Effects of a New Bioceramic Material on Human Apical Papilla Cells

Abstract

Background: The development of materials with bioregenerative properties is critically important for vital pulp therapies and regenerative endodontic procedures. The aim of this study was to evaluate the cytocompatibility and cytotoxicity of a new endodontic biomaterial, PulpGuard, in comparison with two other biomaterials widely used in endodontic procedures, ProRoot Mineral Trioxide Aggregate (MTA) and Biodentine.

Methods: Apical papilla cells (APCs) were isolated from third molars with incomplete rhizogenesis from patients with orthodontic indication for dental extraction. Cultured APCs were incubated for 24, 48, or 72 h with different dilutions of eluates prepared from the three materials. Cellular viability, mobility, and proliferation were assessed in vitro using the Alamar Blue assay and a wound-healing test. The cells were also cultured in direct contact with the surface of each material. These were then analyzed via Scanning Electron Microscopy (SEM), and the surface chemical composition was determined by Energy-Dispersive Spectroscopy (EDS).

Results: Cells incubated in the presence of eluates extracted from ProRoot MTA and PulpGuard presented rates of viability comparable to those of control cells; in contrast, undiluted Biodentine eluates induced a significant reduction of cellular viability. The wound-healing assay revealed that eluates from ProRoot MTA and PulpGuard allowed for unhindered cellular migration and proliferation. Cellular adhesion was observed on the surface of all materials tested. Consistent with their disclosed composition, EDS analysis found high relative abundance of calcium in Biodentine and ProRoot MTA and high abundance of silicon in PulpGuard. Significant amounts of zinc and calcium were also present in PulpGuard discs. Concerning solubility, Biodentine and ProRoot MTA presented mild weight loss after eluate extraction, while PulpGuard discs showed significant water uptake.

Conclusions: PulpGuard displayed a good in vitro cytocompatibility profile and did not significantly affect the proliferation and migration rates of APCs. Cells cultured in the presence of PulpGuard eluates displayed a similar profile to those cultured with eluates from the widely used endodontic cement ProRoot MTA.

Keywords: PulpGuard; SCAPS; biocompatibility; calcium silicate cements; cytotoxicity; regenerative endodontics.

Figure 1

Viability of apical papilla cells incubated in the presence of different endodontic cement eluates. (A–I) Cells were grown in the presence of (A–C) Biodentine, (D–F) ProRoot Mineral Trioxide Aggregate (MTA), or (G–I) PulpGuard eluates at different concentrations (undiluted, 1:2 dilution, and 1:4 dilution). Cellular viability was assess using the Alamar Blue method at 24 h (A,D,G), 48 h (B,E,H), or 72 h (C,F,I). (J) Relative cytotoxicity values of undiluted biocements were compared across time and showed that Biodentine significantly altered the cell viability profile when compared to ProRoot MTA and PulpGuard. These two endodontic cements showed similar influence on cell viability when compared to control conditions. The values for each experiment were normalized to the average of the control cells (not exposed to cement eluate). Results are from n = 3, presented as means ± SEM. Significative differences are indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001; One-way ANOVA with Dunnett’s multiple comparisons test was used in (A–I); two-way ANOVA with Dunnett’s multiple comparisons test was used in (J).

Figure 2

Effect of endodontic cement eluates on cellular migration and proliferation in a wound-healing assay. (A) Representative images of wound closure at the initial time point (0 h), 24, or 48 h after the removal of a cell culture insert and when apical papilla cells were grown in the presence of undiluted eluate extracts; scale bar 250 µm. (B) Following the removal of the wound-generating insert, the percentage of open wound was assessed for Biodentine (red), ProRoot MTA (green), PulpGuard (purple), and control conditions (white). (C,D) Wound closure was re-assessed following 24 h (C) and 48 h (D); the dotted line indicates the percentage of open wound for the control conditions. The results are from n = 4–7, presented as means ± SEM. Significative differences are indicated as * p < 0.05; One-way ANOVA with Dunnett’s multiple comparisons test was used in (B,C)

Figure 3

Surface properties and composition of biomaterials under scanning electron microscopy and energy-dispersive spectroscopy (EDS) analysis. (A) Representative images at different magnifications of the surface characteristics of Biodentine (left), ProRoot MTA (center), and PulpGuard (right), scale bar = 100 µm. (B,C) High-magnification image of the surface characteristics of Biodentine cement (B) revealing a rough surface; EDS analysis (C) displaying the presence of relative high amounts of calcium, oxygen, silicon, and phosphorous; scale bar = 10 µm. (D,E) High-magnification image of the surface characteristics of ProRoot MTA (D) revealing a rough and porous surface; EDS analysis (E) displaying the presence of relative high amounts of calcium, phosphorous, oxygen, and silicon; scale bar = 10 µm. (F,G) High-magnification image of the surface characteristics of PulpGuard (F) revealing a mostly smooth surface with little porosity; EDS analysis (G) displaying the presence of relative high amounts of silicon, zinc, oxygen, and calcium; scale bar = 10 µm.

Figure 4

Relative solubility properties of the individual biomaterials. Weight change in sample discs following incubation of the different materials for 48 h in cell culture medium. The results are from n = 21–26, presented as means ± SEM. Significative differences are indicated as *** p < 0.001; One-sample t-test was used to determine statistically significant changes in weight.