Investigation of the physical, chemical, and biological properties of the cockle shell-derived calcium silicate-based pulp capping material: a pilot study

Abstract

Introduction: Hard-setting calcium hydroxide-based materials, e.g., Dycal and Life, have been widely used for direct pulp capping. However, various studies have shown undesirable effects such as high solubility and unpredictable dentine bridge formation. Bioceramic, mainly composed of tricalcium and dicalcium silicates, e.g., mineral trioxide aggregate and Biodentine, have provided more desirable physical and biological properties. This study aims to measure the physical properties, chemical properties, and biological response of human dental pulp cells (HDPCs) on three dental pulp-capping materials, Dycal, Life, and cockle shell-derived tricalcium silicate pulp capping material (C-Cap).

Methods: C-Cap was prepared from cockle shells and rice husk ash. Its chemical composition was identified using X-ray diffractometry. The setting time, flow, solubility, and radiopacity tests were performed following the International Organization for Standardization 6876:2012. pH and calcium ion release were measured. The materials were subjected to an extraction medium at various concentrations and subsequently measured for cytotoxicity and migration on HDPCs, from three healthy, mature permanent teeth from different donors. Osteogenic differentiation was assessed by examining alkaline phosphatase enzyme activity and alizarin red staining assay. The data were tested for a normal distribution. The differences among groups were statistically analyzed using ANOVA and Tukey's multiple comparison test (p < 0.05).

Results: The setting time of each material was approximately 1-2 min. C-Cap showed the lowest solubility (10.27% ± 1.02%) compared to Dycal (12.67% ± 0.94%) and Life (12.74% ± 1.33%), with a significant difference (p < 0.05). All materials exhibited radiopacity ranging from 2.4 to 2.9 mm of aluminum. C-Cap had the highest flow, alkalinity, and calcium ion release. C-Cap was significantly less cytotoxic than Dycal and Life (p < 0.05). The migration of HDPCs cultured in C-Cap extraction medium (27.74% ± 0.12%) was comparable to that in serum-free medium (27.09% ± 0.08%) with a significant difference (p < 0.05). The mineralization by HDPCs maintained in C-Cap extraction medium was significantly higher than those in Dycal and Life extraction mediums with a significant difference (p < 0.05).

Conclusions: C-Cap, a tricalcium silicate-based pulp capping material has potential for further development. C-Cap exhibited comparable physical properties and superior biological properties when compared to Dycal and Life.

Fig. 1. XRD analysis and particle size of the cockle shell-derived tricalcium silicate powder.

a XRD analysis of the cockle shell-derived tricalcium silicate powder shows the peak of tricalcium silicate(▾), dicalcium silicate (●), and calcium hydroxide (■). b Particle size distribution of tricalcium silicate powder.

Fig. 2. HDPC viability after being treated with 4 concentrations of extraction medium from C-Cap, Dycal, and Life was measured by MTT assay.

The dashed line indicates the reference value of the CM used as control. a relative cell viability (%) 1 day after treated with each extraction medium. b 2 days, c 3 days. d evaluation of cell viability of HDPCs in complete medium and 25% extraction medium of each material at days 1–3. Different uppercase letters indicate statistical differences between each tested material only on the same day (ANOVA and Tukey’s multiple comparison tests, p < 0.05).

Fig. 3. Cell migration was measured by scratch wound assay.

HDPCs were treated with the 25% extraction medium of each material. The control was HDPCs in CM. a The percentage of cell migration at days 1 and 3 after the scratch was made. Different uppercase letters indicate statistical differences between each tested material only on the same day (ANOVA and Tukey’s multiple comparison tests, p < 0.05). b The images represent HDPCs before the scratch was made, the scratch area immediately after making the scratch, and 1, and 3 days after the test. The scratch areas were analyzed by ImageJ. The yellow lines indicate the open wound healing areas. Scale bar: 200 µm.

Fig. 4. The effects of the extracted medium on HDPC osteogenic differentiation.

a ALP activity after being treated with the extracted medium of each material at day 7 and day 14. b quantitative measurement of the relative absorbance of the eluted alizarin red S dye. The dashed line indicates the reference value of the control. Different uppercase letters indicate statistical differences between each tested material only on the same day (ANOVA and Tukey’s multiple comparison tests, p < 0.05). c Mineralized nodules formed by differentiated cells after incubation were stained with Alizarin Red S. HDPCs treated with OM were used as control.