Highlights
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The experimental cement, TCS 50, does re-mineralize demineralized dentin.
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The incorporation of biomimetic analogs promoted re-mineralization upon 6-week storage.
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Re-mineralization was incomplete at 6 weeks, even for TCS 50 with biomimetic analogs.
Abstract
Objectives
To characterize the re-mineralization potential of an experimental zirconium oxide (ZrO 2 ) containing tricalcium silicate (TCS) cement, TCS 50, with the incorporation of biomimetic analogs at demineralized dentin.
Methods
Class-I cavities were prepared in non- carious human third molars. The dentin cavities were demineralized using a pH-cycling protocol, involving 50 cyclic immersions in pH-4.8 and pH-7 baths for 0.5 h and 2.5 h, successively. The cavities were filled with TCS 50 with/without biomimetic analogs (3% polyacrylic acid, 8% sodium trimetaphosphate) being added to the mixed TCS 50 cement prior to application. The commercial hCSCs Biodentine (Septodont) and ProRoot MTA (Dentsply Sirona) served as controls. After 1 and 6 weeks storage in simulated body fluid (SBF), the polished specimen cross-sections were chemically characterized using a field-emission-gun Electron Probe Micro-Analysis (Feg- EPMA).
Results
EPMA line-scans and elemental mappings confirmed early re-mineralization induced by TCS 50 at 1 week. When biomimetic analogs were added to TCS 50, re-mineralization was more efficient after 6 weeks; the relative depth and intensity of re-mineralization were 79.7% and 76.6%, respectively, being significantly greater than at 1 week (pSignificance: The experimental TCS-based cement, TCS 50, proved to be capable of re-mineralizing artificially demineralized dentin. The incorporation of biomimetic analogs promoted re- mineralization upon 6-week SBF storage. However, re-mineralization appeared incomplete, this even for TCS 50 to which biomimetic analogs were added and upon 6-week SBF storage.
1
Introduction
According to the concept of minimal invasive dentistry, caries-infected dentin should be removed, while caries-affected dentin is better preserved and re-mineralized to avoid potential pulp exposure in case of deep carious lesions . In this respect, hydraulic calcium silicate cements (hCSCs), such as the commercially available Biodentine (Septodont, Saint Maur des Fosses, France) and ProRoot MTA (Dentsply Sirona, York, PA, USA), are not only indicated to treat various pulp-related endodontic complications , but could also serve as dentin re-mineralization agents . Previous research revealed that both the hCSCs Biodentine (Septodont) and ProRoot MTA (Dentsply Sirona) re- mineralized artificially demineralized dentin early at 1 week; however, the re-mineralization efficacy was limited, as further down the cavity demineralized dentin remained even upon 6 months . A very plausible explanation for this phenomenon is the fast and densely formed re-mineralization zone that hinders further infiltration of calcium (Ca) deep into the remaining demineralized dentin; hence, re-mineralization remains incomplete . In an attempt to overcome this re-mineralization block and so to improve the re-mineralization potential of hCSCs, the release of Ca from the hydrated cement should be controlled better, i.e. slowed down and prolonged.
An experimental tricalcium silicate (TCS) based cement, being referred as TCS 50, was subsequently developed . The powder of TCS 50 consists of 50 wt% TCS (diameter of ±10 μm; Mineral Research Processing, Meyzieu, France) and 50 wt% zirconium oxide (ZrO 2 ; diameter of ±200 nm; Tosoh, Tokyo, Japan); 1 M CaCl 2 is employed as liquid to accelerate the setting process. The powder and liquid are mixed at a weight ratio of 3:1. A previous study revealed that TCS 50 possessed a mini-fracture toughness comparable to that of the commercial cement Biodentine (Septodont); the Ca release was reduced initially, while reached a prolonged release thereafter . In addition, TCS 50 appeared more biocompatible to human dental pulp fibroblasts than Biodentine (Septodont) . These findings indicated that TCS 50 may be a promising cement formulation not only to serve as a pulp–capping material, but also as a dentin re-mineralization agent.
During the process of dentin mineralization, extracellular matrix proteins play an important role in controlling the nucleation and growth of hydroxyapatite . Recently, biomimetic analogs of the matrix proteins have been applied for re-mineralization of artificial caries-affected dentin lesions . In literature, dual biomimetic analogs, such as polyacrylic acid [PA, (C 3 H 4 O 2 ) n ] in combination with sodium trimetaphosphate (STMP, Na 3 P 3 O 9 ), have been reported to promote ordered deposition of intrafibrillar apatite platelets within collagen fibrils . PA participates in the recruitment of pre-nucleation clusters and also in the stabilization of metastable amorphous calcium-phosphate nano-precursors . The nano-precursors infiltrate into the network of collagen fibrils and transform into intrafibrillar apatite using the fibrils as templates . STMP serves as analog of the matrix phosphoproteins and is so involved in the phosphorylation process of collagen fibrils . Nevertheless, this biomimetic re-mineralization approach should be regarded as a proof-of-principle concept, since the majority of these studies dissolved biomimetic analogs in simulated body fluid (SBF) , and therefore is not directly of clinical relevance. This necessitated the introduction of a more optimal re-mineralization protocol . In a previous study, biomimetic analogs were incorporated into a commercial hCSC cement to re-mineralize caries-like demineralized dentin, and this was shown to enhance the re-mineralization efficacy .
In continuation of previous work , the objective of this study was therefore to characterize the chemical interplay and to quantify the re-mineralization potential of the experimentally synthesized cement TCS 50 when biomimetic analogs were added, this when applied onto demineralized dentin in tooth cavities that are stored in SBF for different time periods. The market-representative hCSCs Biodentine (Septodont) and ProRoot MTA (Dentsply Sirona) were selected as controls. The null hypotheses tested were (1) that TCS 50 is capable of re-mineralizing demineralized dentin, (2) that the incorporation of biomimetic analogs does not affect the re-mineralization potential of TCS 50, (3) that the re- mineralization efficacy does not increase with the storage period, and finally (4) that there is no difference in re-mineralization effectiveness of TCS 50 versus that of the two control hCSCs.
2
Materials and methods
2.1
Specimen preparation
Thirty-six healthy human third molars (gathered as approved by the Commission for Medical Ethics of KU Leuven under the file number S57622) were stored in 0.5% chloramine solution at 4 °C and were used within 3 months after extraction. Each tooth was mounted in a gypsum block to facilitate manipulation. The occlusal third of the crown was removed using a diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA). The dentin surface was verified for absence of enamel and/or pulp tissue under a stereo-microscope (Wild M5A, Heerbrugg, Switzerland). The surface was then coated with a layer of bonding agent using the self-etch adhesive Clearfil SE Bond (Kuraray Noritake, Tokyo, Japan). A standard box- type class-I cavity with the floor ending at mid-coronal dentin (3 × 1.5 mm wide, 0.5 mm deep) was prepared in each tooth using a medium-grit (107 μm) diamond bur (842, Komet, Lemgo, Germany) fixed in a water-cooled high-speed turbine mounted in a custom-adapted Micro Specimen Former (University of Iowa, Iowa City, IA, USA). The cavity bottoms were verified for absence of pulp tissue under the stereo-microscope (Wild M5A), after which the tooth was removed from the gypsum block. All outer surfaces except the occlusal surface of the tooth were coated with a layer of the bonding agent (Clearfil SE Bond, Kuraray Noritake) as well. Each tooth cavity was next demineralized following a pH-cycling protocol, which consisted of 50 cyclic immersions of the tooth in 10 ml demineralization solution for 0.5 h, followed by immersion in 10 ml re-mineralization solution for 2.5 h, both at room temperature; the solutions were refreshed for each cycle. The demineralization solution consisted of 1.5 mM CaCl 2 , 0.9 mM KH 2 PO 4 , and 5 mM NaN 3; the pH was adjusted to 4.8 with acetic acid. The re-mineralization solution consisted of 1.5 mM CaCl 2 , 0.9 mM NaH 2 PO 4 , 0.13 M KCl, and 5 mM NaN 3 ; the pH was adjusted to 7.0 with 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer.
After pH cycling, the tooth was rinsed in the ultrasonic bath with distilled water for 30 min and air-dried, after which they were filled with TCS 50, Biodentine (Septodont) or ProRoot MTA (Dentsply Sirona). TCS 50 is composed of 50 wt% TCS (diameter of ±10 μm; Mineral Research Processing) and 50 wt% ZrO 2 (diameter of ±200 nm; Tosoh) in the powder; 1 M CaCl 2 was used as liquid. The powder was mixed with liquid at the weight ratio of 3:1 for 30 s using a capsule mixer (RotoMix Capsule Mixer, 3 M ESPE, Seefeld, Germany). Biodentine (Septodont) and ProRoot MTA (Dentsply Sirona) were mixed according to the respective manufacturer’s instructions. Biomimetic analogs that include 3% PA powder (molecular weight: 1800 g/mol; Sigma-Aldrich, St. Louis, MO, USA) and 8% STMP powder (molecular weight: 305.89 g/mol; Sigma-Aldrich) were added to the mixed cement prior to application, this for half of the specimens. For both the experimental cement TCS 50 and Biodentine (Septodont), biomimetic analogs were added to the mixed cement immediately after cement mixing using the capsule mixer (RotoMix Capsule Mixer). For ProRoot MTA (Dentsply Sirona), the cement powder was mixed with liquid till achieving a putty-like consistency; biomimetic analogs were then immediately added. All materials used in the present study are listed in Table 1 . A plastic spatula (provided with Biodentine, Septodont) was used to condense the cement, while the tooth was held on a vibrating table (Porex, Aachen, Germany) in order to ensure proper adaptation of the cement to the dentin cavity walls.
| Cement | Manufacturer | Lot number | Composition a |
|---|---|---|---|
| TCS 50 | NA b | NA b | Powder: tricalcium silicate, zirconium oxide |
| Liquid: distilled water, calcium chloride | |||
| Biodentine | Septodont | B07023 | Powder: tricalcium silicate, dicalcium silicate, calcium carbonate and oxide, iron oxide, zirconium oxide |
| Liquid: distilled water, calcium chloride, hydrosoluble polymer | |||
| ProRoot MTA | Dentsply Sirona | 0000102196 | Powder: tricalcium silicate, dicalcium silicate, tricalcium aluminate, bismuth oxide |
| Liquid: distilled water | |||
| Polyacrylic acid (PA) | Sigma-Aldrich | 04610EIV | |
| Sodium trimetaphosphate (STMP) | Sigma-Aldrich | SLBC9410V |
a The composition is based on technical information provided by the respective manufacturer.
Each tooth was immediately immersed in 10 ml SBF at 37 °C for 1 or 6 weeks (n = 3 per storage period). SBF consisted of 136.8 mM NaCl, 4.2 mM NaHCO 3 , 3.0 mM KCl, 1.0 mM K 2 HPO 4 ·3H 2 0, 1.5 mM MgCl 2 ·6H 2 O, 40 mM HCl, 2.5 mM CaCl 2 , 0.5 mM Na 2 SO 4 , 50 mM (CH 2 OH) 3 CNH 2 ; its pH was adjusted to 7.4.
2.2
Electron Probe Micro-Analysis (EPMA)
After storage, the teeth were cross-sectioned perpendicular to the cement–dentin interface by means of a water-cooled diamond saw (Isomet 1000, Buehler). All specimens were subsequently processed for electron microscopy by fixation in 2.5% glutaraldehyde for 24 h, dehydration in ascending concentrations of ethanol (25, 50, 75, and 95% for 30 min each, and finally 100% for 1 h), and finally drying by immersion in hexamethyldisilazane (HMDS) for 10 min. Upon drying, the part of the cement-demineralized dentin interface located at the bottom of the cavity and near the cavity corner was polished using an argon-ion-beam (IB-09010CP Cross Section Polisher, Jeol, Tokyo, Japan) at 5.0 kV for 7 h to achieve an ion-beam polished interfacial area of approximately 1 mm 2 . The ion-beam polished interfaces were coated by a 2-nm thick platinum–palladium (Pt–Pd) layer using a turbomolecular-pumped coater (Q150T S, Quorum, East Sussex, UK). In each specimen, the intensities of chemical elements Ca and phosphorus (P) along the interface were quantified along three 180-μm long line-scans using a field- emission-gun Electron Probe Micro Analyzer (Feg-EPMA; JXA-8530F, Jeol, Tokyo, Japan) at a spatial resolution of ±0.05 μm. The results of the EPMA line-scans analyses were imported into the software package (R3.01, R Foundation for Statistical Computing, Vienna, Austria). The position, from where dentin was demineralized and re-mineralized, was indicated by line I and line II, respectively. The position, until where dentin was re- mineralized and originally demineralized was indicated by lines III and IV, respectively. The depth of demineralization was determined as the distance between lines I and IV in μm using an automated script; the re-mineralization depth, if existed, was determined as the distance between lines II and III, this when the Ca and P concentrations were increased as compared to those within demineralized dentin. The relative re-mineralization depth (D RM ) was calculated as the ratio of the re-mineralization depth to the initial depth of demineralized dentin. To evaluate to which extent re-mineralization was achieved, the relative re-mineralization intensity (I RM ) was calculated as a percentage of the mean P intensity in re- mineralized dentin as compared with that in deeper unaffected sound dentin.
In case chemical changes were detected at the cement–dentin interface, representative points were selected and the elemental composition of these points was quantitatively analyzed, based on which also the Ca/P weight ratio was calculated. In addition, the interface of one representative specimen per experimental group (cement) and storage period was chemically mapped for Ca, P, carbon (C) and zirconium (Zr).
X-ray profiles and element quantifications were performed at 15 kV (voltage) and 15 μA (probe current) under high vacuum. No peak overlapping was detected.
2.3
Statistical analysis
D RM and I RM were statistically analyzed by Kruskal Nemenyi multiple comparison tests to assess the effect of cement type, biomimetic analog as well as storage period on the re-mineralization efficacy. Tests were performed at a significance level of α = 0.05 using a software package (R3.01).
2
Materials and methods
2.1
Specimen preparation
Thirty-six healthy human third molars (gathered as approved by the Commission for Medical Ethics of KU Leuven under the file number S57622) were stored in 0.5% chloramine solution at 4 °C and were used within 3 months after extraction. Each tooth was mounted in a gypsum block to facilitate manipulation. The occlusal third of the crown was removed using a diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA). The dentin surface was verified for absence of enamel and/or pulp tissue under a stereo-microscope (Wild M5A, Heerbrugg, Switzerland). The surface was then coated with a layer of bonding agent using the self-etch adhesive Clearfil SE Bond (Kuraray Noritake, Tokyo, Japan). A standard box- type class-I cavity with the floor ending at mid-coronal dentin (3 × 1.5 mm wide, 0.5 mm deep) was prepared in each tooth using a medium-grit (107 μm) diamond bur (842, Komet, Lemgo, Germany) fixed in a water-cooled high-speed turbine mounted in a custom-adapted Micro Specimen Former (University of Iowa, Iowa City, IA, USA). The cavity bottoms were verified for absence of pulp tissue under the stereo-microscope (Wild M5A), after which the tooth was removed from the gypsum block. All outer surfaces except the occlusal surface of the tooth were coated with a layer of the bonding agent (Clearfil SE Bond, Kuraray Noritake) as well. Each tooth cavity was next demineralized following a pH-cycling protocol, which consisted of 50 cyclic immersions of the tooth in 10 ml demineralization solution for 0.5 h, followed by immersion in 10 ml re-mineralization solution for 2.5 h, both at room temperature; the solutions were refreshed for each cycle. The demineralization solution consisted of 1.5 mM CaCl 2 , 0.9 mM KH 2 PO 4 , and 5 mM NaN 3; the pH was adjusted to 4.8 with acetic acid. The re-mineralization solution consisted of 1.5 mM CaCl 2 , 0.9 mM NaH 2 PO 4 , 0.13 M KCl, and 5 mM NaN 3 ; the pH was adjusted to 7.0 with 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer.
After pH cycling, the tooth was rinsed in the ultrasonic bath with distilled water for 30 min and air-dried, after which they were filled with TCS 50, Biodentine (Septodont) or ProRoot MTA (Dentsply Sirona). TCS 50 is composed of 50 wt% TCS (diameter of ±10 μm; Mineral Research Processing) and 50 wt% ZrO 2 (diameter of ±200 nm; Tosoh) in the powder; 1 M CaCl 2 was used as liquid. The powder was mixed with liquid at the weight ratio of 3:1 for 30 s using a capsule mixer (RotoMix Capsule Mixer, 3 M ESPE, Seefeld, Germany). Biodentine (Septodont) and ProRoot MTA (Dentsply Sirona) were mixed according to the respective manufacturer’s instructions. Biomimetic analogs that include 3% PA powder (molecular weight: 1800 g/mol; Sigma-Aldrich, St. Louis, MO, USA) and 8% STMP powder (molecular weight: 305.89 g/mol; Sigma-Aldrich) were added to the mixed cement prior to application, this for half of the specimens. For both the experimental cement TCS 50 and Biodentine (Septodont), biomimetic analogs were added to the mixed cement immediately after cement mixing using the capsule mixer (RotoMix Capsule Mixer). For ProRoot MTA (Dentsply Sirona), the cement powder was mixed with liquid till achieving a putty-like consistency; biomimetic analogs were then immediately added. All materials used in the present study are listed in Table 1 . A plastic spatula (provided with Biodentine, Septodont) was used to condense the cement, while the tooth was held on a vibrating table (Porex, Aachen, Germany) in order to ensure proper adaptation of the cement to the dentin cavity walls.
| Cement | Manufacturer | Lot number | Composition a |
|---|---|---|---|
| TCS 50 | NA b | NA b | Powder: tricalcium silicate, zirconium oxide |
| Liquid: distilled water, calcium chloride | |||
| Biodentine | Septodont | B07023 | Powder: tricalcium silicate, dicalcium silicate, calcium carbonate and oxide, iron oxide, zirconium oxide |
| Liquid: distilled water, calcium chloride, hydrosoluble polymer | |||
| ProRoot MTA | Dentsply Sirona | 0000102196 | Powder: tricalcium silicate, dicalcium silicate, tricalcium aluminate, bismuth oxide |
| Liquid: distilled water | |||
| Polyacrylic acid (PA) | Sigma-Aldrich | 04610EIV | |
| Sodium trimetaphosphate (STMP) | Sigma-Aldrich | SLBC9410V |
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