Direct pulp capping and pulpotomy are frequently performed as vital pulp therapies for carious primary teeth. Mineral trioxide aggregate (MTA) has been reported as the most suitable material for the same, while other experimental cement are being assessed. ¹ Although MTA has several benefits, it also has certain drawbacks – high material cost, long setting time, tough handling properties, and tooth discoloration. ² To compensate for the MTA's shortcomings, Biodentine was promoted as an alternative. In cases of pulp capping, this material can be used similarly to MTA, to encourage the development of a calcified bridge. ³

A bioceramic repair medication-Bio-C Repair was invented to make the surgical operation even easier. This bioceramic repair agent has exhibited bioactive outcomes similar to MTA. ⁴ To encourage pulp repair and stem cell activity, these protective materials must be biocompatible and bioactive. ⁵ Bioactive pulp-capping cement should possess adequate biocompatibility and bioactivity to promote dental pulp stem cell activity and pulp healing in primary teeth. ⁶ Stem cells derived from human exfoliated deciduous teeth (SHEDs) have the capacity to induce bone formation, generate dentin, and differentiate into other nondental mesenchymal cell derivatives in vitro. In contrast to DPSCs, SHEDs exhibit higher proliferation rate, increased population doublings, osteoinductive capacity in vivo, and an ability to form sphere-like clusters. ⁷ However, various researches have looked at the cytotoxicity of pulp-capping materials with stem cells obtained from permanent tooth pulp tissue; however, none of the studies have employed SHED. As a result, the goal of this research was to assess and compare the responses of SHEDs to MTA Repair HP, Bio-C Repair, and Biodentine. The null hypothesis stated that no significant differences in cytotoxicity and gene expression would exist between these pulp-capping agents.

The present in vitro experimental investigation referred to the research reporting guidelines provided by Nagendrababu et al., 2021. ⁸ The “resource equation” was used to calculate the sample size for the current trial. ⁹ The present research involved three different trials wherein all the tests were performed in triplicate for each experimental group.

Preparation of test specimens

MTA Repair HP (Angelus, Brazil), Biodentine (Septodont, France), and Bio-C Repair (Angelus, Brazil) set cement were evaluated. The International Standard Organization (ISO) 10993-5218 guidelines were used to test these calcium silicate-based cement (CSC), which involved incubating cultured cells with eluates. ¹⁰ One gram of the powder was mixed with an equivalent amount of liquid of each cement to maintain the powder: liquid ratio advised by the manufacturers. To ensure complete polymerization, the set CSC were placed in 12 well plates (3.0 mm height and 314.0 mm ²area) and cultivated for 15 h at 37°C, 95% humidity, and 5% CO _2.Sterilization was maintained for 30 min by exposing the sample to a UV light at 30 W in a laminar flow. The extraction medium was DMEM (HiMedia, India) supplemented with streptomycin. These eluates were collected after passing through a Merck Millipore 0.22 m filter (USA). Following that, three diluted (1/4, 1/2, and 1/1 vol/vol) eluates were tested in order to examine a dose–response correlation. ¹¹

Isolation and characterization of stem cells derived from human exfoliated deciduous teeth

The current experiment, as well as the technique for isolating and collecting SHEDs, had been approved by the Ethical Board of the institution. The parents/guardians of children aged 6–12 years who visited the Department of Pediatric and Preventive Dentistry with exfoliating primary teeth (n = 5) signed a written consent allowing them to be included in the research. These specimens were transferred to India's National Institute of Immunohematology at Parel, Mumbai, after rinsing in normal saline. The pulp tissues of the teeth were extirpated using barbed broaches. Hank's Balanced Salt Solution disinfected the pulp tissues (Gibco, USA). Enzymatic digestion was promoted at 37°C for 60 min using 3 mg mL Collagenase-A (Sigma-Aldrich, USA). Cells (2.0 × 104 cells/cm ²) were collected and cultured for 72 h at 370 C in the presence of 5% CO2 in 25-cm ²plastic culture flasks (BD Biosciences, USA). Nonadherent cells, such as red blood cells, were eliminated. To encourage greater growth, a new medium was used after 3–5 days. Passage zero (P0) was defined as the point at which adherent cells reached 80% confluence. Incubation in a 0.25% trypsin solution for 2–5 min at 37°C was used to detach cells after they had been cleaned in phosphate-buffered saline. The trypsin action was deactivated by the introduction of the culture medium. Centrifuged at 500 g for 5 min, the SHEDs were seeded into 75-cm ²flasks. ¹²

SHED phenotypes were determined using a flow cytometer and specific antibodies for HLA-DR, CD105, CD73, CD45, CD34, and CD90 (BD Biosciences, Pharmingen) prior to the experiments.

Cell viability assay

The viability of SHEDs cultivated in various eluates was examined through MTT cell assay kit after 24 h, 48 h, and 72 h of incubation (EZ count, HiMedia, India). SHEDs cultured in DMEM and 1 mM hydrogen peroxide were used as positive and negative control specimens, according to ISO 10993 standards. ¹⁰ MTT was added to all of the wells in question and cultured for 240 min before the procedure was stopped with the addition of dimethyl sulfoxide. A microplate reader (BioTek Instrument, USA) was used to determine AB _{570 nm}with a reference wavelength at AB ₆₃₀. ¹³ The cement dilution was chosen for gene expression analysis based on the results of the cell viability test.

Gene expression assessment

Quantitative real-time polymerase chain reaction (qRT-PCR) was used to examine the expression levels of osteogenic differentiation markers. 2 × 10 ⁶cells mL ⁻¹were plated in 6-well plates. As per the manufacturer's recommendations, the ribonucleic acid was isolated from SHEDs using the RNeasy Mini Kit (QIAGEN, USA) after 24 h of exposure to the various eluates, diluted in osteogenic (5 mmol L ⁻¹b-glycerophosphate and 50 lg mL ⁻¹L-ascorbic acid) medium. The qPCR gene expression technique was used in the present trial (StepOne, USA). qRT-PCR was performed to identify genes associated with bone formation. Runt-related transcription factor 2 (Runx2), osteocalcin (OCN), dentin matrix protein-1 (DMP-1), alkaline phosphatase (ALP), and glyceraldehyde 3-phosphate Dehydrogenase (control gene) were used. After 7, 14, and 21 days of incubation, the levels of mRNA expression were measured using the ΔΔCt method (fold expression = 2 – [ΔΔCt ± SD]). ¹⁴

Statistical analysis

The data were analyzed using the Statistical Package for the Social Sciences software. The mean measurements of test specimens were analyzed by one-way analysis of variance test. The Bonferroni post hoc test was used to look at multiple pair-wise individual comparisons between groups. A 5% level of significance was used to denote the differences between the means. A mean standard deviation was calculated from the data.

Isolation and characterization of stem cells derived from human exfoliated deciduous teeth

The cultured SHEDs observed under light microscopy showed more round-shaped morphology after 12 days following the third passage Figure 1. In the current trial, flow cytometric examination of SHEDs indicated moderate expression of CD105 (35.60%) and high expression of CD90 (97.50%), and CD73 (93.60%). Negative markers CD45 (0.57%), CD34 (1.60%), and HLA-DR (0.50%) were not expressed in the progeny of SHEDs. Figure 1

Morphology of SHEDs cultured in various eluates after 72 h of incubation. SHEEDs: Stem cells derived from human exfoliated deciduous teeth

Considering that vital pulp treatment comprises direct contact between tissue and treating medicament, biocompatibility characteristics of medicaments must be considered carefully. Several researches have been carried out to look into the possibility of compounds causing toxicity in cells, animals, and humans. For the purpose of replacing animal models, new cell culture methods have been developed. ¹⁵ , ¹⁶ Using extracts has the advantage of being sterile, making it easier to test their response on cells, and they replicate a clinical state where the compounds can dissolve and disperse into the periapical tissue. ¹⁷ , ¹⁸

SHEDs were used in the present trial because of their ability to self-renew, simplicity of isolation and preservation, multi-lineage pluripotency, and clinical importance. ¹⁸ , ¹⁹ MSC population is expected to be positive for CD105, CD73, and CD90 as per the phenotype requirements of ISCT. Furthermore, such cells should not express CD14, CD45, CD34, CD19, HLA class II, CD79a, or CD11b. 17 The present study used CD45, HLA-DR, and CD45 as negative markers, while CD105, CD73, and CD90 were used as positive markers. The results of phenotypical analysis of the present study were in consistent with other studies. ²⁰ , ²¹ , ²² , ²³ The MTT assay, which follows ISO 10993 guidelines, is the gold standard for evaluating the potential cytotoxic effects of pulpotomy materials. ¹⁰ Tetrazolium compounds are employed to produce a quantifiable colorimetric assay to evaluate human cell proliferation and survival. Hence, MTT assay has been used extensively to the extent that it is now the typical technique for assessing cell viability. Therefore, the present study used the same technique for determining cell viability. Cells are exposed to a medication for up to 72 h to evaluate its optimum potency and efficacy. The MTT assay was therefore conducted at 24 h, 48 h, and 72 h of incubation. ²⁴

Cell viability was highest for Biodentine in this trial, followed by Bio-C repair and MTA Repair HP, which was consistent with the findings of Klein-Junior et al., ²⁵ who found that bioceramic material had superior cell viability to MTA when exposed to NIH 3T3 fibroblasts. This could be because Biodentine and Bio-C Repair released much more calcium (Ca) than MTA, as recommended by the manufacturer. The formation of calcium hydroxide is critical for dentin bridge development and cementoblast differentiation but also for its antibacterial action. Other Bio-C Repair components, such as iron oxide, may have altered SHED cell viability, based on the chemical composition and solubility of each constituent of cement. When compared to calcium silicate materials containing bismuth oxide (MTA), those containing zirconium oxide (Bio-C Repair) cause less inflammation. ²⁶

The cell viability of Bio-C Repair was statistically significant after 72 h of incubation when compared to control in the present trial. López-García et al., ²⁷ on the other hand, reported that after 3 days of incubation, Bio-C Repair had comparable cell proliferation to the control with no substantial change. According to Benetti et al., ⁴ when exposed to living cells, MTA Repair HP and Bio-C Repair were found to be biocompatible and had the potential for biomineralization. Cell viability experiments on L929 fibroblasts found that both materials were equally viable in the presence of each other. The variance in outcomes can be described by researches utilizing different forms of cells, each of which responds differently to the substance. ²⁸

Figure 6, Figure 7, Figure 8show the expression of chosen osteogenic differentiation-related gene markers in the current investigation over three exposure durations. Mesenchymal stem cells must adopt the osteoblast/odontoblast phenotype in order to undergo cell proliferation, specialization, and skeletogenesis, and Runx2 is the transcription factor responsible for this. ²⁹ For the 1 ^stand 3 ^rdweeks, the expression level of Runx2 was in the following order: Biodentine > Bio-C Repair > MTA Repair HP. Whereas, for the 2 ^ndweek, the expression level of Runx2 was in the following order: Bio-C Repair > Biodentine > MTA Repair HP. Figure 8

Comparison of mRNA expression levels (mean ± SD) of target genes in SHEDs exposed to eluates of various test specimens in osteogenic media after 21 days of incubation. SD: Standard deviation, SHEEDs: Stem cells derived from human exfoliated deciduous teeth

The response of SHEDs to Bio-C Repair and MTA HP Repair was not cytotoxic within the restrictions of this two-dimensional cell culture trial. Like Biodentine, cell proliferation and odontoblastic differentiation on SHEDs was possible with MTA HP Repair and Bio-C Repair.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article.