Publications by authors named "Mei-Chi Chang"

75 Publications

Effect of butyrate, a bacterial by-product, on the viability and ICAM-1 expression/production of human vascular endothelial cells: Role in infectious pulpal/periapical diseases.

Int Endod J 2021 Aug 22. Epub 2021 Aug 22.

School of Dentistry & Department of Dentistry, National Taiwan University Medical College and National Taiwan University Hospital, Taipei, Taiwan.

Aim: To investigate the effects of butyric acid (BA), a metabolic product generated by pulp and root canal pathogens, on the viability and intercellular adhesion molecule-1 (ICAM-1) production of endothelial cells, which are crucial to angiogenesis and pulpal/periapical wound healing.

Methodology: Endothelial cells were exposed to butyrate with/without inhibitors. Cell viability, apoptosis and reactive oxygen species (ROS) were evaluated using an MTT assay, PI/annexin V and DCF fluorescence flow cytometry respectively. RNA and protein expression was determined using a polymerase chain reaction assay and Western blotting or immunofluorescent staining. Soluble ICAM-1 (sICAM-1) was measured using an enzyme-linked immunosorbent assay. The quantitative results were expressed as mean ± standard error (SE) of the mean. The data were analysed using a paired Student's t-test where necessary. A p-value ≤0.05 was considered to indicate a statistically significant difference between groups.

Results: Butyrate (>4 mM) inhibited cell viability and induced cellular apoptosis and necrosis. It inhibited cyclin B1 but stimulated p21 and p27 expression. Butyrate stimulated ROS production and hemeoxygenase-1 (HO-1) expression as well as activated the Ac-H3, p-ATM, p-ATR, p-Chk1, p-Chk2, p-p38 and p-Akt expression of endothelial cells. Butyrate stimulated ICAM-1 mRNA/protein expression and significant sICAM-1 production (p < .05). Superoxide dismutase, 5z-7oxozeaenol, SB203580 and compound C (p <  .05), but not ZnPP, CGK733, AZD7762 or LY294002, attenuated butyrate cytotoxicity to endothelial cells. Notably, little effect on butyrate-stimulated sICAM-1 secretion was found. Valproic acid, phenylbutyrate and trichostatin (three histone deacetylase inhibitors) significantly induced sICAM-1 production (p < .05).

Conclusion: Butyric acid inhibited proliferation, induced apoptosis, stimulated ROS and HO-1 production and increased ICAM-1 mRNA expression and protein synthesis in endothelial cells. Cell viability affected by BA was diminished by some inhibitors; however, the increased sICAM-1 secretion by BA was not affected by any of the tested inhibitors. These results facilitate understanding of the pathogenesis, prevention and treatment of pulpal/periapical diseases.
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http://dx.doi.org/10.1111/iej.13614DOI Listing
August 2021

Cracked teeth: Distribution and survival at 6 months, 1 year and 2 years after treatment.

J Formos Med Assoc 2021 Apr 12. Epub 2021 Apr 12.

School of Dentistry, College of Medicine, National Taiwan University, Taipei, Taiwan; Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan; School of Dentistry, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Dentistry, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. Electronic address:

Background/purpose: The unpredictable condition of cracked teeth warrants further investigation and clinical experiences. The purpose of this study was to collect and record data on demographics, clinical characteristics, different treatment modalities and survival of cracked teeth at 6-month, 1-year and 2-year recalls.

Methods: 77 cracked teeth from 65 patients were included. Data on demographics, clinical parameters, treatment modalities and recall were collected. Binomial, multinomial and chi square tests were used for statistical analysis.

Results: Most cracked teeth occurred in patients greater than 40 years old (p < 0.01). Cracked teeth themselves were most often molars (79.22%; p < 0.01), a non-terminal tooth in the arch (62.34%; p < 0.05) and nonendodontically-treated teeth (94.81%; p < 0.01). Cracked teeth exhibited pain to percussion (63.64%, p < 0.05) or biting (74.03%; p < 0.01), and no or only positive mobility (76.62%; p < 0.01). Cracks were most often oriented in the mesiodistal direction (68.83%; p < 0.01). Higher survival rates were noted in cracked teeth lacking pre-operative pain to palpation or spontaneous pain, and with no or only positive mobility at 6-month and 1-year recalls. In vital cracked teeth, higher survival rates were noted in teeth lacking pre-operative pain to palpation and with no or only positive mobility at 2-year recalls.

Conclusion: The absence of pre-operative palpation discomfort, spontaneous pain and minimal mobility, as well as the presence of pulp vitality were associated with higher survival rates of cracked teeth at all recall times. Results are useful for diagnosis and outcomes-based treatment planning of cracked teeth.
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http://dx.doi.org/10.1016/j.jfma.2021.03.020DOI Listing
April 2021

Toxic mechanisms of Roth801, Canals, microparticles and nanoparticles of ZnO on MG-63 osteoblasts.

Mater Sci Eng C Mater Biol Appl 2021 Feb 17;119:111635. Epub 2020 Oct 17.

School of Dentistry, National Taiwan University Medical College, Taipei, Taiwan; Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan; School of Dentistry, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Dentistry, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. Electronic address:

ZnO eugenol-based materials are widely used for restoration of caries cavity, apical retrograde filling and root canal sealer. Their effects on apical bone healing await investigation. The toxic mechanisms of ZnO particles and nanoparticles to MG-63 osteoblastic cells were studied. We found the different morphology and size of various particles as observed by scanning electron microscope. Particles of Canals and Roth801 were larger than ZnO-205532 microparticles and ZnO-677450 nanoparticles. Four ZnO particles showed cytotoxicity (>25 μg/ml) as analyzed by MTT. Transmission electron microscope found intracellular vacuoles with particle content. Exposure to ZnO particles induced ROS production and cell cycle arrest as studied by DCF and propidium iodide flow cytometry. ZnO particles activated ATM, ATR, Chk1, Chk2, γ-H2AX, ERK and p38 phosphorylation as detected by immunofluorescent staining and western blotting. The protein expression of cdc2, cyclin B1 and cdc25C were decreased, whereas GADD45α and hemeoxygenase-1 (HO-1) were stimulated. ZnO particles' cytotoxicity to MG63 cells was prevented by N-acetylcysteine (NAC), but not CGK733, AZD7762, U0126 and SB203580. ZnO showed little effect on IL-8 and sICAM-1 secretion. These results indicated that ZnO particles are toxic to osteoblasts. ZnO particles' toxicity were related to ROS, and DNA damage responses, checkpoint kinases, cell cycle arrest, ERK and p38 signaling, but not IL-8 and ICAM-1. These results were useful for materials' development and promote apical healing. Dentists should avoid of extruding ZnO-based sealers excessively over root apex and prevent residual ZnO-based retrograde filling materials in apical area during endodontic practice.
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http://dx.doi.org/10.1016/j.msec.2020.111635DOI Listing
February 2021

Regulation of the regenerative activity of dental pulp stem cells from exfoliated deciduous teeth (SHED) of children by TGF-β1 is associated with ALK5/Smad2, TAK1, p38 and MEK/ERK signaling.

Aging (Albany NY) 2020 11 4;12(21):21253-21272. Epub 2020 Nov 4.

Department of Dentistry, National Taiwan University Hospital, and School of Dentistry, National Taiwan University Medical College, Taipei, Taiwan.

Transforming growth factor-β1 (TGF-β1) regulates wound healing/regeneration and aging processes. Dental pulp stem cells from human exfoliated deciduous teeth (SHED) are cell sources for treatment of age-related disorders. We studied the effect of TGF-β1 on SHED and related signaling. SHED were treated with TGF-β1 with/without pretreatment/co-incubation by SB431542, U0126, 5Z-7-oxozeaenol or SB203580. Sircol collagen assay, 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay, alkaline phosphatase (ALP) assay, RT-PCR, western blotting and PathScan phospho-ELISA were used to measure the effects. We found that SHED expressed ALK1, ALK3, ALK5, TGF-RII, betaglycan and endoglin mRNA. TGF-β1 stimulated p-Smad2, p-TAK1, p-ERK, p-p38 and cyclooxygenase-2 (COX-2) protein expression. It enhanced proliferation and collagen content of SHED that were attenuated by SB431542, 5Z-7-oxozeaenol and SB203580, but not U0126. TGF-β1 (0.5-1 ng/ml) stimulated ALP of SHED, whereas 5-10 ng/ml TGF-β1 suppressed ALP. SB431542 reversed the effects of TGF-β1. However, 5Z-7-oxozeaenol, SB203580 and U0126 only reversed the stimulatory effect of TGF-β1 on ALP. Four inhibitors attenuated TGF-β1-induced COX-2 expression. TGF-β1-stimulated TIMP-1 and N-cadherin was inhibited by SB431542 and 5Z-7-oxozeaenol. These results indicate that TGF-β1 affects SHED by differential regulation of ALK5/Smad2/3, TAK1, p38 and MEK/ERK. TGF-β1 and SHED could potentially be used for tissue engineering/regeneration and treatment of age-related diseases.
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http://dx.doi.org/10.18632/aging.103848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695363PMC
November 2020

Genetic Susceptibility and Protein Expression of Extracellular Matrix Turnover-Related Genes in Oral Submucous Fibrosis.

Int J Mol Sci 2020 Oct 30;21(21). Epub 2020 Oct 30.

School of Dentistry, National Taiwan University Medical College, Taipei 100, Taiwan.

Betel quid (BQ) chewing increased the risk of oral cancer and oral submucous fibrosis (OSMF), an oral premalignant disorder (OPMD) with malignant transformation potential. BQ components such as areca nut (AN), trauma by coarse AN fiber, catechin, copper, alkaloids, stimulated reactive oxygen species (ROS), inflammation and cytotoxicity are suggested to be the contributing factors. They may induce tissue inflammation, proliferation of fibroblasts and collagen deposition, myofibroblast differentiation and contraction, collagen cross-links and inhibit collagen phagocytosis, finally leading to the development of OSMF and oral cancer. These events are mediated by BQ components-induced changes of extracellular matrix (ECM) turnover via regulation of TGF-β1, plasminogen activator inhibitor-1 (PAI-1), cystatin, lysyl oxidase (LOX) and tissue inhibitors of metalloproteinases (TIMPs) and metalloproteinases (MMPs). Genetic susceptibility is also involved in these disease processes. Further understanding the molecular mechanisms of BQ-induced OSMF and oral cancer can be helpful for future disease prevention and treatment.
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http://dx.doi.org/10.3390/ijms21218104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663238PMC
October 2020

Effect of bFGF on the growth and matrix turnover of stem cells from human apical papilla: Role of MEK/ERK signaling.

J Formos Med Assoc 2020 Nov 10;119(11):1666-1672. Epub 2020 Jan 10.

School of Dentistry, National Taiwan University Medical College, Taipei, Taiwan; Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan. Electronic address:

Background/purpose: Basic fibroblast growth factor (bFGF) exhibits multiple biological functions in various tissues. Stem cells from apical papilla (SCAP) can be isolated from human apical papilla tissues in developmental teeth of children. The purposes of this study were to investigate the expression of FGF receptors (FGFRs) and the effects of bFGF on SCAP and related MEK/ERK signaling.

Methods: SCAP cells were treated under different concentrations of bFGF with or without U0126 (an inhibitor of MEK/ERK). Expression of FGFR1 and FGFR2 in SCAP was analyzed by RT-PCR. Cell proliferation was measured by MTT assay. The expressions of type I collagen, cdc 2, cyclin B1, TIMP-1 and p-ERK proteins were examined by Western blot.

Results: SCAP cells expressed FGFR1 and FGFR2. Exposure of SCAP to bFGF enhanced cell proliferation, and the expression cyclinB1, cdc 2, and TIMP-1, but not type I collagen. U0126 pretreatment and co-incubation attenuated the bFGF-induced proliferation, cdc2, cyclin B1 and TIMP-1 proteins' expression, but not type I collagen in SCAP.

Conclusion: SCAP cells express FGFRs. bFGF may stimulate proliferation and affect the matrix turnover of SCAP cells, possibly via stimulation of FGFRs and MEK/ERK signaling pathway. These results are useful for clinical therapies for apexogenesis and regeneration of pulpo-dentin complex.
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http://dx.doi.org/10.1016/j.jfma.2019.12.013DOI Listing
November 2020

Cytotoxicity and genotoxicity of DMABEE, a co-photoinitiator of resin polymerization, on CHO-K1 cells: Role of redox and carboxylesterase.

J Biomed Mater Res B Appl Biomater 2020 07 27;108(5):2088-2098. Epub 2019 Dec 27.

Department of Endodontics, School of Dentistry, College of Medicine, National Taiwan University, Taipei, Taiwan.

The 4-dimethylaminobenzoic acid ethyl ester (DMABEE) is an important co-initiator for resin polymerization in dental resinous materials. As a radical forming chemical with high lipophilicity, the genotoxicity and cytotoxicity of DMABEE deserve prudent investigation. In this study, we found that DMABEE reduced the viability and proliferation of Chinese hamster ovary (CHO-K1) cells in a dose-dependent manner, and altered cell morphology at higher concentrations. G0/G1 cell cycle arrest was induced by DMABEE at 0.25-0.75 mM, and cell proportion of sub-G0/G1 phase was significantly elevated at 1 mM while cell apoptosis was observed. Genotoxic effect was noted when cells were treated by 0.1 mM DMABEE, as revealed by increase of micronucleus formation. Reactive oxygen species overproduction was observed as cells treated with 0.75 and 1 mM, while elevation of intracellular glutathione was noticeable since 0.1 mM. Contrary to our expectation, pretreatment by N-acetyl-l-cysteine enhanced the toxicity of DMABEE on CHO-K1 cells. Catalase mildly reduced the toxic effect and carboxylesterase showed obvious ability to reverse the toxicity of DMABEE. These findings highlight the mechanism of DMABEE toxicity and provide clues for safety improvement of its application in clinical dental treatment.
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http://dx.doi.org/10.1002/jbm.b.34547DOI Listing
July 2020

Stimulation of MMP-9 of oral epithelial cells by areca nut extract is related to TGF-β/Smad2-dependent and -independent pathways and prevented by betel leaf extract, hydroxychavicol and melatonin.

Aging (Albany NY) 2019 12 12;11(23):11624-11639. Epub 2019 Dec 12.

School of Dentistry, National Taiwan University Medical College, and Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan.

Background: There are 200-600 million betel quid (BQ) chewers in the world. BQ increases oral cancer risk. Matrix metalloproteinase-9 (MMP-9) is responsible for matrix degradation, cancer invasion and metastasis. Whether areca nut extract (ANE), a BQ component, stimulates MMP-9 secretion, and the related signaling pathways awaits investigation.

Results: ANE (but not arecoline) stimulated MMP-9 production of gingival keratinocytes and SAS cancer epithelial cells. ANE stimulated TGF-β1, p-Smad2, and p-TAK1 protein expression. ANE-induced MMP-9 production/expression in SAS cells can be attenuated by SB431542 (ALK5/Smad2 inhibitor), 5Z-7-Oxozeaenol (TAK1 inhibitor), catalase, PD153035 (EGFR tyrosine kinase inhibitor), AG490 (JAK inhibitor), U0126 (MEK/ERK inhibitor), LY294002 (PI3K/Akt inhibitor), betel leaf (PBL) extract, and hydroxychavicol (HC, a PBL component), and melatonin, but not by aspirin.

Conclusions: AN components contribute to oral carcinogenesis by stimulating MMP-9 secretion, thus enhancing tumor invasion/metastasis. These events are related to reactive oxygen species, TGF-β1, Smad2-dependent and -independent signaling, but not COX. These signaling molecules can be biomarkers of BQ carcinogenesis. PBL, HC and melatonin and other targeting therapy can be used for oral cancer treatment.

Methods: ANE-induced MMP-9 expression/secretion of oral epithelial cells and related TGF-β1, Smad-dependent and -independent signaling were studied by MTT assay, RT-PCR, western blotting, immunofluorescent staining, and ELISA.
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http://dx.doi.org/10.18632/aging.102565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6932916PMC
December 2019

Analysis of root canal system of maxillary first and second molars and their correlations by cone beam computed tomography.

J Formos Med Assoc 2020 May 5;119(5):968-973. Epub 2019 Oct 5.

School of Dentistry, College of Medicine, National Taiwan University, Taipei, Taiwan; Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan. Electronic address:

Background/purpose: Understanding the root canal systems of molars and the association of root canal system in adjacent or contralateral molars is important for dental practice. This study aimed to use cone-beam computed tomography (CBCT) to analyze the morphology similarity of root canal systems in the maxillary first and second molars.

Methods: CBCT images of 1741 maxillary molars in a total of 519 patients were blindly examined to analyze the correlation of root canal systems between maxillary first and second molars as well as the bilateral first and second molars.

Results: The most common type in maxillary first molars is 3R4C (3 roots/4 canals), whereas in maxillary second molars is 3R3C.The symmetry in type of root canals in bilateral maxillary first and second molars were 87.36% and 79.85%, respectively. The similarities of root canal system in adjacent maxillary first and second molars were 53.07% (right side) and 52.58% (left side). The concurrence of MB2 canal in bilateral maxillary first molars is 77.8%, and 35.97% in maxillary second molars. In the 110 patients with MB2 canal in bilateral maxillary second molars, the chance of bilateral MB2 canals in their maxillary first molar is almost 100%.

Conclusion: Maxillary first molars have higher prevalence of 3R4C than second molars. The symmetry in bilateral maxillary molars is higher than the similarity in adjacent maxillary first and second molars. Application of CBCT analysis of root canal system can improve endodontic treatment outcomes. The correlation of root canal system between teeth is useful for genetic linkage.
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http://dx.doi.org/10.1016/j.jfma.2019.09.012DOI Listing
May 2020

Antiplatelet, antioxidative, and anti-inflammatory effects of hydroquinone.

J Cell Physiol 2019 08 6;234(10):18123-18130. Epub 2019 Mar 6.

School of Dentistry & Department of Dentistry, National Taiwan University Medical College and National Taiwan University Hospital, Taipei, Taiwan.

Platelets play crucial roles in thrombosis and hemostasis through platelet activation and aggregation that are crucial in cardiovascular diseases. Hydroquinone (HQ) and its derivatives are present in many dermatological creams, paints, motor fuels, air, microorganisms, and plant products like wheat bread, fruit, coffee, and red wine. The effect of HQ on humans is not clear. In this study, we found that HQ (>25 μM) inhibited arachidonic acid (AA)-induced platelet aggregation. HQ suppressed AA-induced thromboxane B2 production of platelets. HQ (>10 μM) also attenuated ex vivo platelet-rich plasma aggregation. HQ prevented the interleukin (IL)-1β-induced 8-isoprostane, and PGE2 production, but not IL-8 production of pulp cells. These results indicate that HQ may have an antiplatelet effect via inhibition of thromboxane production. HQ has antioxidative and anti-inflammatory effects, and possible inhibition of COX. Exposure and consumption of HQ-containing products, food or drugs may have antiplatelet, antioxidative, and anti-inflammatory effects.
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http://dx.doi.org/10.1002/jcp.28444DOI Listing
August 2019

Butyrate Stimulates Histone H3 Acetylation, 8-Isoprostane Production, RANKL Expression, and Regulated Osteoprotegerin Expression/Secretion in MG-63 Osteoblastic Cells.

Int J Mol Sci 2018 Dec 17;19(12). Epub 2018 Dec 17.

School of Dentistry and Department of Dentistry, National Taiwan University Hospital and National Taiwan University Medical College, Taipei 100, Taiwan.

Butyric acid as a histone deacetylase (HDAC) inhibitor is produced by a number of periodontal and root canal microorganisms (such as , , etc.). Butyric acid may affect the biological activities of periodontal/periapical cells such as osteoblasts, periodontal ligament cells, etc., and thus affect periodontal/periapical tissue destruction and healing. The purposes of this study were to study the toxic effects of butyrate on the matrix and mineralization marker expression in MG-63 osteoblasts. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cellular apoptosis and necrosis were analyzed by propidium iodide/annexin V flow cytometry. The protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). OPG, soluble RANKL (sRANKL), 8-isoprostane, pro-collagen I, matrix metalloproteinase-2 (MMP-2), osteonectin (SPARC), osteocalcin and osteopontin (OPN) secretion into culture medium were measured by enzyme-linked immunosorbant assay. Alkaline phosphatase (ALP) activity was checked by ALP staining. Histone H3 acetylation levels were evaluated by immunofluorescent staining (IF) and Western blot. We found that butyrate activated the histone H3 acetylation of MG-63 cells. Exposure of MG-63 cells to butyrate partly decreased cell viability with no marked increase in apoptosis and necrosis. Twenty-four hours of exposure to butyrate stimulated RANKL protein expression, whereas it inhibited OPG protein expression. Butyrate also inhibited the secretion of OPG in MG-63 cells, whereas the sRANKL level was below the detection limit. However, 3 days of exposure to butyrate (1 to 8 mM) or other HDAC inhibitors such as phenylbutyrate, valproic acid and trichostatin stimulated OPG secretion. Butyrate stimulated 8-isoprostane, MMP-2 and OPN secretion, but not procollagen I, or osteocalcin in MG-63 cells. Exposure to butyrate (2⁻4 mM) for 3 days markedly stimulated osteonectin secretion and ALP activity. In conclusion, higher concentrations of butyric acid generated by periodontal and root canal microorganisms may potentially induce bone destruction and impair bone repair by the alteration of OPG/RANKL expression/secretion, 8-isoprostane, MMP-2 and OPN secretion, and affect cell viability. However, lower concentrations of butyrate (1⁻4 mM) may stimulate ALP, osteonectin and OPG. These effects are possibly related to increased histone acetylation. These events are important in the pathogenesis and repair of periodontal and periapical destruction.
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http://dx.doi.org/10.3390/ijms19124071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321057PMC
December 2018

IL-1β-induced ICAM-1 and IL-8 expression/secretion of dental pulp cells is differentially regulated by IRAK and p38.

J Formos Med Assoc 2019 Aug 14;118(8):1247-1254. Epub 2018 Dec 14.

Department of Dentistry and School of Dentistry, National Taiwan University Hospital and National Taiwan University Medical College, Taiwan. Electronic address:

Background/purpose: Interleukin 1 beta (IL-1β) is a pro-inflammatory cytokine involved in the acute and chronic inflammatory processes of dental pulp. Intercellular adhesion molecule-1 (ICAM-1) and IL-8 are two major inflammatory mediators. However, the role of interleukin-1 receptor-associated kinases (IRAKs) signaling pathways in responsible for the inflammatory effects of IL-1β on dental pulp cells is not clear.

Methods: Cultured human dental pulp cells were exposed to IL-1β with/without pretreatment and co-incubation with IRAK1/4 inhibitor or SB203580 (p38 inhibitor). IRAK-1 phosphorylation was evaluated by immunno fluorescent staining. The protein expression of ICAM-1 and IL-8 were tested by western blotting. The secretion of soluble ICAM-1 (sICAM-1) and IL-8 was measured by enzyme-linked immunosorbant assay (ELISA).

Results: IL-1β stimulated IRAK-1 phosphorylation of pulp cells within 120 min of exposure. IRAK1/4 inhibitor attenuated the IL-1β-induced ICAM-1, but not IL-8 protein expression. IRAK1/4 inhibitor also prevented the IL-1β-induced sICAM-1, but not IL-8 secretion. SB203580 showed little effect on IL-1β-induced sICAM-1 secretion, but effectively inhibited its induction of IL-8 secretion in pulp cells.

Conclusion: The Results reveal the important role of IL-1β in pulpal inflammatory responses via stimulation of IL-8 and ICAM-1 expression and secretion. Moreover, IL-1β-induced effects on IL-8 and ICAM-1 are differentially regulated by IRAK1/4 and p38 signaling in dental pulp cells. Blocking of IRAKs and p38 signaling may have potential to control inflammation of dental pulp in the future.
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http://dx.doi.org/10.1016/j.jfma.2018.11.015DOI Listing
August 2019

Outcome assessment of apical surgery: A study of 234 teeth.

J Formos Med Assoc 2019 Jun 16;118(6):1055-1061. Epub 2018 Nov 16.

Department of Dentistry and School of Dentistry, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan. Electronic address:

Background/purpose: Apical surgery is an option for management of endodontically-treated tooth with persistent periapical lesions or symptom and sign. The objective of this study was to investigate the correlation between the demography, preoperative, postoperative factors and healed rate of apical surgery.

Methods: Subjects were retrospectively collected from patients who received apical surgery/apicoectomy at the Endodontic Department, National Taiwan University Hospital from January 2013 to June 2015. The standard apical surgery procedures were performed. The demography, preoperative clinical and radiographic examination data as well as postoperative variables were collected. The outcome assessment was carried out after surgery. Statistical analysis was performed by chi square test to evaluate the potential outcome predictors.

Results: Total 187 patients and 234 teeth receiving apical surgery were included. 53 male and 134 female patients were collected. The age was ranged between 17 and 89 years old and the mean age was 43.64 years old. Better healed rate with significant differences were observed in female patient (p < 0.05), age ≤60 years old (p < 0.01), preoperative root canal filling material >2 mm short of apex (p < 0.01), lesion size from ≤2 mm to ≤12 mm (p < 0.05) and follow-up period ≧12 months (p < 0.01) groups.

Conclusion: Gender, age, preoperative root canal filling material extent, lesion size and follow-up period may affect the outcome of apical surgery. Tooth type, post, prosthesis, and lesion area showed no marked effect on apical healing. These results provide more detailed information for the clinical practitioners to make treatment plans and are important for clinical endodontic practices.
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http://dx.doi.org/10.1016/j.jfma.2018.10.019DOI Listing
June 2019

IL-1β induced IL-8 and uPA expression/production of dental pulp cells: Role of TAK1 and MEK/ERK signaling.

J Formos Med Assoc 2018 Aug 27;117(8):697-704. Epub 2018 Apr 27.

Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University Medical College, Taipei, Taiwan; Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan. Electronic address:

Background/purpose: Interleukin 1 beta (IL-1β) is a pro-inflammatory cytokine involved in the inflammatory processes of dental pulp. IL-8 and urokinase plasminogen activator (uPA) are two inflammatory mediators. However, the role of transforming growth factor beta-activated kinase-1 (TAK1) and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways in responsible for the effects of IL-1β on IL-8 and uPA expression/secretion of dental pulp cells are not clear.

Methods: Human dental pulp cells were exposed to IL-1β with/without pretreatment with 5z-7-oxozeaneaeol (a TAK1 inhibitor) or U0126 (a MEK/ERK inhibitor). TAK1 activation was determined by immunofluorescent staining. The protein expression of IL-8 was tested by western blot. The expression of IL-8 and uPA mRNA was studied by reverse transcriptase-polymerase chain reaction (RT-PCR). The secretion of IL-8 and uPA was measured by enzyme-linked immunosorbent assay.

Results: Exposure of dental pulp cells to IL-1β (0.1-10 ng/ml) stimulated IL-8 and uPA expression. IL-1β also induced IL-8 and uPA secretion of dental pulp cells. IL-1β stimulated p-TAK1 activation of pulp cells. Pretreatment and co-incubation of pulp cells by 5z-7oxozeaenol (1 and 2.5 μM) and U0126 (10 and 20 μM) prevented the IL-1β-induced IL-8 and uPA expression. 5z-7oxozeaenol and U0126 also attenuated the IL-1β-induced IL-8 and uPA secretion.

Conclusion: IL-1β is important in the pathogenesis of pulpal inflammatory diseases and repair via stimulation of IL-8 and uPA expression and secretion. These events are associated with TAK1 and MEK/ERK signaling. Blocking of TAK1 and MEK/ERK signaling has potential to control inflammation of dental pulp.
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http://dx.doi.org/10.1016/j.jfma.2018.04.003DOI Listing
August 2018

Lysophosphatidylcholine induces cytotoxicity/apoptosis and IL-8 production of human endothelial cells: Related mechanisms.

Oncotarget 2017 Dec 10;8(63):106177-106189. Epub 2017 Nov 10.

School of Dentistry and Department of Dentistry, National Taiwan University Medical College and National Taiwan University Hospital, Taipei, Taiwan.

Increased levels of oxidized low-density lipoprotein oxLDL) are shown to elevate the risk of cardiovascular diseases such as atherosclerosis, thrombosis, stroke, and myocardial infarction. This is possibly due to the toxic effects of oxLDLs on vascular cells. Various oxLDLs including lysophosphatidylcholine (LPC) and 7-ketocholesterol injure vascular endothelial cells and stimulate inflammatory reaction. However the toxicity of LPC on endothelial cells is not clear. In this study, human endothelial cells were exposed to LPC. Cytotoxicity was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Propidium iodide (PI) staining or PI/Annexin V dual staining flow cytometry were used to determine cell cycle progression and apoptosis. Reactive oxygen species (ROS) level was analyzed by DCFH-DA labeling flow cytometry. RNA and protein expression of endothelial cells was studied by reverse transcriptase-polymerase chain reaction and western blotting. IL-8 secretion was measured by enzyme-linked immunosorbant assay. LPC showed cytotoxicity to endothelial cells (>50 µg/ml). LPC induced cell cycle arrest and apoptosis with concomitant inhibition of cdc2 and cyclin B1 expression. LPC stimulated intracellular ROS production and ATM/Chk2, ATR/Chk1 and Akt activation. IL-8 expression and secretion in endothelial cells were induced by LPC. LPC-induced apoptosis, and IL-8 expression/secretion was attenuated by LY294002, a PI3K/Akt inhibitor. These results reveal that LPC is involved in the pathogenesis of atherosclerosis and vascular diseases by stimulation of inflammation and injury to endothelial cells. These events are related to ROS, ATM/Chk2, ATR/Chk2 and PI3K/Akt signaling. Understanding the toxic mechanisms of LPC is useful for future prevention and treatment atherosclerosis.
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http://dx.doi.org/10.18632/oncotarget.22425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739725PMC
December 2017

Cemental tear: To know what we have neglected in dental practice.

J Formos Med Assoc 2018 Apr 29;117(4):261-267. Epub 2017 Sep 29.

School of Dentistry, National Taiwan University Medical College and Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan. Electronic address:

Cemental tear is a special kind of root surface fracture, contributing to periodontal and periapical breakdown. However, it is a challenge for doctors to diagnose, resulting in delayed or improper treatment. We reviewed the predisposing factors, location, radiographic/clinical characteristics, diagnosis and treatments of cemental tears. From the literature, patients with cemental tear were mainly males, over 60 year-old. Possible predisposing factors include gender, age, tooth type, traumatic occlusal force and vital teeth. Cemental tears were common in upper and lower anterior teeth, single or multiple, and can be present in cervical, middle and apical third of roots. Morphology of cemental tears can be either piece-shaped or U-shaped. Clinically, cemental tear shows a unitary periodontal pocket and signs/symptoms mimicking localized periodontitis, apical periodontitis and vertical root fractures. Treatment of cemental tears include scaling, root planning, root canal treatment, periodontal/periapical surgery, guided tissue regeneration, bone grafting, and intentional replantation. Recurrence of cemental tear is possible especially when the fracture involves root apex. Extraction is recommended for teeth with poor prognosis. In conclusion, cemental tears can involve both periodontal and periapical area. Dentists should understand the predisposing factors and clinical features of cemental tears for early diagnosis/treatment to prevent bone loss/tooth extraction.
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http://dx.doi.org/10.1016/j.jfma.2017.09.001DOI Listing
April 2018

TGF-β1 stimulates cyclooxygenase-2 expression and PGE production of human dental pulp cells: Role of ALK5/Smad2 and MEK/ERK signal transduction pathways.

J Formos Med Assoc 2017 Oct 2;116(10):748-754. Epub 2017 Aug 2.

School of Dentistry, College of Medicine, National Taiwan University Medical College and Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan. Electronic address:

Background/purposes: TGF-β1 is an important growth factor that may influence the odontoblast differentiation and matrix deposition in the reactionary/reparative dentinogenesis to dental caries or other tooth injuries. TGF-β1 exerts its effects through various signaling pathways, such as Smads and MAPKs. Cyclooxygenase-2 (COX-2) is a membrane-associated enzyme that produces prostaglandin E (PGE) at sites of pulpal injury and inflammation, which leads to tissue swelling, redness and pain. The purposes of this study were to investigate the differential signal transduction pathways of TGF-β1 that mediate COX-2 stimulation and PGE production in dental pulp cells.

Methods: Pulp cells were exposed to TGF-β1 with/without SB431542 (an ALK5/Smad2 inhibitor) and U0126 (a MEK/ERK inhibitor). MTT assay was used to estimate cell viability. Enzyme-linked immunosorbent assay (ELISA) was used for measurement of PGE levels. RT-PCR and western blot were used to determined COX-2 mRNA and protein, respectively.

Results: Exposure to TGF-β1 (1-10 ng/ml) increased the COX-2 mRNA and protein level of cultured pulp cells. Exposure to TGF-β1 (0.1-10 ng/mL) significantly stimulated PGE production of dental pulp cells. Under the pretreatment of SB431542, the stimulatory effect of TGF-β1 on COX-2 level of pulp cells was inhibited. Similarly, U0126 also partly inhibited the TGF-β1-induced COX-2 expression.

Conclusion: TGF-β1 increased the COX-2 and PGE level of cultured pulp cells. The effect of TGF-β1 on COX-2 protein expression was associated with ALK5/Smad2/3 and MEK/ERK pathways. These events are important in the early inflammation, repair and regeneration of dental pulp in response to injury.
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http://dx.doi.org/10.1016/j.jfma.2017.07.008DOI Listing
October 2017

Basic Fibroblast Growth Factor Regulates Gene and Protein Expression Related to Proliferation, Differentiation, and Matrix Production of Human Dental Pulp Cells.

J Endod 2017 Jun 14;43(6):936-942. Epub 2017 Apr 14.

School of Dentistry, National Taiwan University Medical College and Department of Dentistry, National Taiwan University Hospital, Taipei City, Taiwan. Electronic address:

Introduction: Basic fibroblast growth factor (bFGF) plays differential effects on the proliferation, differentiation, and extracellular matrix turnover in various tissues. However, limited information is known about the effect of bFGF on dental pulp cells. The purposes of this study were to investigate whether bFGF influences the cell differentiation and extracellular matrix turnover of human dental pulp cells (HDPCs) and the related gene and protein expression as well as the role of the mitogen-activated protein kinase (MEK)/extracellular-signal regulated kinase (ERK) signaling pathway. The expression of fibroblast growth factor receptors (FGFRs) in HDPCs was also studied.

Methods: The expression of FGFR1 and FGFR2 in HDPCs was investigated by reverse-transcription polymerase chain reaction. HDPCs were treated with different concentrations of bFGF. Cell proliferation was evaluated using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Cell differentiation was evaluated using alkaline phosphatase (ALP) staining. Changes in messenger expression of cyclin B1 and tissue inhibitor of metalloproteinase (TIMP) 1 were determined by reverse-transcription polymerase chain reaction. Changes in protein expression of cdc2, TIMP-1, TIMP-2, and collagen I were determined by Western blotting. U0126 was used to clarify the role of MEK/ERK signaling.

Results: HDPCs expressed both FGFR1 and FGFR2. Cell viability was stimulated by 50-250 ng/mL bFGF. The expression and enzyme activities of ALP were inhibited by 10-500 ng/mL bFGF. At similar concentrations, bFGF stimulates cdc2, cyclin B1, and TIMP-1 messenger RNA and protein expression. bFGF showed little effect on TIMP-2 and partly inhibited collagen I expression of pulp cells. U0126 (a MEK/ERK inhibitor) attenuated the bFGF-induced increase of cyclin B1, cdc2, and TIMP-1.

Conclusions: bFGF may be involved in pulpal repair and regeneration by activation of FGFRs to regulate cell growth; stimulate cdc2, cyclin B1, and TIMP-1 expression; and inhibit ALP. These events are partly associated with MEK/ERK signaling.
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http://dx.doi.org/10.1016/j.joen.2017.01.024DOI Listing
June 2017

Clinical and Radiographic Characteristics of Vertical Root Fractures in Endodontically and Nonendodontically Treated Teeth.

J Endod 2017 May 11;43(5):687-693. Epub 2017 Mar 11.

Department of Dentistry and School of Dentistry, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan. Electronic address:

Introduction: A vertical root fracture (VRF) is a root fracture extending along the longitudinal axis of roots and is often noted in endodontically treated teeth. However, the clinical and radiographic characteristics of VRFs are not completely known.

Methods: A total of 65 teeth with 68 vertical fractured roots in 58 Chinese patients were investigated. The clinical examination records and radiographic images were reviewed in detail.

Results: A total of 24 male (41.38%) and 34 female (58.62%) patients aged 25-90 years (average = 57 years) were included; 51 (87.93%) and 7 (12.07%) patients exhibited 1 tooth and 2 teeth with VRFs, respectively, in the dentition. VRFs occurred mainly in the mesial root (20 roots, 57.14%) of the mandibular molars (29 teeth, 44.62%). Clinically, teeth with VRFs usually presented a periodontal probing depth >5 mm (44 teeth, 91.67%, P < .001) with a prosthesis (55 teeth, 84.62%, P < .001) and a relatively intact dentition (42 patients exhibited <4 missing teeth in the dentition, 77.78%, P < .001). Most of the nonendodontically treated VRFs exhibited attrited occlusal surfaces. Radiographic characteristics of the teeth with VRFs were typically associated with prior root canal treatment (56 teeth, 86.15%, P < .001), periodontal bone loss (62 teeth, 95.38%, P < .001), apical bone loss (52 teeth, 80.00%, P < .001), and periodontal ligament widening (61 teeth, 93.85%, P < .001). The mesial roots of the mandibular molars were most susceptible to VRFs in both endodontically and nonendodontically treated teeth.

Conclusions: These results elucidated some clinical and radiographic and diagnostic features that facilitate VRF identification.
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http://dx.doi.org/10.1016/j.joen.2016.12.009DOI Listing
May 2017

Effect of Butyrate on Collagen Expression, Cell Viability, Cell Cycle Progression and Related Proteins Expression of MG-63 Osteoblastic Cells.

PLoS One 2016 28;11(11):e0165438. Epub 2016 Nov 28.

Graduate Institute of Clinical Dentistry and Department of Dentistry, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan.

Aims: Butyric acid is one major metabolic product generated by anaerobic Gram-negative bacteria of periodontal and root canal infection. Butyric acid affects the activity of periodontal cells such as osteoblasts. The purposes of this study were to investigate the effects of butyrate on MG-63 osteoblasts.

Methods: MG-63 cells were exposed to butyrate and cell viability was estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mRNA and protein expression of type I collagen and cell cycle-related proteins were measured by reverse-transcriptase polymerase chain reaction (RT-PCR), western blotting or immunofluorescent staining. Cellular production of reactive oxygen species (ROS) was analyzed by 2',7'-dichlorofluorescein (DCF) fluorescence flow cytometry.

Results: Exposure to butyrate suppressed cell proliferation, and induced G2/M (8 and 16 mM) cell cycle arrest of MG-63 cells. Some cell apoptosis was noted. The mRNA expression of cdc2 and cyclin-B1 decreased after exposure to butyrate. The protein expression of type I collagen, cdc2 and cyclin B1 were decreased, whereas the expression of p21, p27 and p57 was stimulated. Under the treatment of butyrate, ROS production in MG-63 cells markedly increased.

Conclusions: The secretion of butyric acid by periodontal and root canal microorganisms may inhibit bone cell growth and matrix turnover. This is possibly due to induction of cell cycle arrest and ROS generation and inhibition of collagen expression. These results suggest the involvement of butyric acid in the pathogenesis of periodontal and periapical tissue destruction by impairing bone healing responses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0165438PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5125573PMC
June 2017

7-Ketocholesterol induces ATM/ATR, Chk1/Chk2, PI3K/Akt signalings, cytotoxicity and IL-8 production in endothelial cells.

Oncotarget 2016 11;7(46):74473-74483

School of Dentistry and Department of Dentistry, National Taiwan University Medical College and National Taiwan University Hospital, Taipei, Taiwan.

Cardiovascular diseases (atherosclerosis, stroke, myocardiac infarction etc.) are the major systemic diseases of elder peoples in the world. This is possibly due to increased levels of oxidized low-density lipoproteins (oxLDLs) such as 7-ketocholesterol (7-KC) and lysophosphatidylcholine (LPC) that damage vascular endothelial cells, induce inflammatory responses, to elevate the risk of cardiovascular diseases, Alzheimer's disease, and age-related macular degeneration. However the toxic effects of 7-KC on endothelial cells are not known. In this study, 7-KC showed cytotoxicity to endothelial cells at concentrations higher than 10 µg/ml. 7-KC stimulated ATM/Chk2, ATR-Chk1 and p53 signaling pathways in endothelial cells. 7-KC also induced G0/G1 cell cycle arrest and apoptosis with an inhibition of Cyclin dependent kinase 1 (Cdk1) and cyclin B1 expression. Secretion and expression of IL-8 in endothelial cells were stimulated by 7-KC. 7-KC further induced intracellular ROS production as shown by increase in DCF fluorescence and Akt phosphorylation. LY294002 attenuated the 7-KC-induced apoptosis and IL-8 mRNA expression of endothelial cells. These results indicate that oxLDLs such as 7-KC may contribute to the pathogenesis of atherosclerosis, thrombosis and cardiovascular diseases by induction of endothelial damage, apoptosis and inflammatory responses. These events are associated with ROS production, activation of ATM/Chk2, ATR/Chk1, p53 and PI3K/Akt signaling pathways.
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http://dx.doi.org/10.18632/oncotarget.12578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342680PMC
November 2016

Effects of TGF-β1 on plasminogen activation in human dental pulp cells: Role of ALK5/Smad2, TAK1 and MEK/ERK signalling.

J Tissue Eng Regen Med 2018 04 10;12(4):854-863. Epub 2017 Apr 10.

Laboratory of Dental Pharmacology, Toxicology & Material Biocompatibility, Graduate Institute of Clinical Dentistry and Department of Dentistry, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan.

Transforming growth factor-β1 (TGF-β1) plays an important role in the pulpal repair and dentinogenesis. Plasminogen activation (PA) system regulates extracellular matrix turnover. In this study, we investigated the effects of TGF-β1 on PA system of dental pulp cells and its signalling pathways. Dental pulp cells were treated with different concentrations of TGF-β1. MTT assay, reverse transcription-polymerase chain reaction, Western blotting and enzyme-linked immunosorbant assay (ELISA) were used to detect the effect of TGF-β1 on cell viability, mRNA and protein expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) as well as their secretion. The phosphorylation of Smad2 and TAK1 was analysed by Pathscan ELISA or Western blotting. Cells were pretreated with SB431542 (ALK5/Smad2/3 inhibitor), 5z-7-oxozeaenol (TAK1 inhibitor) and U0126 (MEK/ERK inhibitor) for examining the related signalling. TGF-β1 slightly inhibited cell growth that was reversed by SB431542. TGF-β1 upregulated both RNA and protein expression of PAI-1 and uPAR, whereas it downregulated uPA expression. Accordingly, TGF-β1 stimulated PAI-1 and soluble uPAR (suPAR) secretion of pulp cells, whereas uPA secretion was inhibited. TGF-β1 induced the phosphorylation of Smad2 and TAK1. In addition, SB431542, 5z-7-oxozeaenol and U0126 attenuated the TGF-β1-induced secretion of PAI-1 and suPAR. These results indicate that TGF-β1 is possibly involved in the repair/regeneration and inflammatory processes of dental pulp via regulation of PAI-1, uPA and uPAR. These effects of TGF-β1 are related to activation of ALK5/Smad2, TAK1 and MEK/ERK signalling pathways. Clarifying the signal transduction for the effects of TGF-β1 is helpful for pulpo-dentin regeneration and tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/term.2339DOI Listing
April 2018

Transforming growth factor beta 1 increases collagen content, and stimulates procollagen I and tissue inhibitor of metalloproteinase-1 production of dental pulp cells: Role of MEK/ERK and activin receptor-like kinase-5/Smad signaling.

J Formos Med Assoc 2017 May 6;116(5):351-358. Epub 2016 Oct 6.

Graduate Institute of Clinical Dentistry & Department of Dentistry, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan. Electronic address:

Background/purpose: In order to clarify the role of transforming growth factor beta 1 (TGF-β1) in pulp repair/regeneration responses, we investigated the differential signaling pathways responsible for the effects of TGF-β1 on collagen turnover, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinase-1 (TIMP-1) production in human dental pulp cells.

Methods: Pulp cells were exposed to TGF-β1 with/without pretreatment and coincubation by 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenyl mercapto)butadiene (U0126; a mitogen-activated protein kinase kinase [MEK]/extracellular signal-regulated kinase [ERK] inhibitor) and 4-(5-benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H- imidazol-2-yl)-benzamide hydrate (SB431542; an activin receptor-like kinase-5/Smad signaling inhibitor). Sircol collagen assay was used to measure cellular collagen content. Culture medium procollagen I, TIMP-1, and MMP-3 levels were determined by enzyme-linked immunosorbent assay.

Results: TGF-β1 increased the collagen content, procollagen I, and TIMP-1 production, but slightly decreased MMP-3 production of pulp cells. SB431542 and U0126 prevented the TGF-β1-induced increase of collagen content and TIMP-1 production of dental pulp cells.

Conclusion: These results indicate that TGF-β1 may be involved in the healing/regeneration processes of dental pulp in response to injury by stimulation of collagen and TIMP-1 production. These events are associated with activin receptor-like kinase-5/Smad2/3 and MEK/ERK signaling.
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http://dx.doi.org/10.1016/j.jfma.2016.07.014DOI Listing
May 2017

Areca nut components stimulate ADAM17, IL-1α, PGE2 and 8-isoprostane production in oral keratinocyte: role of reactive oxygen species, EGF and JAK signaling.

Oncotarget 2016 Mar;7(13):16879-94

Laboratory of Pharmacology, Toxicology and Chemical Carcinogenesis, School of Dentistry and Department of Dentistry, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan.

Betel quid (BQ) chewing is an etiologic factor of oral submucous fibrosis (OSF) and oral cancer. There are 600 million BQ chewers worldwide. The mechanisms for the toxic and inflammatory responses of BQ are unclear. In this study, both areca nut (AN) extract (ANE) and arecoline stimulated epidermal growth factor (EGF) and interleukin-1α (IL-1α) production of gingival keratinocytes (GKs), whereas only ANE can stimulate a disintegrin and metalloproteinase 17 (ADAM17), prostaglandin E2 (PGE2) and 8-isoprostane production. ANE-induced EGF production was inhibited by catalase. Addition of anti-EGF neutralizing antibody attenuated ANE-induced cyclooxygenase-2 (COX-2), mature ADAM9 expression and PGE2 and 8-isoprostane production. ANE-induced IL-1α production was inhibited by catalase, anti-EGF antibody, PD153035 (EGF receptor antagonist) and U0126 (MEK inhibitor) but not by α-naphthoflavone (cytochrome p450-1A1 inhibitor). ANE-induced ADAM17 production was inhibited by pp2 (Src inhibitor), U0126, α-naphthoflavone and aspirin. AG490 (JAK inhibitor) prevented ANE-stimulated ADAM17, IL-1α, PGE2 production, COX-2 expression, ADAM9 maturation, and the ANE-induced decline in keratin 5 and 14, but showed little effect on cdc2 expression and EGF production. Moreover, ANE-induced 8-isoprostane production by GKs was inhibited by catalase, anti-EGF antibody, AG490, pp2, U0126, α-naphthoflavone, Zinc protoporphyrin (ZnPP) and aspirin. These results indicate that AN components may involve in BQ-induced oral cancer by induction of reactive oxygen species, EGF/EGFR, IL-1α, ADAMs, JAK, Src, MEK/ERK, CYP1A1, and COX signaling pathways, and the aberration of cell cycle and differentiation. Various blockers against ROS, EGF, IL-1α, ADAM, JAK, Src, MEK, CYP1A1, and COX can be used for prevention or treatment of BQ chewing-related diseases.
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http://dx.doi.org/10.18632/oncotarget.7621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941357PMC
March 2016

Prostaglandin E2 Stimulates EP2, Adenylate Cyclase, Phospholipase C, and Intracellular Calcium Release to Mediate Cyclic Adenosine Monophosphate Production in Dental Pulp Cells.

J Endod 2016 Apr 20;42(4):584-8. Epub 2016 Feb 20.

Laboratory of Dental Pharmacology, Toxicology and Pulp Biology, School of Dentistry and Department of Dentistry, National Taiwan University Medical College and National Taiwan University Hospital, Taipei, Taiwan. Electronic address:

Introduction: Prostaglandin E2 (PGE2) plays a crucial role in pulpal inflammation and repair. However, its induction of signal transduction pathways is not clear but is crucial for future control of pulpal inflammation.

Methods: Primary dental pulp cells were exposed to PGE2 and 19R-OH PGE2 (EP2 agonist) or sulprostone (EP1/EP3 agonist) for 5 to 40 minutes. Cellular cyclic adenosine monophosphate (cAMP) levels were measured using the enzyme-linked immunosorbent assay. In some experiments, cells were pretreated with SQ22536 (adenylate cyclase inhibitor), H89 (protein kinase A inhibitor), dorsomorphin (adenosine monophosphate-activated protein kinase inhibitor), U73122 (phospholipase C inhibitor), thapsigargin (inhibitor of intracellular calcium release), W7 (calmodulin antagonist), verapamil (L-type calcium channel blocker), and EGTA (extracellular calcium chelator) for 20 minutes before the addition of PGE2.

Results: PGE2 and 19R-OH PGE2 (EP2 agonist) stimulated cAMP production, whereas sulprostone (EP1/EP3 agonist) shows little effect. PGE2-induced cAMP production was attenuated by SQ22536 and U73122 but not H89 and dorsomorphin. Intriguingly, thapsigargin and W7 prevented PGE2-induced cAMP production, but verapamil and EGTA showed little effect.

Conclusions: These results indicate that PGE2-induced cAMP production is associated with EP2 receptor and adenylate cyclase activation. These events are mediated by phospholipase C, intracellular calcium release, and calcium-calmodulin signaling. These results are helpful for understanding the role of PGE2 in pulpal inflammation and repair and possible future drug intervention.
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http://dx.doi.org/10.1016/j.joen.2015.12.011DOI Listing
April 2016

Effects of Camphorquinone on Cytotoxicity, Cell Cycle Regulation and Prostaglandin E2 Production of Dental Pulp Cells: Role of ROS, ATM/Chk2, MEK/ERK and Hemeoxygenase-1.

PLoS One 2015 14;10(12):e0143663. Epub 2015 Dec 14.

Laboratory of Dental Pharmacology, Toxicology & Material Biocompatibility, Graduate Institute of Clinical Dentistry, and National Taiwan University Medical College, Taipei, Taiwan.

Camphorquinone (CQ) is a popularly-used photosensitizer in composite resin restoration. In this study, the effects of CQ on cytotoxicity and inflammation-related genes and proteins expression of pulp cells were investigated. The role of reactive oxygen species (ROS), ATM/Chk2/p53 and hemeoxygenase-1 (HO-1) and MEK/ERK signaling was also evaluated. We found that ROS and free radicals may play important role in CQ toxicity. CQ (1 and 2 mM) decreased the viability of pulp cells to about 70% and 50% of control, respectively. CQ also induced G2/M cell cycle arrest and apoptosis of pulp cells. The expression of type I collagen, cdc2, cyclin B, and cdc25C was inhibited, while p21, HO-1 and cyclooxygenase-2 (COX-2) were stimulated by CQ. CQ also activated ATM, Chk2, and p53 phosphorylation and GADD45α expression. Besides, exposure to CQ increased cellular ROS level and 8-isoprostane production. CQ also stimulated COX-2 expression and PGE2 production of pulp cells. The reduction of cell viability caused by CQ can be attenuated by N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD), but can be promoted by Zinc protoporphyin (ZnPP). CQ stimulated ERK1/2 phosphorylation, and U0126 prevented the CQ-induced COX-2 expression and prostaglandin E2 (PGE2) production. These results indicate that CQ may cause cytotoxicity, cell cycle arrest, apoptosis, and PGE2 production of pulp cells. These events could be due to stimulation of ROS and 8-isoprostane production, ATM/Chk2/p53 signaling, HO-1, COX-2 and p21 expression, as well as the inhibition of cdc2, cdc25C and cyclin B1. These results are important for understanding the role of ROS in pathogenesis of pulp necrosis and pulpal inflammation after clinical composite resin filling.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143663PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4682794PMC
August 2016

IL-1β-induced MCP-1 expression and secretion of human dental pulp cells is related to TAK1, MEK/ERK, and PI3K/Akt signaling pathways.

Arch Oral Biol 2016 Jan 22;61:16-22. Epub 2015 Oct 22.

Graduate Institute of Clinical Dentistry & Department of Dentistry, National Taiwan University Hospital and National Taiwan University Medical College, Taiwan. Electronic address:

Objective: Interleukin-1β (IL-1β) is an inflammatory molecule of the dental pulp. IL-1β stimulates cyclooxygenase-2 (COX-2) and prostaglandins production of pulp cells and affects the pulpal inflammation and repair. However, the effects of IL-1β on Monocyte Chemotactic Factor-1 (MCP-1) of dental pulp cells and its relation to transforming growth factor β-activated kinase-1 (TAK1), PI3K/Akt, and MEK/ERK signaling and COX activation are not fully clear.

Design: Human dental pulp cells were exposed to IL-1β with/without pretreatment and co-incubation by aspirin (a COX inhibitor), 5z-7-oxozeaenol (a TAK1 inhibitor), LY294002 (a PI3K/Akt inhibitor) or U0126 (a MEK/ERK inhibitor). Viable cell number was evaluated by MTT assay. MCP-1 mRNA expression was tested by reverse transcriptase-polymerase chain reaction (RT-PCR). MCP-1 and COX-2 protein expression was studied by western blot. MCP-1 in the culture medium was measure by ELISA.

Results: IL-1β showed little cytotoxicity to pulp cells. It stimulated MCP-1 mRNA and protein expression and MCP-1 secretion. Aspirin, U0126, LY294002 and 5z-7-oxozeaenol attenuated the IL-1β-induced MCP-1 expression. In addition, 5z-7-oxozeaenol, LY294002, U0126 and aspirin prevented the IL-1β-induced MCP-1 secretion of pulp cells.

Conclusion: These results indicate that IL-1β may be involved in the pulpal inflammatory and healing processes by inducing MCP-1 expression and secretion. These events are related to differential activation of TAK1, PI3K/Akt and MEK/ERK 1/2 signaling and COX activation. These results are important for future pharmacologic intervention of pulpal inflammatory diseases.
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http://dx.doi.org/10.1016/j.archoralbio.2015.10.008DOI Listing
January 2016

Differences in oral habit and lymphocyte subpopulation affect malignant transformation of patients with oral precancer.

J Formos Med Assoc 2016 Apr 26;115(4):263-8. Epub 2015 Sep 26.

School of Dentistry and Department of Dentistry, National Taiwan University Medical College and National Taiwan University Hospital, Taipei, Taiwan. Electronic address:

Background/purpose: In Taiwan, the combination of betel quid chewing, alcohol consumption, and smoking habits increases oral cancer risk by 123-fold compared to persons without these habits. Lymphocyte populations in patients may potentially affect the malignant transformation of oral precancer.

Methods: A total of 28 patients with oral precancer from our previous cohort were enrolled in this study, and their personal information and oral habits were documented. Their lymphocyte populations (CD4+, CD8+, CD19+, and CD56+) and activation markers (CD25 and CD69) were determined by flow cytometry from 1999 to 2004. After follow up till December 2014, data of patients with/without malignant transformation were recorded, and the relation between oral habits and percentage of initial lymphocyte markers was evaluated using the Student t test and Fisher's exact test.

Results: Ten precancer patients developed oral squamous cell carcinoma with a mean period of malignant transformation of 6.8 ± 2.1 years. Patients with malignant transformation had a mean age of 48.4 ± 5.0 years (n = 10), relatively more than that of patients without malignant transformation (41.6 ± 6.3 years, n = 18) (p < 0.05). An increase was noted in the population of peripheral blood mononuclear cells expressing CD4+CD69+, CD19+CD69+, and CD56+CD69+ (p < 0.05) in precancer patients with malignant transformation. Alcohol consumption showed an association with the malignant transformation of patients with precancer (p = 0.030), whereas betel quid and smoking showed little effect.

Conclusion: These results suggest that age, alcohol consumption, and early activation of T cells, B cells, and natural killer cells are crucial in the malignant transformation of oral precancer. Analysis of patient's lymphocyte populations may help predict the malignant transformation of oral precancer.
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http://dx.doi.org/10.1016/j.jfma.2015.07.017DOI Listing
April 2016

Role of ALK5/Smad2/3 and MEK1/ERK Signaling in Transforming Growth Factor Beta 1-modulated Growth, Collagen Turnover, and Differentiation of Stem Cells from Apical Papilla of Human Tooth.

J Endod 2015 Aug 19;41(8):1272-80. Epub 2015 May 19.

Department of Dentistry, National Taiwan University Hospital and Laboratory of Dental Pharmacology, Toxicology and Material Biocompatibility, School of Dentistry, National Taiwan University Medical College, Taipei, Taiwan. Electronic address:

Introduction: Transforming growth factor β1 (TGF-β1) plays an important role in cell proliferation, matrix formation, and odontogenesis. This study investigated the effects of TGF-β1 on stem cells from apical papilla (SCAPs) and its signaling by MEK/ERK and Smad2.

Methods: SCAPs were exposed to TGF-β1 with/without pretreatment and coincubation by SB431542 (an ALK5/Smad 2/3 inhibitor) or U0126 (a MEK/ERK inhibitor). Cell growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay or direct counting of viable cells. Collagen content was determined by using the Sircol collagen assay (Biocolor Ltd, Newtownabbey, Northern Ireland). Cell differentiation was evaluated by measuring alkaline phosphatase (ALP) activity. Smad2 and ERK1/2 phosphorylation was analyzed by Western blotting or PathScan phospho-enzyme-linked immunosorbent assay (Cell Signaling Technology Inc, Danvers, MA).

Results: TGF-β1 stimulated the growth and collagen content of cultured SCAPs. TGF-β1 stimulated ERK1/2 and Smad2 phosphorylation within 60 minutes of exposure. Pretreatment by U0126 and SB431542 effectively prevented the TGF-β1-induced cell growth and collagen content in SCAPs. TGF-β1 stimulated ALP activity at lower concentrations (0.1-1 ng/mL) but down-regulated ALP at higher concentrations (>5 ng/mL). U0126 prevented 0.5 ng/mL TGF-β1-induced ALP activity but showed little effect on 10 ng/mL TGF-β1-induced decline of ALP in SCAPs. Interestingly, SB431542 attenuated both the stimulatory and inhibitory effects on ALP by TGF-β1.

Conclusions: TGF-β1 may affect the proliferation, collagen turnover, and differentiation of SCAPs via differential activation of ALK5/Smad2 and MEK/ERK signaling. These results highlight the future use of TGF-β1 and SCAP for engineering of pulpal regeneration and apexogenesis.
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http://dx.doi.org/10.1016/j.joen.2015.03.022DOI Listing
August 2015

Cytotoxicity and transformation of C3H10T1/2 cells induced by areca nut components.

J Formos Med Assoc 2016 Feb 28;115(2):108-12. Epub 2015 Feb 28.

School of Dentistry and Department of Dentistry, National Taiwan University Medical College and National Taiwan University Hospital, Taipei, Taiwan. Electronic address:

Background/purpose: Betel quid (BQ) chewing is popular in Taiwan and many other countries. There are about 200-600 million BQ chewers in the world. BQ chewing is one major risk factor of oral cancer and oral submucous fibrosis (OSF). While areca nut (AN), a main component of BQ, exhibits genotoxicity, its transformation capacity and its role in the initiation and promotion stages of carcinogenesis are not fully clear.

Methods: Mouse C3H10T1/2 cells were exposed to AN extract (ANE) for 24 hours. Cytotoxicity was evaluated by colony forming efficiency. For the transformation assay, C3H10T1/2 cells were exposed to ANE for 24 hours and then incubated in medium with/without 12-O-tetradecanolylphorbol-13-acetate (TPA; a tumor promoter) for 42 days. Cells were stained with Giemsa and type II and type III transformed foci were counted for analysis of the transformation capacity of ANE.

Results: ANE exhibited cytotoxicity to C3H10T/12 cells at concentrations higher than 320 μg/mL as shown by a decrease in colony numbers. ANE (80-640 μg/mL) alone mildly stimulated the transformed foci formation (p > 0.05). In the presence of TPA, ANE (80-640 μg/mL) markedly stimulated the transformed foci formation. The percentage of dishes with foci increased from 0% in controls to 20% in ANE (80 μg/mL and 320 μg/mL)-treated groups and further increased to 65-94% in ANE plus TPA groups.

Conclusion: These results indicate that ANE is a weak complete carcinogen. ANE is an effective tumor initiator and can induce malignant transformation of C3H10T1/2 cells in the presence of a tumor promoter. ANE may be involved in multistep chemical carcinogenesis by its malignant transformation capacity.
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http://dx.doi.org/10.1016/j.jfma.2015.01.004DOI Listing
February 2016
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