Development of an experimental pulp capping material containing antibacterial and remineralizing peptide: an in vitro study

Abstract

Background

This study aimed to characterize an experimental pulp-capping material prepared by incorporating a hydroxyapatite-binding antimicrobial peptide (HBAMP) into a resin-modified calcium silicate–based material (RMCS), and to evaluate its antibacterial, mineral deposition potential, and cytotoxicity properties.

Methods

The experimental groups were defined as Control, 1% HBAMP-RMCS, and 3% HBAMP-RMCS. Standard polymerized RMCS discs were fabricated for XRD, degree of conversion, microhardness, antibacterial, and cytotoxicity assays. For dentin surface analyses, human mandibular third molars were used. Half of the dentin surfaces were treated with the pulp-capping material, followed by application of a universal adhesive and a nanohybrid composite resin. After 72 h of storage in distilled water, and microhardness and SEM–EDX analyses were performed. Data were statistically analyzed.

Results

The incorporation of HBAMP at 1 wt% and 3 wt% into RMCS caused no significant changes in the degree of conversion or microhardness values (p > 0.05). Regarding mineral deposition potential, HBAMP incorporation resulted in numerical increases in dentin microhardness and Ca/P ratio, although these changes were not statistically significant among the groups (p > 0.05). While HBAMP exhibited potent antibacterial activity against S. mutans and L. casei strains, no inhibition zones were formed when it was incorporated into the RMCS. Furthermore, no statistically significant differences were detected between the mean cell viability of the experimental groups in the MTT assay at the 24-h and 48-h evaluations(p > 0.05).

Conclusions

The incorporation of HBAMP enhanced the early mineral deposition on dentin surfaces of RMCS without adversely affecting its physical properties. The 1% HBAMP-RMCS group demonstrated more consistent outcomes with respect to mineral deposition and cytotoxicity. The antibacterial effect of HBAMP was attenuated when incorporated into RMCS.

Background

In contemporary restorative dentistry, the increasing emphasis on conservative and minimally invasive approaches has led to the frequent adoption of vital pulp therapy in appropriate cases [1–3]. The primary objective of these treatments is to eliminate factors that cause pulp irritation and thereby prevent the progression of infection [4]. Additionally, they aim to stimulate tertiary dentin formation, ensuring the long-term functionality of the tooth [5]. The dental pulp possesses an inherent capacity for healing and regeneration when inflammation is relatively mild and reversible [6]. The pulp-capping material used in these procedures plays a critical role in this process. An ideal pulp-capping material should not only protect the pulp against infection but also provide a biologically favorable environment for tissue repair [7].

Contemporary resin-modified calcium silicate-based (RMCS) pulp capping materials used in vital pulp therapy stimulate odontoblasts by releasing of calcium ions, supporting cell proliferation and differentiation, and promoting hard tissue formation. As a light-cured, resin-based material, RMCS allows for the immediate application of permanent restorations, unlike materials such as Biodentine that require a waiting period. However, the remineralization capacity of RMCS is lower than that of Biodentine. Furthermore, its hydration process is incomplete, resulting in reduced remineralization. While its antibacterial efficacy is lower than or similar to other capping materials, it exhibits lower cytocompatibility due to its resin content [8]. In our study, we aimed to evaluate whether the addition of HBAMP would improve the remineralization and cytotoxicity properties of the material.

In recent years, the increasing prevalence of antibiotic resistance has markedly intensified the need for novel antimicrobial agents, bringing antimicrobial peptides (AMPs) to the forefront as promising alternatives. Owing to their broad-spectrum antibacterial, antifungal, and antiviral activities, multiple mechanisms of action, and structural characteristics that hinder the development of resistance, AMPs have emerged as critical therapeutic candidates in the so-called “post-antibiotic era.” In particular, marine-derived AMPs, characterized by properties such as salt tolerance, protease resistance, and low hemolytic activity, have inspired the development of next-generation synthetic or modified AMP designs [9].

Similarly, proline-rich antimicrobial peptides (PrAMPs) have attracted attention due to their ability to penetrate bacterial cells without disrupting the cell membrane, target essential intracellular components such as the 70S ribosome and DNA, and inhibit protein synthesis through multiple mechanisms [10].

AMPs have gained an increasingly prominent position in translational medicine, not only as direct antimicrobial agents but also as biological templates for the development of peptidomimetics. Contemporary AMP research has shifted toward structural and functional optimization strategies, including the incorporation of D‑amino acids, peptide cyclization, hybrid peptide constructs, nanotechnology‑based delivery systems, and design approaches targeting novel molecular pathways [11].

Peptide-based materials hold considerable potential in caries management owing to their antimicrobial and remineralizing effects [12]. Antimicrobial peptides (AMPs) are regarded as effective agents because they exhibit potent antibacterial activity at low concentrations while maintaining cytocompatibility with mammalian cells. Huang et al. developed a hydroxyapatite-binding antimicrobial peptide (HBAMP) to reduce biofilm formation, combining a broad-spectrum AMP (KSLW) with a hydroxyapatite-binding heptapeptide [13].

The present study aimed to investigate, in vitro, an experimental pulp-capping material obtained by incorporating an antibacterial and remineralizing peptide into RMCS. In addition to antibacterial and early mineral deposition effects, characterization and cytotoxicity parameters were comprehensively evaluated.

The null hypotheses (H₀) of this study were as follows:

1. HBAMP incorporation does not alter the degree of conversion of RMCS.

2. HBAMP incorporation does not change the microhardness of RMCS.

3. HBAMP incorporation does not alter the early mineral deposition effects of RMCS.

4. HBAMP incorporation does not change the antibacterial activity of RMCS against S. mutans and L. casei.

5. HBAMP incorporation does not alter the cytotoxicity of RMCS.

Methods

Before the commencement of this study, the research protocol was approved by the Clinical Research Ethics Committee of Erciyes University (Approval No: 2024/283).

Synthesis of HBAMP

The peptide was synthesized using the standard 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase method. Following synthesis, the peptide was cleaved from the resin with 95% trifluoroacetic acid (TFA) in the presence of appropriate scavengers. Purity and quality were confirmed by reverse-phase high-performance liquid chromatography. The molecular mass of the peptide was determined by matrix-assisted laser desorption/ionization mass spectrometry. The peptide was purified to 95% and lyophilized for further use.

Incorporation of the antibacterial peptide into the experimental pulp-capping material

In our study, the selection of 1% and 3% peptide concentrations was based on previous reports indicating that these concentrations have the potential to enhance the antibacterial efficacy of similar materials while maintaining an optimal balance of biocompatibility and mechanical properties [14, 15]. Moreover, our preliminary tests demonstrated that 3% was the maximum concentration that did not compromise the consistency or homogeneity of the material.

For the control group, a commercially available resin-modified calcium silicate–based pulp-capping material (Theracal LC, Bisco Inc., Schaumburg, IL, USA; Lot No: 2400003923) was selected. For the experimental groups, HBAMP was weighed using a precision balance (Shimadzu AUW-220D, Japan) and incorporated into Theracal LC at concentrations of 1 wt% and 3 wt% under dark conditions. The mixtures were manually blended until homogeneity was achieved and stored at room temperature in light-proof tubes. The experimental groups were defined as follows:

• RMCS without HBAMP (Control)

• RMCS containing 1% HBAMP (1% HBAMP-RMCS)

• RMCS containing 3% HBAMP (3% HBAMP-RMCS)

Specimen preparation

For XRD, FT-IR, and Vickers microhardness tests and agar disk diffusion assays, specimens were fabricated using silicone molds with dimensions of 6 mm in diameter and 1 mm in height. For the MTT assay, silicone molds with a diameter of 4 mm and a height of 1 mm were used. Materials were placed into silicone molds positioned between two sterile glass slabs and polymerized from a single surface for 20 s using a LED light-curing unit (Valo Cordless, 1000 mW/cm², Ultradent, South Jordan, UT, USA) in soft-start mode. The output energy of the LED curing unit was periodically verified using a radiometer. All tests were conducted on the light-exposed surfaces of the specimens.

X-Ray diffraction (XRD)

The crystalline structures of the disc specimens from each group (n = 6/group), as well as HBAMP alone, were analyzed using an X-ray diffractometer (XRD, Panalytical Empyrean, United Kingdom). Measurements were performed at room temperature using a monochromatized Cu Kα radiation source (λ = 1.5406 Å) filtered with nickel. Data were collected in continuous scan mode at a scanning rate of 4°/min over a 2θ range of 10 to 90°. The operating conditions were set at 45 kV and 30 mA.

Fourier transform infrared spectroscopy (FT-IR)

Characterization of the pulp-capping materials and determination of the degree of conversion (DC) (n = 6/group) were performed using a Fourier Transform Infrared Spectrometer (FT-IR, PerkinElmer 400 FT-IR/FT-FIR Spectrometer Spotlight 400 Imaging System, PerkinElmer, Waltham, MA, USA). Spectra were recorded both before and 10 min after polymerization. The FT-IR spectrum of HBAMP was also analyzed separately and converted into a graphical form. Measurements were conducted within the wavelength range of 400–4000 cm⁻¹.

The DC values were calculated using absorbance peaks at approximately 1608 cm⁻¹ (aromatic C = C stretching) and 1638 cm⁻¹ (aliphatic C = C stretching) according to the following formula:

Mineral deposition and surface changes evaluation

Specimen preparation

A total of 24 extracted human mandibular third molars, free of caries, cracks, or fractures, were used. Molar teeth were gathered from the Oral and Maxillofacial surgery clinics of the institution. Prior to extraction, patients were informed about the use of the teeth for research purposes, which was confirmed with their written consents. Following extraction, the teeth were disinfected in 0.5% chloramine-T solution at 4 °C for one week and subsequently stored in distilled water at 37 °C until use. The teeth were sectioned at the cemento-enamel junction using a separator. Occlusal enamel layers were removed under water cooling with a low-speed diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA). All specimens were examined under a stereomicroscope (DV 4; Zeiss, Jena, Germany) at 25 × magnification to confirm the absence of residual enamel. To create a standardized smear layer, dentin surfaces were polished sequentially with 600-, 800-, and 1200-grit silicon carbide papers. The occlusal dentin surfaces were then divided into two halves at a depth of 1 mm using a bur. The teeth were randomly allocated into three groups (Control/RMCS without HBAMP, 1% HBAMP-RMCS, and 3% HBAMP-RMCS) (n = 8/group).

For each specimen, the untreated half of the dentin surface served as an intra-sample control and was protected with Teflon tape to prevent contamination during procedures. A single operator applied the pulp-capping material to one half of the dentin surface at a standardized thickness of 0.5 mm and light-cured for 20 s using a LED curing unit. Subsequently, a universal adhesive resin (Scotchbond Universal Adhesive, 3M ESPE, St. Paul, MN, USA) was applied with a microbrush according to the manufacturer’s instructions. The surfaces were then covered with a nanohybrid universal composite resin (Filtek™ Z550, 3M ESPE, St. Paul, MN, USA) at a thickness of 1 mm and light-cured for 20 s.

The teeth were stored individually in distilled water at 37 °C for 72 h. After storage, the applied materials were carefully removed. Measurements were performed on the central dentin regions that the pulp-capping material had fully covered to ensure reliable results. Surfaces were examined under magnification to confirm the absence of residual material. Microhardness and SEM–EDX analyses were conducted to compare the differences between pre- and post-treatment samples.

Vickers microhardness analysis

The microhardness values of disc specimens from each group and dentin surfaces (to assess remineralization levels) were measured using a microhardness tester (EMCO-TEST Durascan, EMCO-TEST Prüfmaschinen GmbH, Kuchl, Austria). Initially, three indentations were created using a diamond indenter under a 100 g load for 15 s. These indentations were spaced 500 µm apart. The average of the measurement values obtained from three different regions on the specimen surface was recorded as the final value for that specimen. The lengths of the two diagonals of each indentation were measured under a microscope at 40 × magnification, and the Vickers hardness (HV) value was calculated using the following formula:

Field emission scanning electron microscopy (FESEM) analysis

For FESEM analysis, dentin surfaces were sputter-coated with a gold–palladium (Au–Pd) alloy for 15 s at a ratio of 20–80%. The coating was applied in increments of 15 Å every 5 s, reaching a total thickness of 45 Å. The coated surfaces were examined using a field emission scanning electron microscope (GeminiSEM 500, Zeiss, Oberkochen, Germany) operating at 26 kV, at magnifications of 1000 ×, 2500 ×, and 5000 ×.

SEM-Energy dispersive X-Ray spectroscopy (SEM–EDX)

Elemental composition of the specimens was analyzed using SEM coupled with energy-dispersive X-ray spectroscopy (EDX). A secondary electron detector (Zeiss Gemini 500, Zeiss, Jena, Germany) was attached to the SEM for elemental analysis. The system was operated at a 0.5% detector level (by weight), with a 133 eV resolution, a 100 µs amplification time, and a measurement duration of 60 s. Standard resolution was set at 1536 × 1024. EDX microanalyses were performed at 250 × magnification on two randomly selected regions (50 × 50 µm) per specimen. Elemental microanalysis was conducted using the ZAF correction method, which accounts for atomic number (Z), absorption (A), and fluorescence (F) effects. It was performed both in area-based and point-based modes. Graphs and tables displaying the elemental composition and levels of the analyzed regions were obtained. Changes in calcium (Ca) and phosphorus (P) content before and after treatment were statistically evaluated.

Antibacterial activity test

The antibacterial activity of the experimental pulp-capping materials (n = 3/group) and HBAMP was evaluated against Streptococcus mutans (ATCC® 25,175) and Lactobacillus casei (ATCC® 393) strains using the agar disk diffusion method. HBAMP solutions at 1% and 3% concentrations were independently tested. Bacteria were cultured on FAA plates (Fastidious Anaerobe Agar with 5% horse blood; Lab M). From the precultures, bacterial suspensions were prepared at a concentration of 6 × 10⁵ colony-forming units (CFU)/mL. A volume of 150 μL of the bacterial suspension was evenly spread onto FAA agar plates using sterile swabs. An ampicillin antibiotic disc was used as the positive control. Specimens were aseptically placed on the inoculated agar surfaces. Plates were incubated anaerobically at 37 °C for 48 h. Following incubation, inhibition zones formed around the disc specimens were measured. The study was conducted in triplicate and arithmetic means were used for data evaluation.

Evaluation of cytotoxicity

The cytotoxicity of the experimental pulp-capping materials (n = 6/group) was assessed using the Methyl Thiazolyl Tetrazolium (MTT) assay. To eliminate potential surface contaminants, all specimens were rinsed three times with sterile phosphate-buffered saline (PBS) for 10 min each on a shaker platform. The discs were then placed into 96-well plates with the coated surface oriented upward. Human gingival fibroblasts (hGFs, ATCC PCS-201–018) were expanded in Dulbecco’s Modified Eagle Medium (DMEM; supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, and 1% L-glutamine) under standard conditions (37 °C, 5% CO₂, humidified atmosphere) until approximately 80% confluence. Cells were harvested using Trypsin–EDTA, counted with an automated cell counter, and seeded at a density of 10,000 cells in 100 µL medium per well onto the disc-containing plates.

To assess cytocompatibility, cell viability was evaluated by the MTT assay at 24 and 48 h. After each incubation period, the culture medium was aspirated under sterile conditions and replaced with 100 µL of MTT solution (0.5 mg/mL). In the control experiments, a negative control (cells + MTT + solubilizing buffer [10% SDS in 0.1 N HCl]), a positive control (cells + tamoxifen + MTT + solubilizing buffer [10% SDS in 0.1 N HCl]), and a blank reading (cell-free medium + MTT + solubilizing buffer [10% SDS in 0.1 N HCl]) were included. Plates were incubated at 37 °C for 4 h, after which the MTT solution was removed and replaced with 100 µL of dimethyl sulfoxide (DMSO) to dissolve formazan crystals formed by metabolically active cells. The plates were shaken for 5 min on a microplate shaker, and absorbance was measured at 560 nm using a microplate reader (Synergy HT, Biotek, VT, USA). Half maximal inhibitory concentration (IC₅₀) values were estimated using the SPSS software (SPSS Inc., Chicago, IL, USA).

Cell viability was calculated according to the following formula:

Statistical analysis

Data were analyzed using IBM SPSS Statistics v23. Normality of distribution was assessed with the Shapiro–Wilk test. For non-normally distributed data involving three or more groups, the Kruskal–Wallis H test was used, followed by Dunn's test for multiple comparisons. For normally distributed data involving three or more groups, one-way analysis of variance (ANOVA) was performed, with Tukey's or Bonferroni's test used for multiple comparisons.

Differences between dependent groups were evaluated using the Wilcoxon test. For normally distributed paired data measured at different time points, the paired-samples t-test was used. Quantitative data were presented as mean ± standard deviation and median (minimum–maximum). Statistical significance was set at p < 0.05.

Results

Characterization

XRD

Given that HBAMP exhibits an amorphous structural profile, no distinct or well-defined diffraction peaks were detected in its XRD pattern. As shown in Fig. 1, the diffraction peaks obtained from 1% HBAMP-RMCS and 3% HBAMP-RMCS were similar to those of the Control group (Theracal LC). No additional peaks were observed, nor were any existing peaks lost.

Fig. 1.

XRD spectra of experimental groups: HBAMP (pink), Control (green), 1% HBAMP-RMCS (blue), and 3% HBAMP-RMCS (red). RMCS: Resin-Modified Calcium Silicate, HBAMP: Hydroxyapatite-Binding Antimicrobial Peptide. Peak assignments: Ca₃O₅Si: Tricalcium silicate oxide BaO₃Zr: Barium zirconate SrO: Strontium oxide SiO₂: Silicon dioxide Zr₃O: Zirconium oxide Al₂O₃: Aluminum oxide

Within the matrix of Theracal LC, peaks corresponding to zirconium, silicon, strontium, aluminum, and calcium-containing barium particles were identified. The most prominent peak of tricalcium silicate (ICDD: 98–008–1100) was observed at 2θ = 32.193°. The highest peaks for barium zirconate (ICDD: 98–009–7462), aluminum oxide (ICDD: 98–015–1589), and silicon oxide (ICDD: 98–015–8531) were detected at 2θ = 30.073°, 38.593°, and 30.117°, respectively [16].

Fourier transform infrared spectroscopy (FT-IR)

Compared with the Control group (Theracal LC), the 1% HBAMP-RMCS and 3% HBAMP-RMCS groups exhibited distinct peaks in the FT-IR spectra, indicating that HBAMP incorporation produced noticeable differences (Fig. 2). The aliphatic C = C stretching peak at 1638 cm⁻¹ (black arrow) and the aromatic C = C stretching peak at 1608 cm⁻¹ (blue arrow), characteristic of Theracal LC, were affected in the HBAMP-containing groups. These spectral changes are presumed to originate from HBAMP-specific peaks at 1663 cm⁻¹ and 1626.7 cm⁻¹. Collectively, these findings confirm the successful integration of HBAMP into the RMCS matrix.

Fig. 2.

FT-IR transmittance spectra of experimental groups: HBAMP (Peptide), Control (A), 1% HBAMP- RMCS (B), and 3% HBAMP-RMCS (C)

Degree of conversion (DC)

The mean degree of conversion values, standard deviations, and statistical comparisons for each group are presented in Table 1. No statistically significant differences were observed among the groups (p = 0.083).

Table 1.

Comparison of monomer degree of conversion (mean ± standard deviation (SD)) values among experimental groups

Group	Monomer Degree of Conversion mean ± SD	Test Statistic	p
Control	45,343 ± 2,946	2.958	0,083
1% HBAMP-RMCS	48,018 ± 2,8
3% HBAMP-RMCS	44,077 ± 2,85

One-way ANOVA; Mean ± Standard deviation, Median (minimum–maximum)

Vickers microhardness analysis of the pulp-capping material

The median microhardness values, minimum and maximum ranges, and statistical comparisons for each group are presented in Table 2. No statistically significant differences were found among the groups (p = 0.097).

Table 2.

Comparison of Vickers microhardness (median (minimum–maximum)) measurements among experimental groups

Group	Vickers Microhardness		Test Statistic	p
	median	(min–max)
Control	29,31	(19,13—32,93)	4,667	0,097
1% HBAMP-RMCS	24,545	(19,53—27,33)
3% HBAMP-RMCS	27,545	(22,76—28,83)

Kruskal–Wallis H test; Data are presented as mean ± standard deviation, median (minimum–maximum)

Mineral deposition and surface changes evaluation

Evaluation of Vickers microhardness analysis results

The median microhardness values, minimum and maximum ranges, and statistical comparisons of dentin specimens in each group are presented in Table 3. The graph illustrating the Vickers microhardness values of dentin specimens before and after application across the experimental groups is presented in Fig. 3. Post-treatment comparisons revealed a statistically significant difference in median values among the groups (p = 0.049). There was no significant difference between the Control group and the 3% HBAMP-RMCS group (p > 0.05). In contrast, an important difference was found between the Control group and the 1% HBAMP-RMCS group (p < 0.05). Intra-group evaluations demonstrated statistically significant differences in microhardness measurements before and after treatment within the Control, 1% HBAMP-RMCS, and 3% HBAMP-RMCS groups (p < 0.05).

Table 3.

Comparison of Vickers microhardness (median (minimum–maximum)) measurements of tooth specimens before and after application among experimental groups

Group	Before application	After application	After- Before	Test Statistic	p
Control	81,25 (70,93—86,5)	90,55 (83,33—284)	12,81 (3,17–197,77)	2,000	0,023
1% HBAMP-RMCS	80,91 (76,76—211)	99,06 (90,8—276,33)	16,835 (12,74—65,33)	0,000	0,008
3% HBAMP-RMCS	81,965 (77,33—93,2)	92,03 (90,83—100,4)	11,985 (1,6—17,1)	1,000	0,016
Test Statistic	0,305	6,014	5,820
p	0,859	0,049	0,055

^xKruskal–Wallis H test; ^yWilcoxon Test; ^a−bNo statistically significant difference was found between groups sharing the same letter. Data are presented as median (minimum–maximum)

The distinctions between treated and untreated surfaces are expressed in terms of 'after–before

Fig. 3.

Box plot of Vickers microhardness values of dentin specimens before and after application across experimental groups

Evaluation of FESEM analysis results

SEM images revealed that dentin surfaces in the Control group (RMCS without HBAMP) exhibited larger particle sizes and porous deposits. In contrast, HBAMP-containing groups showed reduced particle size and porosity, accompanied by the formation of crystalline and network-like structures (Fig. 4).

Fig. 4.

Scanning electron microscopy (SEM) images at 5000 × magnification, showing the surface morphology of the experimental groups. A1: Control group, untreated dentin surfaces. B1: 1% HBAMP-RMCS group untreated dentin surfaces. C1: 3% HBAMP-RMCS group untreated dentin surfaces. A2: Control group surfaces after material application. B2: 1% HBAMP-RMCS group surfaces after material application. C2: 3% HBAMP-RMCS group surfaces after material application

SEM–EDX (elemental analysis) results

The EDX images obtained from dentin specimens belonging to the experimental groups are shown in Fig. 5. The mean calcium (Ca) and phosphorus (P) values, standard deviations, and statistical comparisons for each group are presented in Table 4. According to EDX analysis, intra-group comparisons revealed no significant differences in phosphorus measurements for the Control and 1% HBAMP-RMCS groups (p > 0.05), whereas the 3% HBAMP-RMCS group exhibited a statistically significant difference (p < 0.05). Intra-group evaluations showed no significant differences in calcium measurements for the Control and 1% HBAMP-RMCS groups (p > 0.05), while the 3% HBAMP-RMCS group demonstrated a statistically significant difference (p < 0.05).

Fig. 5.

EDX images obtained from dentin specimens belonging to the experimental groups. A1: Control group, untreated dentin surfaces. B1: 1% HBAMP-RMCS group untreated dentin surfaces. C1: 3% HBAMP-RMCS group untreated dentin surfaces. A2: Control group surfaces after material application. B2: 1% HBAMP-RMCS group surfaces after material application. C2: 3% HBAMP-RMCS group surfaces after material application

Table 4.

Comparison of elemental analysis results) (mean ± standard deviation (SD)) by element (Ca and P) among experimental group

Group	Before application		After application		After- Before
	Ca	P	Ca	P	Ca	P
Control	33,933 ± 2,472	13,219 ± 0,94	33,843 ± 2,756	12,395 ± 1,223	−0,09 ± 2,569	−0,824 ± 1,188
1% HBAMP-RMCS	34,403 ± 2,097	13,453 ± 0,745	34,965 ± 3,245	13,181 ± 1,34	0,562 ± 1,846	−0,271 ± 0,985
3% HBAMP-RMCS	32,763 ± 2,393	12,779 ± 0,875	32,208 ± 2,644	11,654 ± 0,948	−0,555 ± 0,456	−1,125 ± 0,331

One-way ANOVA; Kruskal Wallis H Test; Wilcoxon Test; ^a−bNo significant difference between groups sharing the same letter. Mean ± Standard deviation, Median (minimum–maximum) ^x–y:No significant difference inter group sharing the same letter.Ca: Calcium P: Phosphorus

The mean Ca/P ratios, standard deviations, and statistical comparisons for each group are presented in Table 5. The graph depicting the Ca/P ratios of dentin specimens before and after application across the experimental groups is presented in Fig. 6. No statistically significant differences were observed between pre- and post-treatment mean Ca/P ratios across groups (p > 0.05). However, intra-group evaluations revealed statistically significant differences in Ca/P measurements for the Control, 1% HBAMP-RMCS, and 3% HBAMP-RMCS groups (p < 0.05).

Table 5.

Comparison of Ca/P ratios (mean ± standard deviation (SD)) among experimental groups

Group	Before application	After application	p
Control	2,57 ± 0,134	2,739 ± 0,186	0,003y
1% HBAMP- RMCS	2,558 ± 0,087	2,656 ± 0,088	0,014y
3%HBAMP-RMCS	2,564 ± 0,089	2,764 ± 0,066	< 0,001y
p	0,976x	0,054x

^xOne-way ANOVA; ^yPaired Sample T Test; Mean ± Standard deviation

Fig. 6.

Graph of Ca/P ratios of dentin specimens before and after application across experimental groups

The mean Ca/P percentage increase values, standard deviations, and statistical comparisons for each group are presented in Table 6. No statistically significant differences were found between the Control group and the HBAMP-containing groups (1% and 3% HBAMP-RMCS) in terms of Ca/P percentage increase values (p = 0.059).

Table 6.

Comparison of mean Ca/P ratio increase values (mean ± standard deviation (SD)) among experimental groups

Group	n	Total	mean ± SD	p
Control	8	47,95	5,99 ± 0,82	0,059
1% HBAMP- RMCS	8	53,86	6,73 ± 2,13
3% HBAMP- RMCS	8	62,68	7,83 ± 3,68

^*One-way ANOVA; Mean ± Standard deviation

Evaluation of antibacterial test results

The test images of the experimental groups are presented in Fig. 7. The mean inhibition zone diameters produced by HBAMP solutions at 1% and 3% concentrations against S. mutans and L. casei strains are presented in Table 7. For S. mutans, 1% HBAMP generated an inhibition zone of 15 mm, while 3% HBAMP produced a zone of 21 mm. For L. casei, inhibition zones of 14 mm and 15 mm were observed with 1% and 3% HBAMP, respectively. In contrast, no inhibition zones were detected in the Control, 1% HBAMP-RMCS, or 3% HBAMP-RMCS groups against either S. mutans or L. casei.

Fig. 7.

Agar disk diffusion test images of the experimental groups. A: HBAMP at concentrations of 1% and 3% against S. mutans; B: HBAMP at concentrations of 1% and 3% against L. casei; C: Control, 1% HBAMP-RMCS, and 3% HBAMP-RMCS against S. mutans; D: Control, 1% HBAMP-RMCS, and 3% HBAMP-RMCS against L. casei

Table 7.

Diameters of inhibition zones (mean ± standard deviation (SD)) produced by HBAMP at concentrations of 1% and 3% against Streptococcus mutans and Lactobacillus casei

	1% HBAMP mean ± SD	3% HBAMP mean ± SD
S.mutans	15 mm ± 0.5a	21 mm ± 0.5b
L.casei	14 mm ± 1a	15 mm ± 1a

xPaired Sample T Test; a−bNo significant difference between groups sharing the same letter. Mean ± Standard deviation

Evaluation of cytotoxicity test results

According to the MTT assay, the mean cell viability rates, standard deviations, and statistical comparisons after 24 h and 48 h of incubation are presented in Table 8. The graph presenting the MTT results of the experimental groups is shown in Fig. 8. The mean effective concentrations of the Control, 1% HBAMP-RMCS, and 3% HBAMP-RMCS groups were determined as 2.08 μg/mL at 24 h and 1.97 μg/mL at 48 h.

Table 8.

Average cell viability rates (mean ± standard deviation (SD)) of experimental groups at the 24 and 48 h time point in the MTT assay

	24 h mean ± SD	48 h mean ± SD
Culture medium control	100,00 ± 5,65A	100,00 ± 4,84A
Control	52,75 ± 4,21B	46,27 ± 2,92B
1%HBAMP-RMCS	45,91 ± 1,77B	45,27 ± 0,84B
3% HBAMP- RMCS	51,17 ± 3,14B	43,15 ± 2,80B

^*Bonferroni Test; ^A−BNo significant difference between groups sharing the same letter within the same column. Mean ± Standard deviation, Median (minimum–maximum)

Fig. 8.

Graph of MTT assay results for the experimental groups

At 24 h and 48 h, statistically significant differences were observed between the culture medium group and all experimental groups in terms of mean cell viability (p < 0.05). However, no statistically significant differences were found among the experimental groups themselves (p > 0.05).

Discussion

This study evaluated the effects of incorporating HBAMP into a resin-modified calcium silicate pulp-capping material. Overall, peptide addition did not negatively affect the physical properties of RMCS and produced measurable early mineral deposition and surface-level changes on dentin within a 72-h period, while its antibacterial activity was attenuated within the RMCS matrix.

A homogeneous polymerization process is a fundamental requirement for the long-term clinical success of resin-based materials [17]. All specimens in the present study were polymerized strictly following the manufacturer’s instructions [8], thereby ensuring both the validity and reproducibility of the polymerization process. In the present study, DC values exceeding 40% were obtained in the experimental groups, which are consistent with previous findings [18, 19].

Demirbuga et al. reported that the DC of experimental materials remained unchanged when the nanocarrier concentration was low or when the nanocarrier or drug did not interfere with the chemical structure of resin monomers [20]. Consistent with the obtained findings, incorporation of HBAMP into RMCS at low concentrations did not compromise polymerization efficiency, while the essential structural characteristics of the material remained intact. Accordingly, the first null hypothesis of this study was accepted.

Surface microhardness can be used as an indirect indicator of the extent of the setting process and the overall resistance to deformation, being a principal parameter for the mechanical properties of the material [21]. The findings obtained in our study indicate that low HBAMP concentrations did not produce a significant effect on microhardness. Given the amorphous nature of HBAMP, the addition of the peptide may not have altered the microhardness of the material. Accordingly, the second null hypothesis of this study was accepted. Consistent with our results, a previous study reported that the addition of functional components such as cHAP and CHX into RMCS formulations at low concentrations did not significantly alter the overall microhardness of the material, yielding values comparable to those observed in our study [5].

Specimens can be subjected to incubation for different time intervals in order to assess the progression of the remineralization process [22, 23]. In our study, a 72-h period was selected as the initial screening step to evaluate early mineral deposition and surface modifications on dentin.

An increase in mineral density within dental tissues is directly associated with enhanced dentin hardness [24]. The Vickers microhardness test is widely used to measure the hardness of dental materials and serves as an effective tool for evaluating remineralization effects [25, 26]. FESEM and SEM–EDX are advanced techniques that not only reveal morphological changes on tooth surfaces but also provide elemental composition analyses, making them frequently employed in dental research [25–29].

Wang et al. reported that initial carious lesions treated with TVH19, exhibited higher surface microhardness values, shallower lesion depths, and increased mineral content [30]. Similarly, Türkay et al. demonstrated that the mechanical properties of caries-affected dentin improved following the application of CaGP [31]. Additionally, a study examining the remineralization capacity of resin-based calcium phosphate cement used for indirect pulp capping found that its application increased the microhardness of residual dentin tissue, thereby supporting remineralization [32]. Consistent with these findings, the incorporation of the peptide was shown to improve dentin microhardness effectively. Notably, the 1% HBAMP-RMCS group showed promising potential as a modification for resin-modified calcium silicate materials.

The efficacy of remineralization agents does not always exhibit a linear relationship with concentration, and excessive concentrations may harm mineralization processes. Moreover, such concentrations were considered impractically high for incorporation into materials, as they inevitably produced some degree of aggregation within the matrix [33]. In accordance with these findings, despite the higher peptide concentration in the 3% HBAMP-RMCS group, the increase in microhardness was not proportional to the peptide concentration.

Calcium and phosphorus are the elements with the highest ionic concentrations in dentin [34]. Accordingly, the Ca/P ratio is frequently employed as an indicator of changes in mineral composition in dental hard tissues [35]. For this reason, the present study evaluated early mineral deposition by comparing changes in calcium and phosphorus contents obtained from EDX analysis.

In our study, although a numerical increase in calcium and phosphorus levels was observed in the 1% HBAMP-RMCS group after the procedure, this difference was not statistically significant. Similar to the findings reported by Gavrila et al. [34], our study showed a remineralization potential with 1% HBAMP, despite the lack of statistically significant differences, suggesting preliminary evidence of early mineral deposition on dentin under short-term conditions.

In a previous study, it was reported that remineralizing nanoparticles were unable to sufficiently bind to the collagen matrix, resulting in ineffective retention of phosphorus within the matrix [36]. In our study, particularly in the 3% HBAMP-RMCS group, structural changes in dentin and insufficient binding of the remineralizing peptide to the collagen matrix may have contributed to mineral dissolution and phosphorus loss.

Similar to the findings reported by Daneshpoor et al. [37], our study also showed numerical increases in Ca/P percentage values despite the lack of statistically significant inter-group differences. This suggests a potential difference among groups, although the current data were insufficient to confirm this distinction definitively.

Kunert et al. [35] reported that dentin surfaces treated with Theracal LC exhibited a porous interface after 24 h, plaque-like precipitates after 7 days of incubation, and sporadic surface deposits of mineral crystallites uniformly covering the surface after 28 days. In the present study, SEM images revealed that dentin surfaces treated with Theracal LC displayed larger particle sizes and a porous structure. In contrast, HBAMP-containing groups exhibited reduced particle size and porosity, accompanied by the formation of crystalline and network-like structures. These findings suggest that HBAMP-containing groups exhibited more pronounced surface changes within a short period (72 h) compared with the control group, supporting the hypothesis that HBAMP may promote mineral deposition on dentin. This phenomenon may be explained by the electrostatic interaction between HBP7 and HAp during the binding process [13]. Accordingly, considering the observed mineral deposition and surface changes, the third null hypothesis of this study was partially rejected.

The agar disk diffusion method is widely used in dentistry to evaluate antibacterial properties, owing to its simplicity and ease of application [38]. In the present study, Streptococcus mutans, recognized as the primary pathogen responsible for dental caries, and Lactobacillus casei, which plays a key role in caries progression, were selected to assess the antibacterial activity of the experimental groups [39]. In our study, although HBAMP alone exhibited antibacterial activity against S. mutans and L. casei strains, no antibacterial effect was observed when it was incorporated into the RMCS formulation. Therefore, the fourth null hypothesis of this study was accepted.

Huang et al. reported that HBAMP was particularly effective against S. mutans and L. acidophilus, and at specific concentrations, it was able to penetrate biofilm layers. Nevertheless, HBAMP-coated HAp surfaces reduced but did not eliminate biofilm formation, suggesting that HBAMP exhibits concentration-dependent antibacterial activity and that its biofilm penetration capacity may be limited [13].

Although the antimicrobial activity of Theracal LC has been reported in previous studies [5, 19], no inhibition zones were observed against S. mutans or L. casei in our research. This inconsistency may be attributed to differences in antibacterial testing methods or bacterial strains used.

Akın et al. also reported that incorporating resveratrol—known for its inhibitory effects on S. mutans—into a calcium hydroxide–based pulp-capping material did not produce significant differences in antibacterial activity among groups [40]. Additionally, the literature has reported that the efficacy and potency of AMPs may be diminished in the presence of salts, metals, variations in pH, and other environmental factors [9]. The elemental composition and pH changes of the RMCS material may have reduced the antibacterial activity of the peptide. These findings collectively indicate that the antibacterial performance of resin-based materials is highly dependent on material composition, release characteristics, and test conditions.

The incorporation of HBAMP into RMCS is intended not to provide rapid release but to achieve controlled and slow release. The polymerized resin–calcium silicate matrix forms a structure that restricts peptide diffusion. Therefore, the absence of inhibition zones in the agar diffusion test does not necessarily indicate a lack of antibacterial activity but may instead reflect the limited diffusion of the peptide from the matrix. Consequently, the agar diffusion method may underestimate antibacterial activity in low-release systems. The literature indicates that such variations in the antibacterial activity of similar materials may arise from factors such as mixing ratios, timing of evaluation, post-curing effects, and differences in test methodologies [40]. The findings of the present study are consistent with these reports. Future studies employing direct contact tests, biofilm viability assays, confocal laser scanning microscopy (CLSM), and peptide release kinetics could further elucidate the antibacterial performance and underlying mechanisms of the material.

The MTT assay employed in this study is a widely preferred, reliable, and effective method for assessing cell viability and cytotoxicity [41]. hGF cells, used in the present tests, are a commonly applied cell line for evaluating the biocompatibility of dental materials [42].

To estimate the IC50 values, a common approach involves fitting the experimental data to a dose–response curve using linear regression. For this purpose, the x–y data representing compound concentrations and corresponding cell viability percentages are plotted, and a straight line is fitted to the data. The equation of the fitted line is expressed as Y = a * X + b, where Y represents the response (cell viability), X represents the concentration of the compound, and 'a' and 'b' are coefficients. The IC50 value can then be calculated from the equation as (0.5 − b)/a [43]. Based on the IC50 values obtained (2.08 μg/ml at 24 h and 1.97 μg/ml at 48 h), we can conclude that the maximum peptide concentration that can be used while maintaining cell viability is approximately 2%. Although no statistically significant differences in cell viability were observed among the groups, concentrations exceeding this threshold were associated with increased cytotoxicity, particularly following prolonged exposure. incubation, the 3% HBAMP-RMCS group exhibited the greatest numerical reduction in cell viability, consistent with the IC₅₀ findings. Therefore, although the intergroup differences were not statistically significant, the IC₅₀ values indicated a concentration-dependent cytotoxic potential of HBAMP, and accordingly, the fifth null hypothesis of this study was partially rejected.

Huang et al. reported that exposure to 250 μg/mL HBAMP for 240 min did not affect the membrane integrity of hGF cells, whereas exposure to 500 μg/mL HBAMP induced time-dependent LDH leakage [13]. These findings indicate that HBAMP at high concentrations and with prolonged exposure may exert adverse effects on cell health. In parallel with our study, it was observed that higher HBAMP concentrations had a greater impact on cell viability during long-term exposure, whereas lower concentrations exhibited less toxic effects.

The low cell viability observed across all groups may be attributed to the intrinsic cytotoxic characteristics of the base RMCS material rather than the presence of HBAMP. However, the MTT assay evaluates only the cytotoxic component of biocompatibility; other critical aspects such as apoptosis, inflammation, oxidative stress, and membrane integrity were not assessed. Therefore, future studies should investigate biocompatibility more comprehensively using different cell lines, 3D cell culture models, and in vivo experiments.

Since this study was conducted under controlled laboratory conditions, the results may not fully reflect clinical circumstances. The evaluation of the materials using a single curing time and a single material thickness represents an important methodological limitation. Additionally, because the study was performed on sound dentin and involved a 72-h incubation period, subtle mineral changes may not have been distinctly observed.

The antibacterial activity assessment was also limited to selected bacterial species and was performed using only the agar diffusion method, which may not fully reflect the clinical behavior of resin-based materials. Furthermore, long-term evaluations of physical properties such as hardness, durability, pH stability, and adhesion should be conducted through extended testing protocols to better determine the material’s clinical applicability. Finally, although the stability, binding behavior, and release kinetics of HBAMP within the matrix were beyond the scope of the present study, these parameters are critically important for future research.

Conclusions

This in vitro study evaluated the preliminary effects of incorporating HBAMP into a resin‑modified calcium silicate–based pulp‑capping material. The addition of HBAMP did not adversely affect the physical properties of RMCS. Although HBAMP exhibited potent antibacterial activity in its isolated form, this effect was limited when incorporated into the RMCS matrix. Numerical increases in dentin microhardness and Ca/P ratios were observed following HBAMP incorporation.

Cytotoxicity results showed no statistically significant differences between the experimental groups. Nevertheless, the IC₅₀ values demonstrated a numerical tendency toward lower cytotoxicity in the 1% HBAMP‑RMCS group, suggesting a possible concentration‑dependent effect.

Overall, the findings suggest that HBAMP incorporation may have the potential to influence early mineral deposition without compromising the basic properties of RMCS; however, these observations remain preliminary. Further studies additional antibacterial assessment methods, and long‑term biological evaluations are required before drawing definitive conclusions regarding the clinical relevance of HBAMP‑modified RMCS in vital pulp therapy.

Abbreviations

AMP

Antimicrobial peptide

PrAMPs

Proline-rich antimicrobial peptides

DNA

Deoxyribonucleic Acid

HBAMP

Hydroxyapatite-binding antimicrobial peptide

RMCS

Resin-modified calcium silicate–based material

HAp

Hydroxyapatite

HBP7

HAp-binding heptapeptide

Authors’ contributions

HÇ: Writing – original draft, Formal analysis, Data curation, Investigation, Conceptualization. HB1: Writing – review & editing, Methodology, Investigation, Material acquisition, Conceptualization. SD: Writing – review & editing, Supervision, Project administration, Funding acquisition. HB2: Methodology, Conceptualization.

Funding

This study was supported by Erciyes University Scientific Research and Project Foundation (Approval No: TDH-2024–13948).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the Clinical Research Ethics Committee of Erciyes University (Approval No: 2024/283), according to the principles of The World Medical Association Helsinki Declaration. Informed written consent forms were obtained from each patient whose extracted teeth were used in the study.

Consent for publication

Not applicable.

Competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.