Cytotoxicity of hydraulic cement dicalcium silicate mixture of eggshell calcium and rice husk silica on fibroblast cells

Abstract

Introduction:

Dicalcium silicate is currently used for vital pulp therapy in dentistry. In this research, a novel dicalcium silicate hydraulic cement is expected to be biocompatible and exhibit low cytotoxicity, making it a promising material for vital pulp therapy. The basis of the cytotoxicity test is the ability of cells to survive in the presence of a given toxic compound.

Objective:

The objective of this study was to determine the cytotoxicity effect of a novel hydraulic cement dicalcium silicate on fibroblast cells.

Methods:

NIH3T3 fibroblast cells that had undergone serum starvation for 24 h were given hydraulic cement culture media of novel dicalcium silica with concentrations of 1:1, 1:2, 1:4, and Dulbecco’s Modified Eagle Medium as a negative control. The cytotoxicity effect was tested at 24 and 48 h using the MTT assay. The results were analyzed using a statistical nonparametric Kruskal–Wallis test, followed by post hoc using Mann–Whitney for the 24-h observation group. In contrast, the 48-h observation was analyzed using a statistical test, the one-way analysis of variance parametric test, followed by a post hoc test using a t-test.

Results:

There was no significant difference in the value of cell viability in the administration of a novel hydraulic cement dicalcium at various concentrations (1:1, 1:2, 1:4) against NIH3T3 fibroblast cells at 24 h of observation, with a mean of the highest viability value at a concentration of 1:4. At 48 h of observation, there was a significant difference in the value of cell viability in the administration of a novel dicalcium silicate hydraulic cement, at various concentrations (1:1, 1:2, 1:4) to NIH3T3 fibroblast cells, with the highest mean viability value treatment group at a concentration of 1:4.

Conclusion:

A novel dicalcium silicate hydraulic cement in various concentrations (1:1, 1:2, 1:4) did not have a cytotoxic effect on NIH3T3 fibroblast cells at 24 h and 48 h. The highest NIH3T3 fibroblast cell viability value at 24 and 48 h was observed at a concentration of 1:4.

INTRODUCTION

Bioactive material based on calcium silicate, known as hydraulic calcium silicate cement (HCSC). HCSC is divided into two types: portland cement (PC) and tricalcium/dicalcium disilicate, comprising several components, including radiopacifiers, additives, and mixing liquid.[1] Calcium silicate hydraulic cement has been widely used due to its good bioactivity and biocompatibility properties. These materials are used in the conservation field in intracoronal, intraradicular, and extraradicular treatment procedures.[2] In addition to being synthesized from chemicals, calcium and silica-based biocompatible materials can be synthesized from abundant natural waste resources such as eggshells and rice husks.[3,4]

Eggshells contain 94%–97% calcium in calcium carbonate (CaCO₃), whereas rice husk ash contains about 90% silica, which has better biocompatibility than synthetic forms of silica.[5] Eggshells have a high calcium content, making them useful as precursors in hydroxyapatite synthesis.[6] The calcium in eggshells is calcium carbonate, which is insoluble in water but easily ionized under low pH conditions. Calcium from eggshells can be extracted to form hydroxyapatite, which is biocompatible and bioactive and has osteoconductive properties, enabling it to integrate with bone tissue.[5] The potential of eggshells as an eco-friendly source of calcium and bioactive compounds for remineralizing demineralized dental substrates.[7]

Rice husk ash mainly contains silica as its main component. Therefore, rice husks and rice husk ash have been used as the primary reinforcing material to improve the mechanical properties of composite metals. Rice husks provide good-quality pozzolanic ash containing silica, which is considered a good substitute for cement. The addition of rice husk ash to cement can enhance compressive strength and durability, reduce porosity within the cement matrix, and lower carbon dioxide emissions. Replacing part of PC with rice husk ash improves workability and mechanical strength while providing economic benefits through lower costs.[8] Dicalcium silicate hydraulic cement is a compound formed from the calcination of an eggshell and rice husk silica at 1100°C with a composition of CaCO₃:SiO₂ = (1.8:1) mol and a heating time of 2 h.[9]

A material considered biologically compatible must have the requirements of not causing damage to the pulp and soft tissue, not containing toxic substances that cause systemic problems, and not triggering allergic reactions or carcinogenic potential. Some adverse biological responses when a material is in contact with living tissue include inflammatory, allergic, toxic, and mutagenic.[10]

A cytotoxicity test is a method for determining whether a material, when placed, is toxic to specific cells. Cytotoxicity is the ability of a material to damage biological and chemical systems in cells, and a decrease in cell viability, cell proliferation, nucleic acid, or protein synthesis characterizes it. Fibroblasts are the most abundant cells in the pulp, especially in the coronal area, and function as basic substance makers and collagen fibers, which are the pulp matrix.[11,12] One of the most frequently used fibroblast cell types in various studies for toxicity testing is NIH3T3, which is derived from embryonic Swiss Webster white mice.[13] This study aims to analyze the cytotoxicity of calcium silicate hydraulic novel cement from a mixture of eggshell calcium and rice husk ash silica on mouse fibroblast cells.

METHODS

Cell culture of NIH3T3 fibroblasts

The cells used in this study were NIH3T3 mouse fibroblasts. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen®, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 U/ml penicillin-G (Gibco, Grand Island, NY, USA), 100 mg/mL streptomycin (Gibco, Grand Island, NY, USA), and 100 μg/mL gentamycin (Gibco, Grand Island, NY, USA). Cells were kept in a humidified incubator at 37°C under 5% CO₂. Serum starvation was performed by reducing the level of FBS in the initial culture medium of NIH3T3 mouse fibroblast cells. Initially, the culture medium was supplemented with 10% FBS, then the cells were harvested, and the culture medium was replaced with DMEMs and 1% FBS for 24 h.

Cell culture exposed to dicalcium silicate hydraulic cement material

Dicalcium silicate hydraulic cement mixed with distilled water at a ratio of 0.3 mL/g was placed on a mold of 2 mm depth and 5 mm diameter. After hardening, the material was ground and dry sterilized. The pulverized powder was taken as 3.5 mg and mixed with 3.5 mL of DMEMs, then incubated for 24 h at 37°C. The surface area ratio was measured to be 1.5 cm²/mL. Solutions were then prepared according to ISO 10993-5 recommendations (surface area ratio of 1.5 cm²/ml). The solution was filtered on a sterile 0.22 μm filter (ISO 10993-5) and then diluted to obtain concentrations of 1:2 (3.5 mg/7 ml) and 1:4 (3.5 mg/14 ml). The cell culture medium in each well was discarded. Then, each well was exposed to 100 μL of the test solution, with three replicates. Then, the cells were reincubated at 37°C and 5% CO₂ for 24 and 48 h – observation of viability of NIH3T3 fibroblast cells with MTT assay.

Viability test with MTT assay

NIH3T3 fibroblast cell culture in each well was added to 15 μl MTT assay and incubated at 37°C and 5% CO₂ for 3 h. Then, each well was added 15 μL of acidified isopropanol and shaken for 1 h. The results were read with a microplate reader at a wavelength of 650 nm. The absorbance value was calculated as a percentage compared to the absorbance value of the control group using the following formula:

Based on the provisions of ISO-10993-5:2009, the lower the % cell viability value, the higher the cytotoxic potential of a material. It has cytotoxic potential if the viability is reduced to <70%.

Data analysis

Quantitative data from the study were analyzed statistically. The total viability of the control and test groups was tested for normality and homogeneity. If the data distribution is expected, the one-way analysis of variance statistical parametric test is performed, followed by the post hoc test using the Bonferroni test if the data are homogeneous or the Tamhane test if the data are not homogeneous. If a significant difference exists, continue the post hoc test with a significance value (P < 0.05). If the data distribution is abnormal, proceed with a nonparametric test, namely the Kruskal–Wallis test, followed by a post hoc test using Mann–Whitney.

RESULTS

Observation result 24 h

Figure 1a-c shows a microscope image of fibroblast cells showing more nuclei after being treated with a hydrolytic cement dicalcium silicate mixture of eggshell calcium and rice husk silica, compared to Figure 1d, which is the control group.

Figure 1.

Microscopic image of NIH3T3 fibroblast cells (red arrow) with ×10 magnification after 24 h of treatment (a) concentration 1:1, (b) concentration 1:2, (c) concentration 1:4, (d) Control

Based on the Kruskal-Wallis test, there was no significant difference in cell viability values in the administration of hydraulic calcium silicate cement mixed with eggshell calcium and rice husk silica at various concentrations (1:1, 1:2, 1:4) to NIH3T3 fibroblasts at 24 hours of observation [Table 1].

Table 1.

Mean values of cell viability and standard deviation of various groups at 24-h observation time

Culture media	n	Mean viability cells±SD	P*
Concentration 1:1	12	102.59±18.52	0.065
Concentration 1:2	12	109.36±8.29
Concentration 1:4	12	110.88±8.90

*Kruskal–Wallis test, with a significance value of P<0.05. SD: Standard deviation

Based on the Mann-Whitney test to determine the differences between the 24-hour observation study groups, it was observed that the 24-hour observation period showed a statistically significant difference in cell viability values between the administration of hydraulic calcium silicate cement mixed with eggshell calcium and rice husk silica at a concentration of 1:1 compared to a concentration of 1:4. However, all concentrations showed no cytotoxic effects because they had cell viability values above the control group [Table 2].

Table 2.

The significance value of cell viability between groups at the 24-h observation time

	Concentration 1:1	Concentration 1:2	Concentration 1:4
Concentration 1:1		0.065	0.038*
Concentration 1:2	0.065		0.507
Concentration 1:4	0.038*	0.507

*Post hoc Mann–Whitney, with a significance value of P<0.05

Observation result in 48 h

Figure 2 shows a microscopic image with ×10 magnification of NIH3T3 fibroblast cells after incubation for 48 h. It can be seen that the number of fibroblast cell nuclei in the control group [Figure 2d] is higher than that of the treatment groups in Figure 2a-c.

Figure 2.

Microscopic image of NIH3T3 fibroblast cells (red arrow) with magnification at ×10 magnification after 48 h treatment (a) concentration 1:1 (b) concentration 1:2, (c) concentration 1:4, (d) Control

Based on the One-way ANOVA statistical test, it was found that there was a significant difference in cell viability values in the administration of calcium silicate hydraulic cement mixed with eggshell calcium and rice husk silica at various concentrations (1:1, 1:2, 1:4) to NIH3T3 fibroblasts at 48 hours of observation [Table 3].

Table 3.

Mean cell viability values and standard deviation of various observation groups at 48 h

Culture media	n	Mean viability cells±SD	P
Concentration 1:1	12	92.83±5.46	0.026*
Concentration 1:2	12	96.53±5.24
Concentration 1:4	12	99.59±6.61

*One-way ANOVA test, with a significance value of P<0.05. SD: Standard deviation

Statistical data analysis was followed by a post hoc test using the T-test. Based on the post hoc test to determine differences between research groups at the 48-hour observation point, a statistically significant difference in cell viability values was observed between the 1:1 concentration and the 1:4 concentration. This indicates that all concentrations were non-toxic, as they had cell viability values above 90% [Table 4].

Table 4.

The significance value of cell viability between groups at the 48-h observation time

	Concentration 1:1	Concentration 1:2	Concentration 1:4
Concentration 1:1		0.381	0.022*
Concentration 1:2	0.381		0.616
Concentration 1:4	0.022*	0.616

*Post hoc t-test, with a significance value of P≤0.05

Observation results comparison between 24 h and 48 h observation results

The highest mean viability of NIH3T3 fibroblasts in the administration of hydraulic calcium silicate semen mixed with eggshell calcium and rice husk silica in the 1:1, 1:2, and 1:4 concentration groups occurred in with a 24-hour observation period [Table 5].

Table 5.

Mean cell viability values and standard deviation between groups at 24 and 48 h of observation

Group	24 h, mean±SD	48 h, mean±SD	P
Concentration 1:1	102.59±18.52	92.83±5.46	0.149
Concentration 1:2	109.36±8.29	96.53±5.24	0.000*
Concentration 1:4	110.88±8.90	99.59±6.61	0.002*

*t-test, with a significance value of P<0.05. SD: Standard deviation

DISCUSSION

Cell culture methods have often been used to test the biological effects (cytotoxicity) of materials in dentistry. Different techniques can be used to assess cytotoxicity, including cell count, determination of proliferation rate, measurement of cell product synthesis, or determination of enzyme activity. Cells used in toxicity tests must be standardized and evaluable and have criteria for measuring toxicity, such as cell viability and function.[14]

Secondary cells (cell lines) can be used according to ISO 10993-5:2009 guidelines for the protocol of initial testing of a new biomaterial in dentistry, specifically in phase 1 testing to evaluate general toxicity in vitro.[15] In this study, to test the cytotoxicity of hydrolytic cement, a dicalcium silicate mixture of eggshell calcium and rice husk silica, NIH3T3 fibroblast cells derived from Swiss Webster white rat embryos were used. Fibroblasts are the most abundant cells in the pulp. NIH3T3 fibroblast cells have been widely used in various toxicity tests because their genetics and biological characteristics can be replicated in mice, as well as in many human conditions. NIH3T3 is also one of the most frequently recommended cells for the MTT assay because it is a rapidly reproducible cell line, has advantages in handling, and is readily available compared to primary cells.[13,16,17]

The basis of cytotoxicity testing is the ability of a cell to survive due to the presence of exposed toxic compounds. Cell viability is the ability of cells to survive without losing metabolic function or proliferation ability. It can be measured by the increase in the number of cells, the increase in the number of proteins, and the DNA synthesized, so cell viability can be a sign of a material’s cytotoxicity.[18] Viability testing can be seen from colorimetric assays. The MTT assay is one of the most commonly used colorimetric assays for assessing cytotoxicity or cell viability. The basic principle of the MTT assay is the reduction of yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically active cells. Living cells contain NAD (P) H-dependent oxidoreductase enzymes that reduce MTT to purple formazan. When cells die, they lose the ability to convert MTT into formazan, so color formation is a marker for cells that remain alive.[19]

Evaluation of hydrolytic cement dicalcium silicate mixture of eggshell calcium and rice husk silica at various concentrations (1:1, 1:2, 1:4) on the viability of NIH3T3 fibroblast cells was observed at 24 and 48 h. Observation at 24 h was done because the time required for a cell to perform one cycle is approximately 24 h, whereas observation at 48 h was done to see if there was an increase or decrease in cell viability after 24 h.[20]

The use of hydrolytic cement dicalcium silicate mixed with eggshell calcium and rice husk silica without dilution (concentration 1:1) and with dilution (concentration 1:2 and 1:4) follows the recommendations of ISO 10993-5:2009 and refers to previous studies.[13,21,22] The concentration with dilution is also done to adjust to the clinical conditions in the oral cavity when the pulp cap material is applied to the remaining dentin with a thickness of 0.01–0.25 mm or in cavities with open pulp where there is extracellular fluid. This condition causes the pulp cap material to dissolve continuously due to fluid elimination, resulting in a lower local concentration of the material. Therefore, dilutions with different concentrations are carried out.[21] A new material that has never been tested for toxicity before can potentially cause cell death if exposed directly to cells without dilution first. The material contains reactive oxygen–nitrogen species that can trigger this.[21,22]

In Table 1 and Figure 1 of the 24-h observation results, the highest mean viability of NIH3T3 fibroblast cells was found in the 1:4 concentration group (110.88%), followed by the 1:2 concentration group (109.36%) and the 1:1 concentration group (102.59%). At 24 h of observation, there was an increase in fibroblast cell viability after the administration of hydrolytic cement, dicalcium silicate mixture of eggshell calcium and husk silica at various concentrations (1:1, 1:2, 1:4), where the value was higher than that of the control group. Hydrolytic cement, a calcium silicate mixture of eggshell calcium and rice husk silica, contains calcium and silica ions so that it can stimulate growth factors that play a role in the process of cell growth, proliferation, proinflammation, and angiogenesis; this can be seen in the 24-h observation with cell viability values increasing to above 100%. This research is in line with the research of Alshatwi et al. regarding pure biogenic silica nanoparticles synthesized from rice husk ash of various concentrations, which can increase fibroblast cell proliferation at 24 h of observation.[23] Pintado et al. found similar results: dicalcium silicate, contained in mineral trioxide aggregate, has no toxic effect on NIH3T3 or human pulp fibroblast cells.[24]

Based on the Mann-Whitney test to determine the differences between the 24-hour observation groups [Table 2], it can be seen that the 24-hour observation shows a statistically significant difference in cell viability values between the administration of hydraulic calcium silicate cement mixed with eggshell calcium and rice husk silica at a ratio of 1:1 compared to 1:4. However, all concentrations showed no cytotoxic effects because they had cell viability values above the control group.

In Table 3 and Figure 2 of the 48-h observation results, the mean value of NIH3T3 fibroblast cell viability after the administration of hydrolytic cement dicalcium silicate mixture of eggshell calcium and silica husk of various concentrations (1:1, 1:2, 1:4), where the value is lower when compared to the control group. The 1:4 concentration had an average value of fibroblast cell viability (92.83%), in the 1:2 concentration group (96.53%), while in the 1:1 concentration (99.59%). This is in line with the research of Benetti et al., who conducted a cytotoxicity test of bioceramic materials of various concentrations with the MTT test with the results of rat fibroblast cell viability at 48 h observation decreased when compared to 24-h observation, but the decrease in cell viability was <30% when compared to the control group. Thus, bioceramic materials are not toxic to fibroblast cells.[22]

In Table 5, cell viability decreases in the treatment group after 48 h of observation compared to 24 h. However, because the decrease in cell viability is not >30% compared to the control group, this material does not exhibit a cytotoxicity effect according to ISO 10993-5:2009 recommendations.[14] A decrease in cell viability may occur due to a reduction in the Ca²⁺ component and an increase in PH in a hydrolytic cement dicalcium silicate mixture of eggshell calcium and rice husk silica. Mohamed et al. research shows that a reduction in the Ca²⁺ component and an increase in PH can cause cell lysis and affect cell growth. The initial cytotoxic effect decreases gradually over time.[25,26] This finding is also in line with research conducted by Silalahi et al., which states that changes in pH will affect the solubility and availability of mineral ions, thereby impacting the absorption of nutrients by cells and decreasing the viability of rat pulp cells.[27]

Dicalcium silicates hydrate to form calcium silicate gel, producing calcium hydroxide. During this setting process, various ions are released, including Si⁴⁺, Ca⁺⁺, and OH⁻. The freshly mixed material elutes a significant amount of Ca⁺⁺ and OH⁻, increasing pH and a high concentration of Ca⁺⁺ ions. This alteration in calcium homeostasis around cells can negatively affect cellular metabolism, resulting in apoptosis and cell death, thereby decreasing cell viability.[28]

Based on the post hoc test to determine differences between research groups at the 48-hour observation point, a statistically significant difference in cell viability values was observed between the 1:1 concentration and the 1:4 concentration. This indicates that all concentrations were non-toxic, as they had cell viability values above 90% [Table 4], so it can be concluded that the overall administration of hydrolytic cement calcium silicate mixture of eggshell calcium and rice husk silica of various concentrations (1:1, 1:2, 1:4) did not have a toxic effect on NIH3T3 fibroblast cells at 24 h or 48 h observation time. This follows the results of research by Palakurthy et al. (2019), which states that calcium silica synthesized from rice husk ash and eggshells by the sol gel method has no cytotoxicity effect even at high concentrations, with cell viability values above 70%. In addition, this material exhibits good biocompatibility properties.[29]

Based on the provisions of ISO-10993-5:2009, if viability is reduced to <70%, then a material has cytotoxic potential. From the results of this study, it was found that the cell viability of all treatment groups was above 90%, so it can be concluded that the overall administration of hydrolytic cement calcium silicate mixture of eggshell calcium and rice husk silica of various concentrations (1:1, 1:2, 1:4) did not have a toxic effect on NIH3T3 fibroblast cells at 24 h or 48 h observation time. This follows the results of research by Palakurthy et al., which states that calcium silica synthesized from rice husk ash and eggshells by the sol-gel method has no cytotoxicity effect even at high concentrations, with cell viability values above 70%. In addition, this material exhibits good biocompatibility properties.[29] Research by Sadrabad et al. states that mineral trioxide aggregates containing calcium silica can be categorized as noncytotoxic materials on human dental pulp stem cells.[30]

The limitation of this study is that the setting time of the hydrolytic cement of dicalcium silicate mixed with eggshell calcium and rice husk silica is unknown. The release of calcium ions during the setting time will affect cell activity, so there is a correlation between high cell viability values and shorter setting times

CONCLUSION

Hydrolytic cement, a dicalcium silicate mixture of eggshell calcium and rice husk silica at various concentrations (1:1, 1:2, 1:4), has no cytotoxic effect on NIH3T3 fibroblast cells at 24 and 48 h of observation time. The highest viability value of NIH3T3 fibroblast cells at 24 and 48 h was found at a concentration of 1:4.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.