblast was observed after 6 days in 0.01% CP medium. Using the wound healing model, it was also found that CP could enhance the migratory ability of fibroblasts. Conclusion: The results confirm the feasibility of CP be a supplemental compound in mouthwash for treatment of periodontal diseases in dental application to improve wound healing in the mouth. Copyright ª 2015, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.
Conflicts of interest: The authors have no conflicts of interest relevant to this article. * Corresponding author. Tai-Horng Young, Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, No 1, Sec. 1, Jan-Ai Rd., Taipei 100, Taiwan, ROC. ** Corresponding author. Min-Huey Chen, Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, No 1, Changde St., Taipei, Taiwan, ROC. E-mail addresses: [email protected] (T.-H. Young), [email protected] (M.-H. Chen). http://dx.doi.org/10.1016/j.jfma.2015.01.003 0929-6646/Copyright ª 2015, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.
Please cite this article in press as: Tsai H-C, et al., Citrus polyphenol for oral wound healing in oral ulcers and periodontal diseases, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/j.jfma.2015.01.003
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Introduction Oral ulceration is a common disease in adults. Ulcers are a common oral condition, and cause pain and discomfort to patients.1 There are three clinical forms: small aphthous, large, and herpetiform ulceration.2 In general, small ulceration will regenerate in < 1 week, but, when the ulcer is > 1 cm in diameter, the healing time may be up to 1 month.3 Thus, rapidly reducing wounds in the mouth is an important issue.4,5 Previous studies show that oral physical injury, or vitamin or folic acid, etc. deficiency are the etiologic factors causing oral ulceration.6e8 Traditionally, chemical cauterization, supplying vitamins, or local corticosteroids are used to reduce the ulcer size and shorten wound healing time.1,9,10 Recently, some plant compounds, such as Astragalus membranaceus and Rehmannia glutinosa, have also been regarded as new supplements for different diseases, enhancing fibroblast proliferation in wound healing,11,12 Sinomenine has an anti-inflammatory function.13 Noticeably, various polyphenolic compounds from plants have been confirmed to have different pharmaceutical functions.14,15 Previous studies show that: high molecular weight polyphenol from black tea can reverse the epithelialemesenchymal transition of human oral cancer cells16; polyphenols extracted from pu-erh tea or blueberry regulate the proliferation and differentiation of 3T3-L1 preadipocytes17,18; tea polyphenol has an antioxidant function and induces neuron-like differentiation of mesenchymal stem cells19; and polyphenol-rich strawberry extracts increase mitochondrial activity and regenerative capacity for cells.20 In addition, previous studies show that flavonoids present in citrus polyphenol (CP) could prevent inflammatory, cardiovascular, or neurodegenerative diseases.21e29 Therefore, we want to understand whether CP could be a supplement compound of mouthwash to affect wound healing for oral ulceration. In this study, a citrus polyphenolic compound was used as a supplement to improve the proliferation and migration capacities of fibroblast. The metabolic activity and cell toxicity of CP were investigated. The effect of CP on cellular motility was also evaluated via a scratch assay model.
Materials and methods CP The CP used in this study was purchased from company (Sinetrol-WS SWS130218, Fytexia, France).
Culture of Hs68 cell with or without CP The protocols were modified for cell culture from previous studies.30,31 The cell line used in this study was a human newborn foreskin fibroblast cell line, Hs68 (BCRC number: 60 038). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS; Biological Industries, Beit-Haemek, Israel), with the culture dish incubated at 37 C in an atmosphere of 95% air and 5% CO2. The cells reached confluence within 3e5 days, and were
H.-C. Tsai et al. then rinsed in phosphate buffered saline (PBS), detached by trypsinization with 0.05% trypsin for 5 minutes at 37 C, and centrifuged (1500 rpm). Subsequently, cells were suspended in culture medium, and seeded at approximately 10,000 cells/cm2 in a 24-well culture plate (TCPS; Corning, New York, NY, USA). Four hours after seeding, when all cells adhered to the culture plate, the culture medium was changed to DMEM containing 10% FBS in the presence or absence of 1%, 0.1%, or 0.01% CP. The fresh medium was changed every day for all experiments in this study. Cell morphology was observed under a DMI600 inverse phase contrast microscope (Leica, Wetzlar, Germany).
Analyzing the cell activity effect of CP on Hs68 by MTT assay The effect of CP on cell survival was evaluated by cellular ability to reduce 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, SigmaeAldrich, St Louis, MO, USA).32 Since mitochondrial dehydrogenases of viable cells cleave selectively to the tetrazolium ring, yielding blue/ purple formazan crystals, the level of the reduction of MTT into formazan can reflect the level of cell metabolism.33 For the MTT assay, the culture medium was removed after a predetermined culturing time, and then cells were incubated with 0.2 mL of MTT (5 mg/mL in PBS) for 3 hours at 37 C. After incubation, the medium was aspirated and the formazan reaction products were dissolved in dimethyl sulfoxide in PBS and shaken for 15 minutes. The optical density of the formazan solution was read on an enzymelinked immunosorbent assay plate reader (M2e, Molecular Devices) at 570 nm. All samples were assayed in six experiments with comparable results.
Analyzing the cell toxicity of CP on Hs68 by lactate dehydrogenase assay In order to evaluate the cell toxicity of CP, Hs68 cells with a density of 1 104/cm2 were plated in 24-well cell culture plates (TCPS) in DMEM medium in the presence or absence of CP under 37 C incubation. After 4 hours, 3 days, and 6 days of incubation, about 100 mL/well supernatants were collected and added to a new 96-well plate and then 100 mL of lactate dehydrogenase (LDH) assay solution was added to each well for 30 minutes at 37 C. Then, 50 mL of 1N HCl was added. The absorbance was recorded by ELISA plate reader (M2e, Molecular Devices) at 490 nm and 630 nm. All samples were assayed in six experiments with comparable results.
The cell motility of Hs68 evaluated by scratch assay According to a protocol detailed previously, scratch assays were prepared using confluent cell monlolayers.34e37 Hs68 cells were cultured in DMEM containing 10% FBS and cultured until confluence. In order to create a rectangular scratch, the monolayer was gently scratched with a pipette tip. Then, culture medium and detached cells were removed. The scratched well was washed twice with PBS and fresh medium in the presence or absence of CP were added; and then, the cells would be cultured for 16 hours (short term) or 3 days (long term). Cell motility was
Please cite this article in press as: Tsai H-C, et al., Citrus polyphenol for oral wound healing in oral ulcers and periodontal diseases, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/j.jfma.2015.01.003
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Citrus polyphenolic compound for dentistry measured using an inverse phase-contrast time-lapse microscopic system (Leica DMI600).
Statistical analysis All statistical analysis was performed using one-way analysis of variance followed by post hoc procedure (Duncan’s test) with p < 0.05 or p < 0.01 considered significant for all tests.
3 started to attach on TCPS and the morphology of Hs68 cells after 4 hours’ incubation. After 3 days’ incubation, a growth of Hs68 cell in the medium without or with CP (0.1% and 0.01%) could be observed and it displayed a flat and polygon shape; it revealed that the number of Hs68 cells increased when cultured on TCPS in medium containing 0.1% or 0.01% CP. By contrast, in the medium containing 1% CP, the number of HS68 cells did not increase. Furthermore, apart from the 1% CP group, Hs68 cells could continuously grow and then reach confluence after 6 days of incubation.
Results
MTT and LDH assays
Behavior of Hs68 cells cultured on TCPS in the presence or absence of CP
The MTT assay relies on the ability of the viable cells to reduce a water-soluble yellow dye to a water-insoluble purple formazan product. Fig. 2A shows the MTT conversion of Hs68 cells cultured on TCPS in the presence or absence of CP medium. The effects of medium with different concentration on the MTT reduction activity of cells was observed that the MTT conversion of Hs68 cells in medium with 0.01% CP was significantly higher than that in medium
The effects of Hs68 cells cultured on TCPS in the presence or absence of CP were investigated. As shown in Fig. 1, the behaviors of Hs68 cells cultured on TCPS in the presence or absence of CP were compared. The results show that Hs68 cells in medium containing different concentrations of CP
Figure 1 Optical photomicrographs of Hs68 cells cultured on TCPS in medium in the absence or presence of citrus polyphenol at (A) 0, (B) 3, and (C) 6 days of incubation. Scale bar Z 100 mm. Please cite this article in press as: Tsai H-C, et al., Citrus polyphenol for oral wound healing in oral ulcers and periodontal diseases, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/j.jfma.2015.01.003
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without CP at 6 days of incubation. The MTT reduction activity of cells in the presence or absence of CP exhibited a significantly stable increase at all time points, respectively. These results indicate that medium with 0.01% CP is favorable for the proliferation of Hs68 cells. Next, the toxic effect of CP on the cultured cell was quantitatively determined by measuring the release of LDH into the culture medium in the same experiments described for the reduction of MTT. As shown in Fig. 2B, the release of LDH of HS68 in medium containing different concentrations of CP (0.01% and 0.1 %) was lower than that in medium without CP. The significant difference (p < 0.01) was observed after 6 days in culture. This was consistent with the result of Fig. 2A that the growth of Hs68 cells on TCPS was greatly increased in the presence of CP. This suggests that medium with CP increases the cell number and is not toxic for HS68 cell. MTT 0% 1% 0.1% 0.01%
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Scratch assay for Hs68 cells in the presence or absence of CP The wound healing ability of Hs68 cells was tested by the scratch assay. We characterized the movement of confluent Hs68 cells in a TCPS well in the presence or absence of CP after being scratched. After scratching a confluent cell monolayer, it could be observed that the first cell of Hs68 cells in CP-free medium or medium containing 0.1% CP started to migrate into the scratched site after 6 hours (Fig. 3A and B, white arrow). The first migratory Hs68 cells in 0.01% CP medium in scratched site were also observed after 4 hours’ incubation (Fig. 3C, white arrow). By contrast, the Hs68 cells would start to migrate in 6 hours after scratching. Subsequently, the movement of Hs68 cells continued to be observed from 6 hours to 16 hours. Fig. 3A also shows that there were a few cells to be observed in the scratched site at 16 hours. Compared with CP-free medium, this test clearly revealed that more mixed-shape (polygonal and spindly) Hs68 cells in CP medium migrated from edge of scratched site during 16 hours’ incubation, as shown in Fig. 3B and C. Quantitative analysis showed that the migratory cell numbers in CP-free, 0.1%CP, and 0.01% CP medium were 18 1, 21 2, and 58 2, respectively. In addition, the number of migratory Hs68 cells in 0.01% CP medium was three times more than that in the absence of CP, as shown in Fig. 3D. Fig. 4 shows that Hs68 cells in 0.01% CP medium not only gradually migrated into scratch site but also was faster than in CP-free medium to close the scratch after 3 days of incubation. The scratch assay suggests that Hs68 cells cultured in medium containing 0.01% CP have better migratory ability than those in CP-free medium.
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Figure 2 (A) Metabolic activity and (B) LDH activity of Hs68 cells cultured in the absence or presence of CP on TCPS after 6 days of incubation was assayed by MTT and LDH. Data were determined from six independent cultures and expressed as mean standard deviation. Asterisks indicate significant difference from the medium without CS (p < 0.01) as determined by one-way ANOVA followed by Duncan’s test.
Fibroblasts play an important role in oral wound healing. In wound healing, fibroblasts proliferate and migrate into the wound site to repair it.38 Previous studies show that the behavior of fibroblasts could be regulated by different nutrition, growth factors, and cytokines to induce proliferation, differentiation, and migration during wound healing. It is also reported that increasing the number of fibroblasts could improve wound healing in an experimental model.39 Therefore, promoting proliferation and migration of fibroblasts is very important. In the present study, CP was used to supplement Hs68 cells. After 6 days of incubation, most Hs68 cells in the 0.01% CP medium displayed a polygon (active state), reaching a confluent morphology, transferring into a spindle fibroblast phenotype (inactive state; Fig. 1). The effects of CP on metabolic activity and toxicity of Hs68 cell were evaluated. In these experiment conditions, apart from the 1% CP group, the MTT reduction activity of cells in other conditions exhibited a significantly stable increase at all time points (Fig. 2A). Subsequently, the LDH assay showed that 0.1% or 0.01% CP treatment did not have toxicity to cells (Fig. 2B). These results are consistent with the morphology of Fig. 1, showing that Hs68 cell growth is inhibited at a high concentration of CP (1%). However, it is reasonable to suggest that Hs68 cells cultured in medium
Please cite this article in press as: Tsai H-C, et al., Citrus polyphenol for oral wound healing in oral ulcers and periodontal diseases, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/j.jfma.2015.01.003
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Figure 3 Dynamical process of in vitro scratch assay with Hs68 cells in medium in the (A) absence or presence of (B) 0.1% or (C) 0.01% CP on the scratched well in 16 hours of incubation. Scale bar Z 100 mm. (D) Average numbers of migratory Hs68 cells were counted in the scratch site at 16 hours of incubation. Please cite this article in press as: Tsai H-C, et al., Citrus polyphenol for oral wound healing in oral ulcers and periodontal diseases, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/j.jfma.2015.01.003
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Figure 4 Optical photomicrographs of in vitro scratch assay with Hs68 cells in medium in the (A) absence or presence of (B) 0.1% or (C) 0.01% CP on the scratched well after 3 days of incubation. Scale bar Z 100 mm.
containing 0.1% and 0.01% CP could survive and it did not inhibit proliferation of cells. In addition, many studies show that polyphenols, such as from strawberries,20 have an antioxidant function for cells.40e42 CP could also have effect against oxidative stress43; this suggests that the antioxidant property is one of the reasons for CP enhancing mitochondrial activity. Previous studies showed that fibroblasts would proliferate, migrating into the wound site in the short term.44,45 Therefore, the wound healing abilities of Hs68 cells was analyzed by a scratch assay. After scratching confluent monolayer cells, the first migratory cell of Hs68 cells in 0.01% CP medium migrated into the scratched site at 4 hours; the cell morphology at the scratched edge appeared as a polygonal shape (Fig. 3). According to previous study, after sensing the wound created, Hs68 cells will transfer into an active polygonal phenotype.37 This suggests that 0.01% CP medium could induce fibroblast into an active state earlier than medium without CP. Following the wound healing process, more Hs68 cells migrate into the scratched site after 16 hours of incubation. Compared with medium without CP, the cell number in the scratched site in CP medium (21 2 and 58 2) was higher than that in medium without CP (18 1); indeed with 0.01% CP, the cell number was three times as high as that without CP, as shown in Fig. 3. These short-term scratch assay results suggests that 0.01% CP could be a medium for promoting migration of fibroblasts in a wound healing model in vitro.
Previous study showed that, in many cases, oral ulceration usually heals without any treatment in 7e14 days.46,47 Therefore, we continuously observed the wound healing of medium with or without CP for 3 days. Clearly, the wound in the medium with 0.01% CP were nearly filled only after 2 days (Fig. 4). Compared with CP-free medium, this reveals that 0.01% CP increases wound repair rate. Only in the 0.01% CP medium group was the wound site closed completely. This suggests that 0.01% CP could accelerate the fibroblast healing time.
Conclusion This study demonstrated that as a supplement for fibroblast culture, CP could induce the proliferation and migration of fibroblast cells. In addition, CP also accelerated the wound healing time when a wound was created in vitro. These results are very encouraging since this information should be useful for the growth of strategies for enhancing the healing of wound in fibroblast in oral injury.
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Journal of the Formosan Medical Association (2015) xx, 1e8
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.jfma-online.com
ORIGINAL ARTICLE
Citrus polyphenol for oral wound healing in oral ulcers and periodontal diseases Hsiao-Cheng Tsai a,b, Yi-Chen Li a, Tai-Horng Young a,*, Min-Huey Chen b,** a Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan, ROC b Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, Taipei, Taiwan, ROC
Received 16 September 2014; received in revised form 8 November 2014; accepted 5 January 2015
KEYWORDS citrus polyphenol; proliferation; wound healing
Background/purpose: Various polyphenolic compounds from plants have been confirmed to have different pharmaceutical functions. The purpose of this study was to evaluate citrus polyphenol (CP) for dental applications. A medium with CP was developed to improve oral wound healing. The CP could be used as a supplemental compound in mouthwash for periodontal diseases. Method: In this study, the metabolic activity and cell toxicity of CP (1%, 0.1%, and 0.01%) for fibroblasts were investigated by MTT and lactate dehydrogenase assays (n Z 6). The effect of CP on motility of fibroblast was also evaluated via a wound healing model. Results: The growth of Hs68 cells on TCPS was greatly increased in the presence of 0.01% CP. In addition, the significant difference (p < 0.01) of cell toxicity of fibroblast was observed after 6 days in 0.01% CP medium. Using the wound healing model, it was also found that CP could enhance the migratory ability of fibroblasts. Conclusion: The results confirm the feasibility of CP be a supplemental compound in mouthwash for treatment of periodontal diseases in dental application to improve wound healing in the mouth. Copyright ª 2015, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.
Conflicts of interest: The authors have no conflicts of interest relevant to this article. * Corresponding author. Tai-Horng Young, Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, No 1, Sec. 1, Jan-Ai Rd., Taipei 100, Taiwan, ROC. ** Corresponding author. Min-Huey Chen, Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, No 1, Changde St., Taipei, Taiwan, ROC. E-mail addresses: [email protected] (T.-H. Young), [email protected] (M.-H. Chen). http://dx.doi.org/10.1016/j.jfma.2015.01.003 0929-6646/Copyright ª 2015, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.
Please cite this article in press as: Tsai H-C, et al., Citrus polyphenol for oral wound healing in oral ulcers and periodontal diseases, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/j.jfma.2015.01.003
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Introduction Oral ulceration is a common disease in adults. Ulcers are a common oral condition, and cause pain and discomfort to patients.1 There are three clinical forms: small aphthous, large, and herpetiform ulceration.2 In general, small ulceration will regenerate in < 1 week, but, when the ulcer is > 1 cm in diameter, the healing time may be up to 1 month.3 Thus, rapidly reducing wounds in the mouth is an important issue.4,5 Previous studies show that oral physical injury, or vitamin or folic acid, etc. deficiency are the etiologic factors causing oral ulceration.6e8 Traditionally, chemical cauterization, supplying vitamins, or local corticosteroids are used to reduce the ulcer size and shorten wound healing time.1,9,10 Recently, some plant compounds, such as Astragalus membranaceus and Rehmannia glutinosa, have also been regarded as new supplements for different diseases, enhancing fibroblast proliferation in wound healing,11,12 Sinomenine has an anti-inflammatory function.13 Noticeably, various polyphenolic compounds from plants have been confirmed to have different pharmaceutical functions.14,15 Previous studies show that: high molecular weight polyphenol from black tea can reverse the epithelialemesenchymal transition of human oral cancer cells16; polyphenols extracted from pu-erh tea or blueberry regulate the proliferation and differentiation of 3T3-L1 preadipocytes17,18; tea polyphenol has an antioxidant function and induces neuron-like differentiation of mesenchymal stem cells19; and polyphenol-rich strawberry extracts increase mitochondrial activity and regenerative capacity for cells.20 In addition, previous studies show that flavonoids present in citrus polyphenol (CP) could prevent inflammatory, cardiovascular, or neurodegenerative diseases.21e29 Therefore, we want to understand whether CP could be a supplement compound of mouthwash to affect wound healing for oral ulceration. In this study, a citrus polyphenolic compound was used as a supplement to improve the proliferation and migration capacities of fibroblast. The metabolic activity and cell toxicity of CP were investigated. The effect of CP on cellular motility was also evaluated via a scratch assay model.
Materials and methods CP The CP used in this study was purchased from company (Sinetrol-WS SWS130218, Fytexia, France).
Culture of Hs68 cell with or without CP The protocols were modified for cell culture from previous studies.30,31 The cell line used in this study was a human newborn foreskin fibroblast cell line, Hs68 (BCRC number: 60 038). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS; Biological Industries, Beit-Haemek, Israel), with the culture dish incubated at 37 C in an atmosphere of 95% air and 5% CO2. The cells reached confluence within 3e5 days, and were
H.-C. Tsai et al. then rinsed in phosphate buffered saline (PBS), detached by trypsinization with 0.05% trypsin for 5 minutes at 37 C, and centrifuged (1500 rpm). Subsequently, cells were suspended in culture medium, and seeded at approximately 10,000 cells/cm2 in a 24-well culture plate (TCPS; Corning, New York, NY, USA). Four hours after seeding, when all cells adhered to the culture plate, the culture medium was changed to DMEM containing 10% FBS in the presence or absence of 1%, 0.1%, or 0.01% CP. The fresh medium was changed every day for all experiments in this study. Cell morphology was observed under a DMI600 inverse phase contrast microscope (Leica, Wetzlar, Germany).
Analyzing the cell activity effect of CP on Hs68 by MTT assay The effect of CP on cell survival was evaluated by cellular ability to reduce 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, SigmaeAldrich, St Louis, MO, USA).32 Since mitochondrial dehydrogenases of viable cells cleave selectively to the tetrazolium ring, yielding blue/ purple formazan crystals, the level of the reduction of MTT into formazan can reflect the level of cell metabolism.33 For the MTT assay, the culture medium was removed after a predetermined culturing time, and then cells were incubated with 0.2 mL of MTT (5 mg/mL in PBS) for 3 hours at 37 C. After incubation, the medium was aspirated and the formazan reaction products were dissolved in dimethyl sulfoxide in PBS and shaken for 15 minutes. The optical density of the formazan solution was read on an enzymelinked immunosorbent assay plate reader (M2e, Molecular Devices) at 570 nm. All samples were assayed in six experiments with comparable results.
Analyzing the cell toxicity of CP on Hs68 by lactate dehydrogenase assay In order to evaluate the cell toxicity of CP, Hs68 cells with a density of 1 104/cm2 were plated in 24-well cell culture plates (TCPS) in DMEM medium in the presence or absence of CP under 37 C incubation. After 4 hours, 3 days, and 6 days of incubation, about 100 mL/well supernatants were collected and added to a new 96-well plate and then 100 mL of lactate dehydrogenase (LDH) assay solution was added to each well for 30 minutes at 37 C. Then, 50 mL of 1N HCl was added. The absorbance was recorded by ELISA plate reader (M2e, Molecular Devices) at 490 nm and 630 nm. All samples were assayed in six experiments with comparable results.
The cell motility of Hs68 evaluated by scratch assay According to a protocol detailed previously, scratch assays were prepared using confluent cell monlolayers.34e37 Hs68 cells were cultured in DMEM containing 10% FBS and cultured until confluence. In order to create a rectangular scratch, the monolayer was gently scratched with a pipette tip. Then, culture medium and detached cells were removed. The scratched well was washed twice with PBS and fresh medium in the presence or absence of CP were added; and then, the cells would be cultured for 16 hours (short term) or 3 days (long term). Cell motility was
Please cite this article in press as: Tsai H-C, et al., Citrus polyphenol for oral wound healing in oral ulcers and periodontal diseases, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/j.jfma.2015.01.003
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Citrus polyphenolic compound for dentistry measured using an inverse phase-contrast time-lapse microscopic system (Leica DMI600).
Statistical analysis All statistical analysis was performed using one-way analysis of variance followed by post hoc procedure (Duncan’s test) with p < 0.05 or p < 0.01 considered significant for all tests.
3 started to attach on TCPS and the morphology of Hs68 cells after 4 hours’ incubation. After 3 days’ incubation, a growth of Hs68 cell in the medium without or with CP (0.1% and 0.01%) could be observed and it displayed a flat and polygon shape; it revealed that the number of Hs68 cells increased when cultured on TCPS in medium containing 0.1% or 0.01% CP. By contrast, in the medium containing 1% CP, the number of HS68 cells did not increase. Furthermore, apart from the 1% CP group, Hs68 cells could continuously grow and then reach confluence after 6 days of incubation.
Results
MTT and LDH assays
Behavior of Hs68 cells cultured on TCPS in the presence or absence of CP
The MTT assay relies on the ability of the viable cells to reduce a water-soluble yellow dye to a water-insoluble purple formazan product. Fig. 2A shows the MTT conversion of Hs68 cells cultured on TCPS in the presence or absence of CP medium. The effects of medium with different concentration on the MTT reduction activity of cells was observed that the MTT conversion of Hs68 cells in medium with 0.01% CP was significantly higher than that in medium
The effects of Hs68 cells cultured on TCPS in the presence or absence of CP were investigated. As shown in Fig. 1, the behaviors of Hs68 cells cultured on TCPS in the presence or absence of CP were compared. The results show that Hs68 cells in medium containing different concentrations of CP
Figure 1 Optical photomicrographs of Hs68 cells cultured on TCPS in medium in the absence or presence of citrus polyphenol at (A) 0, (B) 3, and (C) 6 days of incubation. Scale bar Z 100 mm. Please cite this article in press as: Tsai H-C, et al., Citrus polyphenol for oral wound healing in oral ulcers and periodontal diseases, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/j.jfma.2015.01.003
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without CP at 6 days of incubation. The MTT reduction activity of cells in the presence or absence of CP exhibited a significantly stable increase at all time points, respectively. These results indicate that medium with 0.01% CP is favorable for the proliferation of Hs68 cells. Next, the toxic effect of CP on the cultured cell was quantitatively determined by measuring the release of LDH into the culture medium in the same experiments described for the reduction of MTT. As shown in Fig. 2B, the release of LDH of HS68 in medium containing different concentrations of CP (0.01% and 0.1 %) was lower than that in medium without CP. The significant difference (p < 0.01) was observed after 6 days in culture. This was consistent with the result of Fig. 2A that the growth of Hs68 cells on TCPS was greatly increased in the presence of CP. This suggests that medium with CP increases the cell number and is not toxic for HS68 cell. MTT 0% 1% 0.1% 0.01%
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Scratch assay for Hs68 cells in the presence or absence of CP The wound healing ability of Hs68 cells was tested by the scratch assay. We characterized the movement of confluent Hs68 cells in a TCPS well in the presence or absence of CP after being scratched. After scratching a confluent cell monolayer, it could be observed that the first cell of Hs68 cells in CP-free medium or medium containing 0.1% CP started to migrate into the scratched site after 6 hours (Fig. 3A and B, white arrow). The first migratory Hs68 cells in 0.01% CP medium in scratched site were also observed after 4 hours’ incubation (Fig. 3C, white arrow). By contrast, the Hs68 cells would start to migrate in 6 hours after scratching. Subsequently, the movement of Hs68 cells continued to be observed from 6 hours to 16 hours. Fig. 3A also shows that there were a few cells to be observed in the scratched site at 16 hours. Compared with CP-free medium, this test clearly revealed that more mixed-shape (polygonal and spindly) Hs68 cells in CP medium migrated from edge of scratched site during 16 hours’ incubation, as shown in Fig. 3B and C. Quantitative analysis showed that the migratory cell numbers in CP-free, 0.1%CP, and 0.01% CP medium were 18 1, 21 2, and 58 2, respectively. In addition, the number of migratory Hs68 cells in 0.01% CP medium was three times more than that in the absence of CP, as shown in Fig. 3D. Fig. 4 shows that Hs68 cells in 0.01% CP medium not only gradually migrated into scratch site but also was faster than in CP-free medium to close the scratch after 3 days of incubation. The scratch assay suggests that Hs68 cells cultured in medium containing 0.01% CP have better migratory ability than those in CP-free medium.
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Figure 2 (A) Metabolic activity and (B) LDH activity of Hs68 cells cultured in the absence or presence of CP on TCPS after 6 days of incubation was assayed by MTT and LDH. Data were determined from six independent cultures and expressed as mean standard deviation. Asterisks indicate significant difference from the medium without CS (p < 0.01) as determined by one-way ANOVA followed by Duncan’s test.
Fibroblasts play an important role in oral wound healing. In wound healing, fibroblasts proliferate and migrate into the wound site to repair it.38 Previous studies show that the behavior of fibroblasts could be regulated by different nutrition, growth factors, and cytokines to induce proliferation, differentiation, and migration during wound healing. It is also reported that increasing the number of fibroblasts could improve wound healing in an experimental model.39 Therefore, promoting proliferation and migration of fibroblasts is very important. In the present study, CP was used to supplement Hs68 cells. After 6 days of incubation, most Hs68 cells in the 0.01% CP medium displayed a polygon (active state), reaching a confluent morphology, transferring into a spindle fibroblast phenotype (inactive state; Fig. 1). The effects of CP on metabolic activity and toxicity of Hs68 cell were evaluated. In these experiment conditions, apart from the 1% CP group, the MTT reduction activity of cells in other conditions exhibited a significantly stable increase at all time points (Fig. 2A). Subsequently, the LDH assay showed that 0.1% or 0.01% CP treatment did not have toxicity to cells (Fig. 2B). These results are consistent with the morphology of Fig. 1, showing that Hs68 cell growth is inhibited at a high concentration of CP (1%). However, it is reasonable to suggest that Hs68 cells cultured in medium
Please cite this article in press as: Tsai H-C, et al., Citrus polyphenol for oral wound healing in oral ulcers and periodontal diseases, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/j.jfma.2015.01.003
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Figure 3 Dynamical process of in vitro scratch assay with Hs68 cells in medium in the (A) absence or presence of (B) 0.1% or (C) 0.01% CP on the scratched well in 16 hours of incubation. Scale bar Z 100 mm. (D) Average numbers of migratory Hs68 cells were counted in the scratch site at 16 hours of incubation. Please cite this article in press as: Tsai H-C, et al., Citrus polyphenol for oral wound healing in oral ulcers and periodontal diseases, Journal of the Formosan Medical Association (2015), http://dx.doi.org/10.1016/j.jfma.2015.01.003
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Figure 4 Optical photomicrographs of in vitro scratch assay with Hs68 cells in medium in the (A) absence or presence of (B) 0.1% or (C) 0.01% CP on the scratched well after 3 days of incubation. Scale bar Z 100 mm.
containing 0.1% and 0.01% CP could survive and it did not inhibit proliferation of cells. In addition, many studies show that polyphenols, such as from strawberries,20 have an antioxidant function for cells.40e42 CP could also have effect against oxidative stress43; this suggests that the antioxidant property is one of the reasons for CP enhancing mitochondrial activity. Previous studies showed that fibroblasts would proliferate, migrating into the wound site in the short term.44,45 Therefore, the wound healing abilities of Hs68 cells was analyzed by a scratch assay. After scratching confluent monolayer cells, the first migratory cell of Hs68 cells in 0.01% CP medium migrated into the scratched site at 4 hours; the cell morphology at the scratched edge appeared as a polygonal shape (Fig. 3). According to previous study, after sensing the wound created, Hs68 cells will transfer into an active polygonal phenotype.37 This suggests that 0.01% CP medium could induce fibroblast into an active state earlier than medium without CP. Following the wound healing process, more Hs68 cells migrate into the scratched site after 16 hours of incubation. Compared with medium without CP, the cell number in the scratched site in CP medium (21 2 and 58 2) was higher than that in medium without CP (18 1); indeed with 0.01% CP, the cell number was three times as high as that without CP, as shown in Fig. 3. These short-term scratch assay results suggests that 0.01% CP could be a medium for promoting migration of fibroblasts in a wound healing model in vitro.
Previous study showed that, in many cases, oral ulceration usually heals without any treatment in 7e14 days.46,47 Therefore, we continuously observed the wound healing of medium with or without CP for 3 days. Clearly, the wound in the medium with 0.01% CP were nearly filled only after 2 days (Fig. 4). Compared with CP-free medium, this reveals that 0.01% CP increases wound repair rate. Only in the 0.01% CP medium group was the wound site closed completely. This suggests that 0.01% CP could accelerate the fibroblast healing time.
Conclusion This study demonstrated that as a supplement for fibroblast culture, CP could induce the proliferation and migration of fibroblast cells. In addition, CP also accelerated the wound healing time when a wound was created in vitro. These results are very encouraging since this information should be useful for the growth of strategies for enhancing the healing of wound in fibroblast in oral injury.
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