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The calcium dynamics of human dental pulp stem cells stimulated with tricalcium silicate-based cements determine their differentiation and mineralization outcome.

ABSTRACT: Calcium (Ca²⁺) signalling plays an indispensable role in dental pulp and dentin regeneration, but the Ca²⁺ responses of human dental pulp stem cells (hDPSCs) stimulated with tricalcium silicate-based (TCS-based) dental biomaterials remains largely unexplored. The objective of the present study was to identify and correlate extracellular Ca²⁺ concentration, intracellular Ca²⁺ dynamics, pH, cytotoxicity, gene expression and mineralization ability of human dental pulp stem cells (hDPSCs) stimulated with two different TCS-based biomaterials: Biodentine and ProRoot white MTA. The hDPSCs were exposed to the biomaterials, brought in contact with the overlaying medium, with subsequent measurements of extracellular Ca²⁺ and pH, and intracellular Ca²⁺ changes. Messenger RNA expression (BGLAP, TGF-?, MMP1 and BMP2), cytotoxicity (MTT and TUNEL) and mineralization potential (Alizarin red and Von Kossa staining) were then evaluated. Biodentine released significantly more Ca²⁺ in the ?-MEM medium than ProRoot WMTA but this had no cytotoxic impact on hDPSCs. The larger Biodentine-linked Ca²⁺ release resulted in altered intracellular Ca²⁺ dynamics, which attained a higher maximum amplitude, faster rise time and increased area under the curve of the Ca²⁺ changes compared to ProRoot WMTA. Experiments with intracellular Ca²⁺ chelation, demonstrated that the biomaterial-triggered Ca²⁺ dynamics affected stem cell-related gene expression, cellular differentiation and mineralization potential. In conclusion, biomaterial-specific Ca²⁺ dynamics in hDPSCs determine differentiation and mineralization outcomes, with increased Ca²⁺ dynamics enhancing mineralization.

SUBMITTER: Rathinam E

PROVIDER: S-EPMC7804324 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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The calcium dynamics of human dental pulp stem cells stimulated with tricalcium silicate-based cements determine their differentiation and mineralization outcome.

Rathinam Elanagai E Govindarajan Srinath S Rajasekharan Sivaprakash S Declercq Heidi H Elewaut Dirk D De Coster Peter P Martens Luc L Leybaert Luc L

Scientific reports 20210112 1

Calcium (Ca2+) signalling plays an indispensable role in dental pulp and dentin regeneration, but the Ca2+ responses of human dental pulp stem cells (hDPSCs) stimulated with tricalcium silicate-based (TCS-based) dental biomaterials remains largely unexplored. The objective of the present study was to identify and correlate extracellular Ca2+ concentration, intracellular Ca2+ dynamics, pH, cytotoxicity, gene expression and mineralization ability of huma ...[more]

PMID: 33436827

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Project description:The purpose of this study was to compare the cytotoxic effects and mineralization activity of three calcium silicate-based root canal sealers to those of a conventional resin-based sealer. Experiments were performed using human dental pulp stem cells grown in a monolayer culture. The root canal sealers tested in this study were EndoSequence BC Sealer (Brasseler), BioRoot RCS (Septodont), Endoseal MTA (Maruchi), and AH Plus (Dentsply DeTrey). Experimental disks 6 mm in diameter and 3 mm in height were made and stored in a 100% humidity chamber at 37 °C for 72 h to achieve setting. The cytotoxicity of various root canal sealers was evaluated using a methyl-thiazoldiphenyl-tetrazolium (MTT) assay. To evaluate cell migration ability, a scratch wound healing method was used, and images of the scratch area were taken using a phase-contrast microscope. Cell morphology was evaluated by a scanning electron microscope after direct exposure for 72 h to each sealer disk. In the cell viability assay, there were no significant differences between the EndoSequence BC, BioRoot RCS, Endoseal MTA, and control groups in any experimental period (p > 0.05). In the cell migration assay, there were no significant differences between the EndoSequence BC, Endoseal MTA, and control groups in any experimental period (p > 0.05). BioRoot RCS exhibited slower cell migration relative to EndoSequence BC and Endoseal MTA for up to 72 h (p < 0.05). Conversely, it showed a similar wound healing percentage at 96 h (p > 0.05). In an evaluation of cell morphology, cells in direct contact with EndoSequence BC, BioRoot RCS, and Endoseal MTA disks showed superior spreading compared to those in contact with the AH Plus disk. In an Alizarin red staining assay, EndoSequence BC, BioRoot RCS, and Endoseal MTA showed a significant increase in mineralized nodule formation compared to the AH Plus group (p < 0.05). In conclusion, all calcium silicate-based root canal sealers tested in this study showed good biological properties and mineralization activity compared to conventional resin-based sealer.

Project description:Objective:The aim of this study was to histologically examine the tissue reaction of three different calcium silicate cements in the closure of perforations in rat incisor teeth. Material and Methods. An experimental lateral root perforation with pulp exposure was performed in 32 lower incisors of 16 male Wistar albino rats. They were randomly assigned into three test groups (each including eight teeth) that were filled either by Biodentine (BD) or MicroMega mineral trioxide aggregate (MM-MTA) or EndoSequence root repair material putty (ESRRM putty), besides eight unperforated incisors from the other four rats (control group). The inflammatory response and healing process were evaluated histologically and scored after one and four weeks. Differences among groups were tested by Kruskal-Wallis tests at P ? 0.05. Results:In the first week, BD produced more inflammatory response in the pulpal (score 3) than other materials (score 2). Only ESRRM putty showed odontoblast-like cells in 50%, 25% dentine-like deposit, 25% evidence of bone deposition in the drilling site (score 2), and minimum periodontal ligament (PDL) necrosis and disorganization (25%, score 2). After one month, all groups had healthy pulpal tissue, but 25% of ESRRM putty retained score 1 inflammatory response, and 50% of the BD case had an incomplete palisading odontoblast layer (score 3). A thick and regular dentine bridge deposition was seen in the ESRRM putty group in comparison with MM-MTA and BD cases. The cortical plate healing in all ESRRM putty samples was complete (score 3), while an incomplete closure was seen in MM-MTA and BD groups (score 2). Both the MM-MTA and ESRRM putty groups had fully organized PDL (score 2), while in 50% of BD cases, a necrotizing area and disorganized PDL with inflammatory cells infiltration were still present. Statistically significant differences in the scores of any histologic parameters among the three tested materials were observed neither in the 1st nor in the 4th weeks of the experimental period. Conclusion:Better tissue compatibility and repair of pulpal and periodontal tissue have been detected after lateral perforation in the root of rat incisors when treated with ESRRM putty than MM-MTA and BD. However, the difference was not significant.

Project description:X-linked hypophosphatemia (XLH) is a skeletal disease caused by inactivating mutations in the PHEX gene. Mutated or absent PHEX protein/enzyme leads to a decreased serum phosphate level, which cause mineralization defects in the skeleton and teeth (osteomalacia/odontomalacia). It is not yet altogether clear whether these manifestations are caused solely by insufficient circulating phosphate availability for mineralization or also by a direct, local intrinsic effect caused by impaired PHEX activity. Here, we evaluated the local role of PHEX in a 3-dimensional model of extracellular matrix (ECM) mineralization. Dense collagen hydrogels were seeded either with human dental pulp cells from patients with characterized PHEX mutations or with sex- and age-matched healthy controls and cultured up to 24 d using osteogenic medium with standard phosphate concentration. Calcium quantification, micro-computed tomography, and histology with von Kossa staining for mineral showed significantly lower mineralization in XLH cell-seeded scaffolds, using nonparametric statistical tests. While apatitic mineralization was observed along collagen fibrils by electron microscopy in both groups, Raman microspectrometry indicated that XLH cells harboring the PHEX mutation produced less mineralized scaffolds having impaired mineral quality with less carbonate substitution and lower crystallinity. In the XLH cultures, immunoblotting revealed more abundant osteopontin (OPN), dentin matrix protein 1 (DMP1), and matrix extracellular phosphoglycoprotein (MEPE) than controls, as well as the presence of fragments of these proteins not found in controls, suggesting a role for PHEX in SIBLING protein degradation. Immunohistochemistry revealed altered OPN and DMP1 associated with an increased alkaline phosphatase staining in the XLH cultures. These results are consistent with impaired PHEX activity having local ECM effects in XLH. Future treatments for XLH should target both systemic and local manifestations.

Project description:ObjectiveThis study evaluated the bioactivity and physicochemical properties of three commercial calcium silicate-based endodontic materials (MTA, EndoSequence Root Repair Material putty, and Biodentine™). Material and Methods. Horizontal sections of 3 mm thickness from 18 root canals of human teeth were subjected to biomechanical preparation with WaveOne Gold large rotary instruments. The twelve specimens were filled with three tested materials (MM-MTA, EndoSequence Root Repair Material putty, and Biodentine™) and immersed in phosphate-buffered saline for 7 and 30 days. After this period of time, each specimen of each material was processed for morphological observation, surface precipitates, and interfacial dentin using SEM. In addition, the surface morphology of the set materials, without soaking in phosphate-buffered solution after one day and after 28 days stored in phosphate-buffered saline, was evaluated using SEM; also, the pH of the soaking water and the amount of calcium ions released from the test materials were measured by using an inductively coupled plasma-optical emission spectroscopy test. Data obtained were analyzed using one-way analysis of variance and Tukey's honest significant difference test with a significance level of 5%.ResultThe formation of precipitates was observed on the surfaces of all materials at 1 week and increased substantially over time. Interfacial layers in some areas of the dentin-cement interface were found from one week of immersion. All the analyzed materials showed alkaline pH and capacity to release calcium ions; however, the concentrations of released calcium ions were significantly more in Biodentine and ESRRM putty than MM-MTA (P < 0.05). ESRRM putty maintained a pH of around 11 after 28 days.ConclusionCompared with MM-MTA, Biodentine and ESRRM putty showed significantly more calcium ion release. However, exposure of three tested cements to phosphate-buffered solution resulted in precipitation of apatite crystalline structures over both cement and dentin that increased over time. This suggests that the tested materials are bioactive.

Dataset Information

The calcium dynamics of human dental pulp stem cells stimulated with tricalcium silicate-based cements determine their differentiation and mineralization outcome.

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The calcium dynamics of human dental pulp stem cells stimulated with tricalcium silicate-based cements determine their differentiation and mineralization outcome.

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