Development and Analysis of a Hydroxyapatite Supplemented Calcium Silicate Cement for Endodontic Treatment

Abstract

Aim: To develop an endodontic cement using bovine bone-derived hydroxyapatite (BHA), Portland cement (PC), and a radiopacifier. Methods: BHA was manufactured from waste bovine bone and milled to form a powder. The cements were developed by the addition of BHA (10%/20%/30%/40% wt), 35% wt, zirconium oxide (radiopacifier) to Portland cement (PC). A 10% nanohydroxyapatite (NHA) cement containing PC and a radiopacifier, and a cement containing PC (PC65) and a radiopacifier were also manufactured as controls. The cements were characterised to evaluate their compressive strength, setting time, radiopacity, solubility, and pH. The biocompatibility was assessed using Saos-2 cells where ProRoot MTA acted as the control. Compressive strength, solubility and pH were evaluated over a 4-week curing period. Results: The compressive strength (CS) of all cements increased with the extended curing times, with a significant CS increase in all groups from day 1 to day 28. The BHA 10% exhibited significantly higher CS compared with the other cements at all time points investigated. The BHA 10% and 20% groups exhibited significantly longer setting times than BHA 30%, 40% and PC65. The addition of ZrO₂ in concentrations above 20% wt and Ta₂O₅ at 30% wt resulted in a radiopacity equal to, or exceeding that of, ProRoot MTA. The experimental cements exhibited relatively low cytotoxicity, solubility and an alkaline pH. Conclusions: The addition of 10% and 20% BHA to an experimental PC-based cement containing 35% ZrO₂ improved the material's mechanical strength while enabling similar radiopacity and biocompatibility to ProRoot MTA. Although BHA is a cost-effective, biomimetic additive that can improve the properties of calcium silicate endodontic cements, further studies are now warranted to determine its clinical potential.

Keywords: biocompatibility; bovine derived hydroxyapatite; calcium silicate cement; compressive strength; radiopacity.

Figure 1

FTIR Spectra. (a) NHA (blue) and BHA (red). (b) Zirconia dioxide. (c) Portland cement. (d) BHA10% (blue) and BHA 20% (red).

Figure 2

SEM images and their respective EDX analysis of the cements after initial set and 21 days immersion in H₂O. Yellow arrows indicate apatite formation. (Error bar = 10 µm).

Figure 3

Compressive strength after 1, 7 and 28 days ageing in distilled water using ISO 9917-1 (2007) (n = 10). Significant differences between materials from day 1–28 (p < 0.001), on day 1 between BHA 10% and PC 65, NHA, BHA 30% & 40% groups (*** p < 0.001), on day 7 between BHA 10% and NHA, BHA 30% and BHA 40% (*** p < 0.001), and day 28 between BHA 10 and PC65, NHA, BHA 30% and BHA 40% (* p < 0.05).

Figure 4

Optical density expressed in equivalent thickness of aluminium (Al) of MTA, Portland cement (PC) and PC with different radiopacifiers, as determined by ImageJ software (n = 3). All BHA groups contained 35% ZrO₂. Red line depicts ISO 9917-1 (2007) cut off at 3 mm Al equivalent thickness. Significant differences between MTA and BHA 10%, BHA 30% and BHA 40%, ZR 35%, Zr 40% and Ta40 (*** p < 0.001) and MTA and BHA 20% (* p < 0.05).

Figure 5

Average setting time of the tested cements (n = 3). Time recorded based on average of three tests using Vicat apparatus. The final time recorded when needle was unable to make complete circular indentation in setting cement. Significant differences between NHA and all other groups *** p < 0.001, BHA 10% & BHA 20%, and between BHA 30%, BHA 40% & PC 65. *** p < 0.001. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

(A) Average pH value of the tested cements for the duration of 4 weeks. (B) Mean weight loss of the materials studied over the four-week period. (n = 3).

Figure 7

(A) Proliferation (MTS assay) of Saos-2 cells after seeding onto BHA cements and MTA after 24, 48 and 72 h. Error bars represent ± SE of the mean after 1-way ANOVA with Tukey’s multiple comparison test (n = 3, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and error bars represent the SE of the mean). (B) Fluorescent images showing cell viability of Saos-2 cells seeded on the BHA cements and MTA discs after 48 h (n = 3). Green = live cells (calcein), Red = dead cells (ethidium homodimer-1). Scale bar = 100 μm.