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Research Articles
Cytocompatibility and cell proliferation evaluation of calcium phosphate-based root canal sealers
Letícia Boldrin Mestieri, Ivana Maria Zaccara, Lucas Siqueira Pinheiro, Fernando Branco Barletta, Patrícia Maria Polli Kopper, Fabiana Soares Grecca
Restor Dent Endod 2020;45(1):e2.   Published online November 15, 2019
DOI: https://doi.org/10.5395/rde.2020.45.e2
AbstractAbstract PDFPubReaderePub
Objectives

This study aimed to evaluate the cell viability and migration of Endosequence Bioceramic Root Canal Sealer (BC Sealer) compared to MTA Fillapex and AH Plus.

Materials and Methods

BC Sealer, MTA Fillapex, and AH Plus were placed in contact with culture medium to obtain sealers extracts in dilution 1:1, 1:2 and 1:4. 3T3 cells were plated and exposed to the extracts. Cell viability and migration were assessed by 3-(4,5-dimethyl-thiazoyl)-2,5-diphenyl-tetrazolium bromide (MTT) and Scratch assay, respectively. Data were analyzed by Kruskal-Wallis and Dunn's test (p < 0.05).

Results

The MTT assay revealed greater cytotoxicity for AH Plus and MTA Fillapex at 1:1 dilution when compared to control (p < 0.05). At 1:2 and 1:4 dilutions, all sealers were similar to control (p > 0.05) and MTA Fillapex was more cytotoxic than BC Sealer (p < 0.05). Scratch assay demonstrated the continuous closure of the wound according to time. At 30 hours, the control group presented closure of the wound (p < 0.05). At 36 hours, only BC Sealer presented the closure when compared to AH Plus and MTA Fillapex (p < 0.05). At 42 hours, AH Plus and MTA Fillapex showed a wound healing (p > 0.05).

Conclusions

All tested sealers demonstrated cell viability highlighting BC Sealer, which showed increased cell migration capacity suggesting that this sealer may achieve better tissue repair when compared to other tested sealers.

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Effects of four novel root-end filling materials on the viability of periodontal ligament fibroblasts
Makbule Bilge Akbulut, Pembegul Uyar Arpaci, Ayce Unverdi Eldeniz
Restor Dent Endod 2018;43(3):e24.   Published online May 25, 2018
DOI: https://doi.org/10.5395/rde.2018.43.e24
AbstractAbstract PDFPubReaderePub
Objectives

The aim of this in vitro study was to evaluate the biocompatibility of newly proposed root-end filling materials, Biodentine, Micro-Mega mineral trioxide aggregate (MM-MTA), polymethylmethacrylate (PMMA) bone cement, and Smart Dentin Replacement (SDR), in comparison with contemporary root-end filling materials, intermediate restorative material (IRM), Dyract compomer, ProRoot MTA (PMTA), and Vitrebond, using human periodontal ligament (hPDL) fibroblasts.

Materials and Methods

Ten discs from each material were fabricated in sterile Teflon molds and 24-hour eluates were obtained from each root-end filling material in cell culture media after 1- or 3-day setting. hPDL fibroblasts were plated at a density of 5 × 103/well, and were incubated for 24 hours with 1:1, 1:2, 1:4, and 1:8 dilutions of eluates. Cell viability was evaluated by XTT assay. Data was statistically analysed. Apoptotic/necrotic activity of PDL cells exposed to material eluates was established by flow cytometry.

Results

The Vitrebond and IRM were significantly more cytotoxic than the other root-end filling materials (p < 0.05). Those cells exposed to the Biodentine and Dyract compomer eluates showed the highest survival rates (p < 0.05), while the PMTA, MM-MTA, SDR, and PMMA groups exhibited similar cell viabilities. Three-day samples were more cytotoxic than 1-day samples (p < 0.05). Eluates from the cements at 1:1 dilution were significantly more cytotoxic (p < 0.05). Vitrebond induced cell necrosis as indicated by flow cytometry.

Conclusions

This in vitro study demonstrated that Biodentine and Compomer were more biocompatible than the other root-end filling materials. Vitrebond eluate caused necrotic cell death.

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White mineral trioxide aggregate mixed with calcium chloride dihydrate: chemical analysis and biological properties
Hany Mohamed Aly Ahmed, Norhayati Luddin, Thirumulu Ponnuraj Kannan, Khairani Idah Mokhtar, Azlina Ahmad
Restor Dent Endod 2017;42(3):176-187.   Published online April 17, 2017
DOI: https://doi.org/10.5395/rde.2017.42.3.176
AbstractAbstract PDFPubReaderePub
Objectives

This study aimed to evaluate the chemical and biological properties of fast-set white mineral trioxide aggregate (FS WMTA), which was WMTA combined with calcium chloride dihydrate (CaCl2·2H2O), compared to that of WMTA.

Materials and Methods

Surface morphology, elemental, and phase analysis were examined using scanning electron microscope (SEM), energy dispersive X-ray microanalysis (EDX), and X-ray diffraction (XRD), respectively. The cytotoxicity and cell attachment properties were evaluated on human periodontal ligament fibroblasts (HPLFs) using methyl-thiazol-diphenyltetrazolium (MTT) assay and under SEM after 24 and 72 hours, respectively.

Results

Results showed that the addition of CaCl2·2H2O to WMTA affected the surface morphology and chemical composition. Although FS WMTA exhibited a non-cytotoxic profile, the cell viability values of this combination were lesser than WMTA, and the difference was significant in 7 out of 10 concentrations at the 2 time intervals (p < 0.05). HPLFs adhered over the surface of WMTA and at the interface, after 24 hours of incubation. After 72 hours, there were increased numbers of HPLFs with prominent cytoplasmic processes. Similar findings were observed with FS WMTA, but the cells were not as confluent as with WMTA.

Conclusions

The addition of CaCl2·2H2O to WMTA affected its chemical properties. The favorable biological profile of FS WMTA towards HPLFs may have a potential impact on its clinical application for repair of perforation defects.

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Basic Research
The effect of tumor necrosis factor (TNF)-α to induce matrix metalloproteinase (MMPs) from the human dental pulp, gingival, and periodontal ligament cells
Eun-Mi Rhim, Sang-Hyuk Park, Duck-Su Kim, Sun-Young Kim, Kyoung-Kyu Choi, Gi-Woon Choi
J Korean Acad Conserv Dent 2011;36(1):26-36.   Published online January 31, 2011
DOI: https://doi.org/10.5395/JKACD.2011.36.1.26
AbstractAbstract PDFPubReaderePub
Objectives

In the present study, three kinds of tissues cells (pulp, gingiva, and periodontal ligament) were investigated if those cells express MMP and TIMP when they were stimulated with neuropeptides (substance P, CGRP) or proinflammatory cytokine, TNF-α.

Materials and Methods

The cells cultured from human dental pulp (PF), gingiva (GF) and periodontal ligament were (PDLF) stimulated with Mock, SP, TNF-α, and CGRP for 24 hrs and 48 hrs. for an RNase protection assay and Enzyme Linked Immunosorbent Assay.

Cells (PF, GF and PDLF) seeded in 100 mm culture dish were stimulated with SP (10-5, 10-8 M) or only with medium (Mock stimulation) for 4hrs and for 24 hrs for RNase Protection Assay, and they were stimulated with CGRP (10-5 M) and TNF-α (2 ng/mL) for 24 hrs and with various concentraion of TNF-α (2, 10, and 100 ng/mL) for Rnase Protection Assay with a human MMP-1 probe set including MMP 1, 2, 8, 7, 8, 9, 12, and TIMP 2, 3.

In addition, cells (PF, GF and PDLF) were stimulated with Mock and various concentraion of TNF-α (2, 10, and 100 ng/mL) for 24 hrs and with TNF-α (10 ng/mL) for 48 hrs, and the supernatents from the cells were collected for Enzyme Linked Immunosorbent Assay (ELISA) for MMP-1 and MMP-13.

Results

The expression of MMPs in PF, GF, PDLF after stimulation with SP and CGRP were not changed compared with Mock stimulation for 4 hrs and 24 hrs. The expression of MMP-1, -12, -13 24 hrs after stimulation with TNF-α were upregulated, however the expression of TIMP-3 in PF, GF, PDLF after stimulation with TNF-α were downregulated. TNF-α (2 ng/mL, 10 ng/mL, 100 ng/mL) increased MMP-1 and MMP-12 expression in PF dose dependently for 24 hrs.

Conclusions

TNF-α in the area of inflammation may play an important role in regulating the remodeling of dentin, cementum, and alveolar bone.

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Original Articles
The effect of neuropeptides on secretion of Interleukin-8 (IL-8)
Kyung-Jun Kim, Sang-Hyuk Park, Kyoung-Kyu Choi, Sang-Jin Park
J Korean Acad Conserv Dent 2006;31(3):153-160.   Published online May 31, 2006
DOI: https://doi.org/10.5395/JKACD.2006.31.3.153
AbstractAbstract PDFPubReaderePub

We investigated the secretion of Interleukin-8 (IL-8) from ginviva and periodontal ligament stimulated with Substance P (SP) and Calcitonin Gene-related Peptide (CGRP). Gingiva (GF), periodontal ligament (PDLF) and pulp (PF) tissues were collected from extracted intact 3rd molars.

Cultured cells were stimulated with different concentrations of SP for 4 hrs, and stimulated with SP, CGRP and Tumor Necrosis Factor-α (TNF-α) for 8 hrs. Then RNase Protection Assay was carried out. ELISA was performed using supernatants of stimulated cells for quantitative analysis of IL-8. Results were assessed using student t-test with significance of P < 0.05.

According to this study, the results were as follows:

IL-8 mRNA was detected in all type of cells studied (PF, GF and PDLF).

IL-8 mRNA expression was not increased after stimulating 4 hrs with SP (10-5M) and SP (10-8M) compared with Mock stimulation in all type of cells studied.

IL-8 mRNA expression was not increased after stimulating 8 hrs with SP (10-4M) and CGRP (10-6M) compared with Mock stimulation in all type of cells studied.

TNF-α(2 ng/ml) increased the expression of IL-8 mRNA in all kind of cells studied.

The secretion of IL-8 from GF was increased 8 hrs after the stimulation with CGRP (10-6M) (p < 0.05).

The secretion of IL-8 from PDLF was increased 8 hrs after the stimulation with SP (10-4M) (p < 0.05).

Calcitonin Gene-related Peptide (CGRP) increased Interleukin-8 (IL-8) which plays an important role in chemotaxis of neutrophil in Calcitonin Gene-related Peptide (CGRP) gingival tissue, whereas Substance P increased the secretion of IL-8 from periodontal ligament.

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MMP and TIMP production in periodontal ligament fibroblasts stimulated by Prevotella nigrescens lipopolysaccharide
Won-Kyung Yang, WooCheol Lee, Mi-Ri Kim, Ho-Hyun Son
J Korean Acad Conserv Dent 2005;30(5):372-384.   Published online September 30, 2005
DOI: https://doi.org/10.5395/JKACD.2005.30.5.372
AbstractAbstract PDFPubReaderePub

The purpose of this study was to monitor the secretion of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) by human periodontal ligament (PDL) fibroblasts stimulated with Prevotella nigrescens lipopolysaccharide (LPS), and to examine the effect of calcium hydroxide treatment on P. nigrescens LPS.

LPS was extracted and purified from anaerobically cultured P. nigrescens. PDL fibroblasts were stimulated by the LPS (0, 0.1, 1, 10 ug/ml) or LPS (10 ug/ml) pretreated with 12.5 mg/ml of Ca(OH)2 for 3 days, for various periods of time (12, 24, 48 h). Immunoprecipitation were performed for protein level analysis of MMP-1, MMP-2 and TIMP-1. Total RNA was isolated and real-time quantitative polymerase chain reaction (PCR) was performed for quantification of MMP-1 mRNA.

According to this study, the results were as follows:

1. The production of MMP-1 by stimulation with P. nigrescens LPS increased in time-dependent manner, and showed maximum value at 48 h in both protein and mRNA level. But there was no dose-dependent increase.

2. MMP-2 production time-dependently increased when stimulated with 1 and 10 ug/ml LPS, but there was no dose-dependent increase.

3. TIMP-1 production increased to 24 h, but decreased at 48 h. It increased when stimulated with 0.1 and 1 ug/ml LPS, but suppressed at 10 ug/ml.

4. P. nigrescens LPS pretreated with Ca(OH)2 markedly downregulated MMP-1 gene expression.

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Tissue engineering of dental pulp on type I collagen
Gwang-Hee Lee, Sung-Yoon Huh, Sang-Hyuk Park
J Korean Acad Conserv Dent 2004;29(4):370-377.   Published online July 31, 2004
DOI: https://doi.org/10.5395/JKACD.2004.29.4.370
AbstractAbstract PDFPubReaderePub

The purpose of this study was to regenerate human dental pulp tissues similar to native pulp tissues. Using the mixture of type I collagen solution, primary cells collected from the different tissues (pulp, gingiva, and skin) and NIH 3T3 (1 × 105 cells/ml/well) were cultured at 12-well plate at 37℃ for 14 days. Standardized photographs were taken with digital camera during 14 days and the diameter of the contracted collagen gel matrix was measured and statistically analyzed with student t-test. As one of the pulp tissue engineering, normal human dental pulp tissue and collagen gel matrix cultured with dental pulp cells for 14 days were fixed and stained with Hematoxyline & Eosin.

According to this study, the results were as follows:

1. The contraction of collagen gel matrix cultured with pulp cells for 14 days was significantly higher than other fibroblasts (gingiva, skin) (p < 0.05).

2. The diameter of collagen gel matrix cultured with pulp cells was reduced to 70.4% after 7 days, and 57.1% after 14 days.

3. The collagen gel without any cells did not contract, whereas the collagen gel cultured with gingiva and skin showed mild contraction after 14 days (88.1% and 87.6% respectively).

4. The contraction of the collagen gel cultured with NIH 3T3 cells after 14 days was higher than those cultured with gingival and skin fibroblasts, but it was not statistically significant (72.1%, p > 0.05).

5. The collagen gel matrix cultured with pulp cells for 14 days showed similar shape with native pulp tissue without blood vessels.

This approach may provide a means of engineering a variety of other oral tissue as well and these cell behaviors may provide information needed to establish pulp tissue engineering protocols.

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