Enhanced bioactive properties of BiodentineTM modified with bioactive glass nanoparticles

Abstract Objective To prepare nanocomposite cements based on the incorporation of bioactive glass nanoparticles (nBGs) into BiodentineTM (BD, Septodent, Saint-Maur-des-Fosses Cedex, France) and to assess their bioactive properties. Material and Methods nBGs were synthesised by the sol-gel method. BD nanocomposites (nBG/BD) were prepared with 1 and 2% nBGs by weight; unmodified BD and GC Fuji IX (GIC, GC Corporation, Tokyo, Japan) were used as references. The in vitro ability of the materials to induce apatite formation was assessed in SBF by X-ray diffraction (XRD), attenuated total reflectance with Fourier transform infrared spectroscopy (ATR-FTIR), and scanning electron microscopy (SEM) with energy dispersive X-ray (EDX) analysis. BD and nBG/BD were also applied to dentine discs for seven days; the morphology and elemental composition of the dentine-cement interface were analysed using SEM-EDX. Results One and two percent nBG/BD composites accelerated apatite formation on the disc surface after short-term immersion in SBF. Apatite was detected on the nBG/BD nanocomposites after three days, compared with seven days for unmodified BD. No apatite formation was detected on the GIC surface. nBG/BD formed a wider interfacial area with dentine than BD, showing blockage of dentine tubules and Si incorporation, suggesting intratubular precipitation. Conclusions The incorporation of nBGs into BD improves its in vitro bioactivity, accelerating the formation of a crystalline apatite layer on its surface after immersion in SBF. Compared with unmodified BD, nBG/BD showed a wider interfacial area with greater Si incorporation and intratubular precipitation of deposits when immersed in SBF.


Introduction
Biodentine TM (BD), a tricalcium silicate-based cement, was developed as a dentine substitute with clinical applications, including direct and indirect pulp capping 9 and the restoration of coronal dentine 16 . For some of these applications, the material may come into direct contact with pulpal tissues or with deeply carious dentine, making its biocompatibility and ability to seal in moist environments relevant clinical properties. It is well established that the placement of a permanent, properly sealed restoration is crucial to clinical success in indirect and direct pulp therapies 11 , a property that closely relates to the bioactivity of the applied restorative material.
of the material which results in the formation of a bond between the tissues and the material" 2 . BD has been shown in vitro to induce the formation of calcium and phosphorous surface precipitates after immersion 7 and allows the formation of an interfacial layer with dentine 8,13 . The mechanism of a degradation of collagenous components occurs due to an alkaline caustic effect, which forms a porous structure that facilitates the permeation of Ca 2+ , OH -, and CO 3 2-ions, mineralising this substrate 26 . In vivo studies demonstrated the formation of reparative dentine after BD pulp capping, which is an evidence for its bioactivity, resulting in a bond with the tissue 19 ; however, there are concerns about the stability of this interfacial layer, since only amorphous-calciumphosphate has been identified, not dentine-like hydroxyapatite 13 .
Bioactive glass (BG) is a well-known bioactive ceramic material that has gained attention due to its ability to chemically bond with hard tissues through the formation of an apatite layer on its surface 12 .
This apatite layer forms following solution-mediated dissolution of the glass 12  In vitro bioactivity assay The ability of the cement materials to induce the formation of apatite was assessed in acellular SBF, which was prepared as described by Kokubo,et al. 15 (1990) using the standard ion composition (Na + 142.0,   (Figure 1a). In the case of BD, the appearance of this apatite peak was only detected after seven days of incubation (Figure 1c).

Materials and surface deposit characterisation
On the other hand, no XRD peaks were detected for the GIC before or after SBF incubation. SEM/EDX images of samples before and after SBF soaking for seven days are presented in Figure 3. In vitro bioactivity of the dentine-cement interface Cross-sectional SEM images of the cement-treated dentine interfaces after seven days of immersion  In addition, there was a distinctive mineral-rich interfacial layer within the dentine in contact with BD and the nanocomposite cements, which was thicker and had greater Si uptake in the dentine treated with nanocomposites have more prominent mineralisation silicate cements to mineralise dentine when immersed 8,21 . It is believed that the formation of this interfacial layer could be related to the good marginal seal of calcium silicate cements 8 , supported by reports in which immersion plugs 19 and increased push-out strengths 22 . For BD, this interfacial layer has also been named the "mineral dual effects of an alkaline caustic etching followed by mineral exchange 13 . This leads to the belief that spaces along the interface and via interactions with 7 . The precise role of Si uptake remains unclear, but that silica uptake in dentine may increase its acid resistance and physical strength 8 .
In the present study, this interfacial layer was on dentine with exposed tubules. It was found that when nano-BG is applied, a rod-like apatite structure is formed within the tubules 4 , whereas only a surface layer apatite onto the tubule opening was detected with micro-BG. Therefore, the use of BG with nanometric dimensions strongly favours the BG diffusion into the tubules and its consequent transformation into apatite phase. In addition, demineralised dentine can be faster remineralised by nBGs than with micron-sized BG as consequence of the substantially higher rate of dissolution of nBGs 3,24 .
It would be interesting to study the long term stability of this interfacial layer in conditions that mimic the dynamics of its possible clinical applications.
When BG has been added to other carriers, such as toothpastes, it has been demonstrated that the dentin tubule occlusion layer formed is resistant to acid challenge 5 permeability under simulated oral environment 30 .
In addition, it has been suggested that the apatite rods formed into dentine tubules after brushing with nBGs slurries would have excellent retention 4 . This is based on the observation that the continuous occluding apatite rods are tightly bonded to the dentine tubules, therefore the mechanical retention of these rods may be ensured as the angling and contours of the dentin tubule will avoid dislodgement of this interface 4 . Nevertheless, the study of the stability of the mineralised layer observed when nBG/BD was applied onto dentine would be of interest.
with nBGs to accelerate the formation of dentine-like crystalline apatite inside of dentine tubules could cement could generate a strongly mineralised seal dentinal tissue in restorative therapies.

Conclusions
The incorporation of nBGs into BD enhances BD's in vitro bioactive properties, accelerating the formation of a crystalline apatite layer on its surface after a short period of immersion in SBF and greatly enhances the formation of a mineral-rich interfacial layer when in contact with dentine.