Elsevier

Dental Materials

Volume 24, Issue 11, November 2008, Pages 1486-1494
Dental Materials

Induction of specific cell responses to a Ca3SiO5-based posterior restorative material

https://doi.org/10.1016/j.dental.2008.02.020Get rights and content

Abstract

Objectives

A Ca3SiO5-based cement has been developed to circumvent the shortcomings of traditional filling materials. The purpose of this work was to evaluate its genotoxicity, cytotoxicity and effects on the target cells’ specific functions.

Methods

Ames’ test was applied on four Salmonella typhimurium strains. The micronuclei test was studied on human lymphocytes. The cytotoxicity (MTT test), the Comet assay and the effects on the specific functions by immunohistochemistry were performed on human pulp fibroblasts.

Results

Ames’ test did not show any evidence of mutagenicity. The incidence of lymphocytes with micronuclei and the percentage of tail DNA in the Comet assay were similar to the negative control. The percentage of cell mortality with the new cement as performed with the MTT test was similar to that of biocompatible materials such as mineral trioxide aggregate (MTA) and was less than that obtained with Dycal. The new material does not affect the target cells’ specific functions such as mineralization, as well as expression of collagen I, dentin sialoprotein and Nestin.

Significance

The new cement is biocompatible and does not affect the specific functions of target cells. It can be used safely in the clinic as a single bulk restorative material without any conditioning treatment. It can be used as a potential alternative to traditionally used posterior restorative materials.

Introduction

Commonly used direct restorative materials for Class I and II cavities are resin composites and amalgams [1], [2]. In the early 1980s, amalgam restorations were reported to release mercury vapors which may be harmful to the environment, the dentist as well as the patient [3].

Direct composite restorations have gradually been used to replace amalgam for anterior restorations and small- to moderate-sized posterior restorations. In contrast to amalgam, resin composites enable micro-mechanical retention by the use of different bonding techniques. Yet there is still some concern with composite resin wear resistance in high-stress situations, polymerization shrinkage, microleakage, and unreacted monomer and toxic ingredient release [4], [5], [6].

Search for a replacement for amalgam and resin composites has been ongoing for many years. Calcium hydroxide (Dycal®) is one of the most widely used pulp capping agents. Its basic pH is the main reason for its apparent toxicity in vitro [7]. However, it has been demonstrated that a dentin bridge formation can be obtained with this material 3 months after capping human teeth with mild to moderate chronic inflammation, mild hyperemia and necrosis [7], [8].

Recent research focused on the use of biocompatible materials such as Portland cement. Mineral trioxide aggregate developed in the 1990s as a root-end filling material has a similar constitution to Portland cement and is composed primarily of tricalcium and dicalcium silicate [9]. It is known as a biocompatible material. In vitro, a high rate of cell viability was reported with MTA extracts with a methyltetrazoilum (MTT) assay [10], [11], [12]. Additionally, MTA used for pulp capping or partial pulpotomy stimulates reparative dentin and complete bridge formation in vivo after 2 months with no signs of inflammation [8], [13], [14]. However, the setting time of MTA is 2 h 45 min which is too long for a material to be used as a dental restorative material [15]. Moreover, the mechanical properties of both Dycal and MTA are not compatible for use as dental restorative material.

Tricalcium silicate is the main constituent of MTA, and the main raw material in Portland cement. It is known that Ca3SiO5 possesses hydraulic property and the spontaneous development of strength on hydration. But its setting time is too long and its compressive strength hardly reaches 20.2 MPa after 28 days to meet the need of clinical applications as a restorative material [16]. Calcium chloride is one of the most effective accelerators of hydration and setting in Portland cement pastes. Although the addition of CaCl2 up to 15% in the liquid phase into Ca3SiO5 decreased the final setting time from 180 to 90 min, the compressive strength remained weak (23.46 MPa) at 7 days [17]. The use of superplasticisers as very effective dispersing agents to reduce the water content was used in fast setting Portland cements. This has been shown to lower the setting time to 7 min but the compressive resistance did not exceed 50 MPa even after 28 days [18].

Based on Portland cement properties, a Ca3SiO5-based material for direct restorative posterior fillings has been developed in the authors’ laboratory. The material is inorganic and non-metallic. It is composed of Ca3SiO5, CaCO3, ZrO2, water and a superplasticising admixture to reduce the water content of the mix and to retain its workability. This material is presented in the form of a powder and a liquid and can be prepared by mixing with an amalgamator. The new Ca3SiO5 cement is compatible with working in the clinic. It has a setting time of 10 min and was developed to be used in direct and indirect pulp capping procedures as a single application bulk restorative material without any cavity conditioning treatment. Since it may be directly applied to the dental pulp, its biological properties were compared to biomaterials usually used in pulp capping procedures such as MTA and Dycal.

Since this material belongs to a new class of restorative materials, its biocompatibility is questioned and in this paper its cytotoxicity and genotoxicity are investigated. The effect it may have on the specific functions of target cells was also evaluated.

Section snippets

Reagents

All materials used for culture media preparation were purchased from Gibco BRL (Life Technologies Inc., Grand Island, NY, USA) unless otherwise specified. Minimum Essential Medium (MEM) was supplemented with 10% fetal bovine serum; 100 UI/ml penicillin; 100 μg/ml streptomycin (Biowhittaker, Gagny, France) and 0.25 μg/ml amphotericin B (Fungizone®). Chemicals were obtained from Sigma–Aldrich (Sigma Chemicals Corp., St. Louis, MO) unless otherwise stated.

Teeth

For pulp cell cultures, normal immature third

Determination of the toxicity with or without dentin disc interposition

When the toxicity was evaluated indirectly through a dentin slice, the analysis of variance failed to show a statistical difference between the new cement, Pro Root MTA, and Dycal (ns) (Table 1). None of the materials was cytotoxic. However, when the toxicity was evaluated without dentin slice interposition, the analysis of variance showed a statistically significant difference among the three materials (P < 0.001). The Duncan test showed that Dycal displayed a higher cytotoxicity than MTA and

Discussion

The biocompatibility of the new cement is shown in this study by the absence of cytotoxicity and genotoxicity and the fact that the new material does not affect the cytodifferentiation of human pulp fibroblasts in odontoblastic cells.

Although Portland cements are known as non-toxic, in this work, 3 tests were performed to evaluate the genotoxicity of the new Ca3SiO5 cement after solubilisation in hydrophilic or hydrophobic conditions. These tests were performed because the cement developed here

Conclusions

The results of the current study need to be confirmed in vivo and suggest that this new Ca3SiO5 cement could be used as a direct pulp capping agent but also as a lining agent. This material would possibly induce the secretion of reactionary dentin often considered as a preliminary step for pulp healing after caries removal. The good handling properties of this material associated with its biological, mechanical and physical properties let us think that this material could be used as a pulp

Acknowledgements

This work was supported by institutional funding from the French “Ministère de l’éducation nationale, de l’enseignement supérieur et de la recherche”. The authors wish to thank Dr. Jean-Charles Gardon for providing the third molars used in this work.

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