# In silico and in vitro characterization of GH12, an innovative peptide for dental pulp regeneration

Lisa Reiniche, Marjorie Faure, Marianne Leveque, Cédric Orelle, Audrey Ziverec, Jean-Christophe Farges, François Virard, Jean-Daniel Malcor,

Mourad Bekhouche, Maxime Ducret

► To cite this version:

Lisa Reiniche, Marjorie Faure, Marianne Leveque, Cédric Orelle, Audrey Ziverec, et al.. In silico and in vitro characterization of GH12, an innovative peptide for dental pulp regeneration. Journal of Dentistry, 2025, 163, pp.106105. 10.1016/j.jdent.2025.106105. hal-05343278

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# *In silico* and *in vitro* characterization of GH12, an innovative peptide for dental pulp regeneration

Lisa Reiniche<sup>a</sup>, Marjorie Faure<sup>a,c,d</sup>, Marianne Leveque<sup>a</sup>, Cédric Orelle<sup>b</sup>, Audrey Ziverec<sup>a</sup>, Jean-Christophe Farges<sup>a,c,d</sup>, François Virard<sup>c,d,e</sup>, Jean-Daniel Malcor<sup>a</sup>, Mourad Bekhouche<sup>a</sup>, Maxime Ducret<sup>a,c,d,*</sup> <sup>IFC</sup>

## ARTICLE INFO

### Keywords:

GH12

Dental pulp

Cytotoxicity

Pore

Lysosome

Fibrin hydrogel

## ABSTRACT

**Objective:** To characterize the mechanism of action of GH12 and to assess its cytotoxicity on dental pulp mesenchymal stem cells (DP-MSCs).

**Methods:** GH12 mechanism of action was characterized using *in silico* predictions and lipid bilayer liposome assays. GH12 cytotoxicity was assessed in 2D culture using DP-MSCs through Live/Dead assay and lysosome staining after 2, 4, and 6 h of incubation. Finally, GH12 cytotoxicity was assessed in a fibrin-based 3D hydrogel after 48 h.

**Results:** *In silico* predictions suggested a trimeric or tetrameric pore-forming mechanism for GH12, and its capacity to destabilize membranes was further validated by liposome permeabilization assays. In 2D cultures, GH12 significantly decreased DP-MSCs viability and increased lysosomal staining at 50 µg/mL after 2, 4, and 6 h (*p* < 0.05). In the 3D model, GH12 did not exhibit a significant effect on DP-MSCs viability (92 % at 50 µg/mL after 48 h; *p* = 0.18).

**Conclusion:** *In silico* predictions suggested that GH12 may adopt trimeric or tetrameric assemblies consistent with a pore-forming mechanism. While cytotoxicity and cellular stress were observed in 2D cultures of DP-MSCs, no toxicity was detected in the fibrin-based 3D model.

**Clinical Relevance:** These findings support the potential integration of the promising antimicrobial peptide GH12 into advanced regenerative endodontic models, paving the way for further preclinical investigations and its eventual translation into clinical applications.

## 1. Introduction

Damage of the dental pulp (DP) may require pulpectomy and subsequent filling using a sterile root material. Due to the several limitations related to conventional therapies [1–4] regenerative endodontic procedures (REPs) have been developed [1]; they can be composed of scaffolds, growth factors, or DP-mesenchymal stem cells (DP-MSCs) [3, 5]. However, it has been observed that tissues obtained using such procedures do not always display the properties of a pulp tissue and may even present scarring fibrosis or heterotopic mineralization, therefore

failing to allow tissue function [5]. This outcome can be explained by the infectious context of the endodontic space, where disinfection is never entirely effective inside the tooth, allowing residual pathogens to persist and compromise the success of the regeneration procedure [2,6]. Of note, oral bacteria have developed antimicrobial resistance (AMR) to antibiotics such as clindamycin and amoxicillin [7], which is a significant challenge in dentistry [8].

A promising alternative to antibiotics in REPs is the use of antimicrobial peptide (AMP) such as GH12, known to reduce the risk of AMR compared to conventional strategy [8]. Recent studies have reported its

<footnote><sup>a</sup> Laboratoire de Biologie Tissulaire et Ingénierie thérapeutique, UMR 5305 CNRS/Université Claude Bernard Lyon 1, UMS 3444 BioSciences Gerland-Lyon Sud, Lyon, France</footnote>

<footnote><sup>b</sup> Molecular Microbiology and Structural Biochemistry (MMSB), UMR 5086 CNRS/Université Claude Bernard Lyon 1, Lyon, France</footnote>

<footnote><sup>c</sup> Faculté d'Odontologie, Université Claude Bernard Lyon 1, Lyon, France</footnote>

<footnote><sup>d</sup> Service d'Odontologie, Hospices Civils de Lyon, Lyon, France</footnote>

<footnote><sup>e</sup> Cancer Research Center of Lyon (CRCL), INSERM 1052, CNRS 5286, Lyon, France</footnote>

<footnote>* Corresponding author at: LBTI, 7 passage du vercors, 69007 Lyon, France.</footnote>

<footnote>E-mail addresses: ducret.maxime@gmail.com, maxime.ducret@univ-lyon1.fr (M. Ducret).</footnote>
strong antibacterial activity against cariogenic bacteria [9], as well as mono-species [10] and multi-species endodontic biofilms [11], and its potential use as an irrigant [10], making it a promising candidate for REPs. The present study investigates the potential of GH12 to be incorporated into a scaffold, where it could prevent endodontic bacterial colonization. This antibacterial protection is particularly relevant as bacteria can compromise pulp regeneration [12,13]. In this study, a fibrin matrix was selected to provide a reproducible *in vitro* model, reflecting key features of blood-derived scaffolds used in REPs [14].

According to the literature, the mechanism of action of GH12 may involve pores formation in the bacterial membranes, disrupting their integrity, and leading to lysis and death [15]. However, the effect of GH12 on DP-MSCs has not been explored and could provide insights into its suitability for REPs.

This study aimed to investigate GH12's mechanism of action by predicting its potential oligomeric assembly at the membrane using *in silico* modeling, and by evaluating its pore-forming activity through a liposome leakage assay. GH12 cytotoxicity was assessed in DP-MSCs using 2D and fibrin-based models.

# 2. Materials and methods

## 2.1. Structural representation and predictive modeling of GH12 assembly

The predictive models were generated using AlphaFold 3 [16] and Boltz-2 (version 2.1.1), [17,18]. For Boltz-2, predictions were performed using the default parameters, including 5 samples, 3 recycles, a step scale of 1.638, and a batch size of 1. AlphaFold 3 predictions were also obtained using default parameters, including the use of PDB templates restricted to entries prior to the default cut-off date (30/09/2021) and 10 recycles. Predictions were performed in both software using increasing numbers of GH12 peptides (from 2 to 7 copies) with the following GH12 sequence: GLLWHLLHHLLHH. The software based its results on 2 parameters: predicted Template Modeling (pTM) score and interface predicted Template Modeling (ipTM) score. The pTM score is a metric used to assess the overall quality of a predicted protein structure. It typically ranges from 0 to 1, and a higher score indicates a more reliable and accurate model. A score close to 1 suggests that the predicted structure is very similar to the potential native structure, whereas a lower score indicates less confidence in the prediction. The ipTM score evaluates the quality of the predicted interface between protein chains or within protein complexes. Like the pTM score, it ranges from 0 to 1, with higher values indicating better and more reliable interface predictions. The 3D visualization was performed in OpenPyMOL (version 3.0) [19].

## 2.2. GH12 production and assessment of membrane permeabilization via liposome leakage assay

### 2.2.1. GH12 production

GH12 is a 12-amino acid (GLLWHLLHHLLHH), cationic, amphipathic α-helical AMP characterized by its high stability. Peptides were synthesized using Fmoc/tert-butyl solid phase strategy at the Protein Science Facility of the SFR Biosciences platform (Lyon, France). 9-Fluorenylmethoxycarbonyl (Fmoc) protected amino acids were supplied from Merck. All other reagents were purchased from Sigma-Aldrich. Briefly, peptides were assembled on a LibertyTM microwave peptide synthesizer (CEM) at a 0.1 mmol scale on Fmoc-Rink amide aminomethyl resin, using HCTU as a coupling reagent, DIEA as base, and 20 % piperidine for Fmoc deprotection. Peptides were cleaved from the resin with a mixture of trifluoroacetic acid (TFA)/triisopropylsilane/H2O 95/2.5/2.5 v/v/v at room temperature for 3 h. The cleavage solution was concentrated, precipitated in cold diethyl ether, dissolved in an H2O/acetonitrile (ACN) 95/5 v/v (0.1 % TFA) mixture and freeze dried. Crude products were purified by preparative reverse-phase high performance liquid chromatography using a C18 column with a gradient

of ACN in water with 0.1 % TFA. Purified peptides were characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry at the Protein Science Facility of the SFR Biosciences platform (Lyon, France).

### 2.2.2. Liposome leakage assay

To characterize GH12's mechanism of action on membranes, its ability to permeabilize liposomes was investigated with a carboxy-fluorescein leakage assay. A solution of 100 mM 6-carboxyfluorescein (6-CF) was prepared in distilled water at pH 7-7.5 [20]. Then, 10 mg of *Escherichia coli* Extract Total (Avanti Polar Lipids, AL, USA) were resuspended in 1 mL of 100 mM 6-CF. The solution was submitted to 7 cycles of freezing/thawing (in liquid nitrogen and 37 °C water bath, respectively). Then, the multilamellar liposomal suspension was extruded 15 times through a 400-nm polycarbonate membrane and 19 times through a 100-nm membrane using a mini extruder (Avanti Polar Lipids). This process resulted in the formation of unilamellar vesicles, which were then collected using a PD-10 desalting column to remove the excess of free 6-CF. Fluorescence experiments were performed using a final volume of 1 mL of distilled water containing 2 µL of liposomes or free 6-CF. Fluorescence was monitored using a Photon Technology International Quanta Master I fluorimeter (excitation was set at 492 nm and emission between 500 and 550 nm). In the assays, the n-dodecyl-β-d-maltoside (DDM) detergent was used at 0.5 % (W/V) as a positive control of permeabilization, while 40 µg/mL GH12 was added at multiple time points.

## 2.3. Isolation, expansion, and amplification of DP-MSCs

### 2.3.1. Isolation and expansion of DP-MSCs

DP-MSCs isolation and expansion were performed as previously described [21]. Briefly, healthy human third molars were collected from donors aged 12-17 years who provided (and their parents) informed consent, in accordance with the World Medical Association's Declaration of Helsinki and the French Public Health Code (Article R1211-49), and following a protocol approved by the French Ministry of Higher Education, Research and Innovation (CODECOH: DC-2014-2325). After extraction, the teeth were kept in a PBS solution with Penicillin/Streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). The tooth's apical papilla was removed with a scalpel to allow the access to the dental pulp, which was then carefully extracted and cut into fragments of 0.5-2 mm³. Then, explants were seeded in a 6-well plate, that has been previously coated with human placental collagen I and III at 1 mg/mL (CTI Biotech, Lyon, France) and cultured in serum-free medium (Univercell Biosolutions, Toulouse, France) at 37 °C and 5 % CO₂. After 2 weeks, cells outgrowing from the explants were harvested using TrypLE (Thermo Fischer Scientific, Odense, Denmark), resuspended in Stem Cryo3 (Stem alpha, Saint-Laurent-de-Chamouset, France) with 10 % DMSO medium at a density of $1 \times 10^6$ cells and stored at -196 °C.

### 2.3.2. Amplification of DP-MSCs

Collagen I and III coatings were applied to T75 flasks at least two hours before cell seeding. Frozen aliquots containing DP-MSCs were thawed in warm SPE IV medium (CTI Biotech, Lyon, France) and seeded onto the collagen coated flasks for amplification. The culture medium was replaced every 2 to 3 days, and once the cells reached an 80-90 % confluence, they were seeded into a new flask at a density of 40 % confluence. The use of DP-MSCs did not exceed 5 passages for any of the experiment to maintain the mesenchymal stem cell phenotype [22-24].

## 2.4. Cytotoxicity of GH12 on DP-MSCs: 2D model

### 2.4.1. Live/Dead assay

For the 2D culture, the viability of the cells in presence of 0, 10, 25, and 50 µg/mL of GH12 was assessed over a short period of time (2, 4, 6
h) using the Live/Dead™ kit (04,511–1KT-F, Sigma-Aldrich, St. Louis, MO, USA). This kit comprises calcein-AM and propidium iodide (PI) solutions to stain viable and dead cells, respectively. Briefly, 10 µL of calcein-AM and 5 µL of PI were diluted into 5 mL of PBS. Medium was removed and 300 µL of the prepared solution was added in each well. After 15 min of incubation at 37 °C, photos were taken with an eclipse TE300 (Nikon, Japan) microscope using FITC and TRICT filters. Images were analyzed using the Fiji software (1.53q version) following the BRTI Life Science (Bioactive Regenerative Therapeutics Inc, USA, MN) protocol to measure viability. The following formulas for quantification were used: 1) Total Cell Number = Live Cells + Dead Cells; and 2) Percentage of Live Cells = (Live Cells/Total Cell Number) *100.

The experiment was conducted on samples from three different donors (N = 3, two females aged 16 and 15 years, and one male aged 15 years) with duplicates performed for each condition. The mean percentage of live cells was calculated for each condition and used for statistical analysis.

### 2.4.2. LysoTracker staining

Lysosomes were stained using the LysoTracker™ Deep Red marker (Thermo Fisher Scientific, Waltham, MA, USA). The GH12 solution was added at concentrations of 0, 10, 25, and 50 µg/mL, and lysosome staining in DP-MSCs was observed after 2, 4, and 6 h. DP-MSC's media was removed and replaced by 75 nM LysoTracker™ Deep Red diluted into PBS and incubated during 45 min at 37 °C. Microscopy images were acquired using an Eclipse TE300 (Nikon, Japan) inverted microscope with a Cy5 filter: for all images, the exposure time was set at 1s, and the gain was adjusted to 5.4 to optimize signal intensity. The Fiji software was established to analyze the fluorescence intensity. The experiment was conducted on samples from three different donors (N = 3; one female aged 15 years, and 2 males aged 15 and 13 years) with duplicates performed for each condition. The mean intensity of fluorescence for each condition was calculated and used for statistical analysis.

## 2.5. Cytotoxicity of GH12 on DP-MSCs: fibrin-based model

### 2.5.1. Fabrication of fibrin hydrogel for DP-MSCs encapsulation

Fibrin hydrogels were synthesized following established protocols [25]. Human fibrinogen (ref. F3879) and thrombin (ref. T6884) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The formulations of fibrin-GH12 hydrogels were prepared by combining fibrinogen with a final concentration of 3 mg/mL, 3 M sodium chloride, 0.4 M calcium chloride, and 50 µg/mL GH12 in PBS. DP-MSCs were added to the mix at a final concentration of 22,000 cells/µL quantified using a Cellometer T4 (Nexcelom Bioscience Ltd, Lawrence, MA, USA). For the negative control (-) no GH12 was added, while for the positive control of death (+), 1 µL of Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) was incorporated into the gel, reaching a final concentration of 1 % (V/V). To induce polymerization, thrombin was used at a final concentration of 4 U/mL. Subsequently, 20 µL of this mixture was injected into small cylindrical PTFE tubing (1.32 mm diameter, ref. TS2204453, Adtech Polymer Engineering Ltd, Aston Down, United Kingdom). After 15 min of polymerization at room temperature, cylinders were cut to obtain a 5-mm length and immerged into SPE medium (CTI Biotech, Lyon, France) in 96-well plates (1 cylinder/well). Hydrogels were cultured at 37 °C for 48 h. The experiment was conducted on samples from three different donors (N = 3; two females aged 16 and 15 years, and one male aged 12 years) with duplicates performed for each condition. The mean percentage of live cells was calculated for each condition and used for statistical analysis.

### 2.5.2. Live/Dead assay

For the fibrin-based model, samples were collected, cut in half lengthways to increase the accessibility of the gel and promote fluorescent marker diffusion. The assay was then performed following the same procedure as for the 2D culture. Images were acquired using an

Eclipse TE300 (Nikon, Japan) inverted microscope from the exposed inner surface of the hydrogel. Although stacking effects may occur in 3D matrices, all samples were imaged using the same standardized approach to ensure consistency across conditions. Analysis was performed on a selected 660 µm² square area, ensuring it was free of blurring. The same viability analysis used for 2D culture was applied.

### 2.5.3. Technical validation of GH12 bioactivity in the fibrin-based model

This antibacterial assay was performed to confirm that the synthesized peptide remained active within the fibrin scaffold. The corresponding results are presented in the Supplementary Data (Supplementary Figure 1). Briefly, *S. aureus* (RN4220) and *E. faecalis* (ATCC 29212) cultures were grown in a controlled environment until they reached the stationary growth phase. The bacterial suspension was then diluted to an optical density of 0.6 at 600 nm. A total of 50 µL of the diluted culture was seeded onto a 9-cm plate containing agar-BHI (2.5 %, W/V, Brain Heart Infusion, Dutscher, Bernolsheim, France). Afterward, 30 µL of a pre-polymerized fibrin hydrogel was placed onto the agar surface. Fibrin hydrogels were prepared as described previously. For the negative control (-) GH12 was not added, while for the positive control of antibacterial activity (+) clindamycin (75 µg/mL) was added into the gel. GH12 was tested at a concentration of 50 µg/mL. Plates were incubated at 37 °C for 48 h under 10 % CO₂. Following incubation, images of the plates were taken using a standardized imaging setup, and the diameters of the bacterial inhibition zones were measured using the Fiji software.

## 2.6. Statistical analysis

All statistical analyses were conducted using GraphPad PRISM software (La Jolla, CA, USA). Variables were expressed as mean ± standard deviation (SD). The normal distribution of the variables was confirmed using a Shapiro test. A two-way ANOVA was used to assess the effects of treatment concentration and exposure time, followed by Tukey's multiple comparison test. A p-value < 0.05 was considered statistically significant.

# 3. Results

## 3.1. Structural representation and predictive modeling of GH12 assembly

Boltz and AlphaFold modeling were performed to predict GH12 potential oligomeric arrangements. Increasing numbers of GH12 monomers (from 2 to 7) were tested in silico (Fig. 1B). The prediction scores showed consistent tendencies between the two software tools, with both identifying the trimeric and tetrameric forms as the most stable configurations. These results suggest that these assemblies may represent the optimal multimeric states of GH12, and possibly its native conformation when interacting with membranes. The trimeric form showed the highest confidence score with Boltz, while the tetrameric form was preferred by AlphaFold. In both cases, the arrangement in trimer and tetramer appears to be uniform, since monomers share the same orientation. When more than four monomers were involved, the configurations became less uniform, displaying less defined interactions and reduced stability (Fig. 1A).

## 3.2. GH12 effect on liposome membrane

Liposome leakage was used to assess GH12 ability to form a pore, the potential mechanism involved in its interactions with cellular membranes [15]. The large unilamellar vesicles were prepared using 6-CF at sufficiently high concentration to induce the auto quenching of 6-CF and attenuate its fluorescence intensity. First, the effect of the DDM detergent on liposome permeability and dye dilution was tested. As expected, DDM addition resulted in a substantial increase in the fluorescence (Fig. 2A). Moreover, DDM did not alter the fluorescence of free 6-CF
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Predictive modeling of GH12 assembly. (A) 3D representations of Boltz and AlphaFold predictions using an increasing number of GH12 peptides ranging from 2 to 7 (number of peptides indicated at the bottom right). (B) Predicted Template Modeling (pTM) and Interface Predicted Template Modeling (ipTM) scores for each assembly prediction ranging from 2 to 7 peptides.</figcaption></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 2.** Liposomes leakage assay to assess the interaction of GH12 with lipid membranes. (A) Addition of the n-dodecyl-β-D-maltoside (DDM) detergent to the liposomes induces 6-CF leakage and an increase in its fluorescence. (B) The addition of DDM to free 6-CF did not substantially alter its fluorescence. (C) The addition of 40 µg/mL GH12 to liposomes likely caused a time-dependent decrease in 6-CF fluorescence. (D) The addition of 40 µg/mL GH12 to free 6-CF caused a strong quenching of its fluorescence.</figcaption></figure>

molecules (Fig. 2B), which indicated that DDM permeabilized the vesicles.

Afterwards, the effect of GH12 on the permeability of the liposomes was investigated. Surprisingly, the addition of GH12 seemed to induce a time-dependent decrease in 6-CF fluorescence (Fig. 2C). Then, the effect of GH12 was analyzed when incubated with free 6-CF. GH12 strongly and immediately quenched the fluorescence of free 6-CF (Fig. 2D). Altogether, these data indicate that GH12 first permeabilizes the liposomal membrane, allowing the release of the encapsulated dye, and then

interacts with 6-CF, leading to quenching. Such a time-dependent decrease is consistent with membrane permeabilization, since the dye is initially confined within the liposomes. This result also suggests that GH12 acts fairly rapidly, since its effects occurred within 10 min.

### 3.2.1. Cytotoxicity of GH12 on DP-MSCs in 2D models

The level of GH12 cytotoxicity was analyzed through three concentrations (10, 25, and 50 µg/mL) over three time points: 2, 4, and 6 h post-treatment (Fig. 3). A significant decrease in cell viability was found only
<figure><img src="image_10.png" /><figcaption>**Fig. 3.** Cytotoxicity of GH12 and lysosome staining in DP-MSCs following short-term GH12 treatment (2,4, and 6 h) in a 2D model. (A) Percentages of living cells after GH12 treatment over time (N = 3). (B) Microscopy images after 4 h of GH12 treatment showing cytotoxicity assessed with the Live/Dead assay. Live cells are marked in green and dead cells in red. Scale bar of 500 µm. (C) Lysosome staining of DP-MSCs after GH12 treatment over time (N = 3). (D) Microscopy images of the lysosomes staining in cells after 4 h of GH12 treatment. Scale bar of 100 µm. Statistical significance: ns, not significant, *, p < 0.05.</figcaption></figure>

using the highest concentration of 50 µg/mL GH12 in all the three time points (p < 0.05 for all), which indicated a rapid cellular response. At 6 h post-treatment, the mean percentage of living cells decreased from 99.17 % (± 0.33 %) in the control, to 86.70 % (± 7.7 %) at 50 µg/mL of GH12 (Fig. 3A; p < 0.05). In addition, lysosomal staining was performed to assess whether GH12 induces a cellular stress response potentially linked to cytotoxic effects. A significant increase in mean lysosome staining was found; 22.21 % (± 9.11 %) at 2 h, 35.82 % (± 11.42 %) at 4 h, and 24.25 % (± 5.95 %) at 6 h with 50 µg/mL of GH12 (Fig. 3C; p < 0.05 for all). Although no significant decrease in cell viability was observed with 25 µg/mL of GH12, the lysosome staining was significantly increased with this concentration at 4 h (22.76 % ± 7.78%; p < 0.05) and at 6 h (21.40 % ± 10.84%; p < 0.05).

The staining intensity was particularly high in small and round cells, which are likely to be dying cells (Fig. 3D). No significant differences were observed between the different exposure times, suggesting that the effect of GH12 does not vary over time, except in the case of lysosomes, where a significant difference was detected at 25 µg/mL after 4 h.

### 3.2.2. Cytotoxicity of GH12 in fibrin-based models

Following the initial results in the 2D culture, a more advanced fibrin hydrogel model was used (Fig. 4). No significant difference was found in cell viability between high concentrations of GH12 (50 µg/mL) and the negative control group without GH12 (Fig. 4A and 4B). This finding suggests that, in the fibrin-based model, GH12 was less cytotoxic.

## 4. Discussion

The present results suggest that GH12 acts through a trimeric or tetrameric pore-forming mechanism at the membrane. In the 2D culture, GH12 impaired DP-MSCs viability at 50 µg/mL and induced increased lysosomal staining at 25 and 50 µg/mL. In the fibrin-based model, GH12 was not cytotoxic, even at 50 µg/mL.

Insight into the oligomerization structure and stability of GH12 was obtained through *in silico* analysis, and the liposome leakage assay allowed to report GH12's ability to interact with lipid bilayers and induce membrane permeabilization, a key mechanism in antibacterial activity. Of note, this pore-forming mechanism at the membrane is comparable to other structurally similar AMPs [26–31]. While the

<figure><img src="image_11.png" /><figcaption>**Fig. 4.** Assessment of cytotoxicity of GH12 in a fibrin-based model after 48 h. (A) Percentage of living DP-MSCs following GH12 treatment (N = 3). (B) Microscopy images showing DP-MSCs cytotoxicity after 48 h of GH12 treatment assessed with Live/Dead assay. Living cells are shown in green, and dead cells are shown in red. Scale bar: 500 µm. Statistical significance: ns, not significant, *, p < 0.05.</figcaption></figure>
liposome leakage assay does not confirm the specific oligomeric struc-
ture, previous studies suggest that some AMPs can form trimeric or
tetrameric pores at the membrane [26,28,31]. In addition, the present
findings are consistent with the pore-forming activity observed in a
previous study using electron microscopy, in which GH12 caused bac-
terial membrane disruption on *Streptococcus mutans*, *Lactobacillus aci-*
*dophilus*, and *Actinomyces viscosus* [15]. While the results suggesting a
pore-forming ability of this innovative peptide align with the litera-
ture [15], as for other antimicrobial peptides, the precise configuration
of the pore (e.g., barrel-stave, toroidal, or other [32]) requires further
investigations. Importantly, GH12 is expected to self-assemble into its
most stable conformation, making the trimeric and tetrameric pre-
dictions considered as indicative rather than conclusive, and further
experimental validation, such as NMR, or molecular dynamics simula-
tions [31,33–35], will be required to confirm the oligomeric state and
the mechanism of GH12 activity.

In the 2D model, GH12 had a rapid cytotoxic effect at 50 µg/mL. This concentration is lower than the 256 µg/mL reported as non-cytotoxic in human gingival fibroblasts after short exposure [15], highlighting a tissue-specific response [36], its potential use in endodontics would involve prolonged contact with pulp cells, a context with limited safety data. In addition, cytotoxicity was associated with elevated lysosome staining, suggesting a stress response that potentially leads to cell death at higher concentrations. Although no study has directly demonstrated an increase in lysosomal activity in pulpal cells with GH12, it has been established that AMPs can be stored within lysosomes that are key organelles involved in waste disposal and cellular degradation [37–39]. At the highest concentration, GH12 might have induced lysosomal membrane permeabilization (LMP), resulting in the release of lysosomal contents into the cytosol and potentially leading to cell death [40,41]. Regarding the absence of cytotoxicity along with lysosome staining at 25 µg/mL, this concentration may be below the cytotoxicity threshold or the cellular stress and might have triggered autophagy, a protective mechanism that removes damaged macromolecules and organelles, mitigating prolonged cytotoxicity [42]. The observed increase in lysosomal activity suggests that GH12 may be internalized and directed to the lysosomal compartment. This is consistent with findings on a structurally similar AMP, which localizes to lysosomes and targets endocytosed bacteria [43–45]. This localization may reflect either a cellular clearance pathway or indicate that GH12 acts as an intracellular AMP. Therefore, GH12 is of particular interest, as oral and endodontic bacteria can migrate into systemic circulation and potentially colonize distant organs [46]; this AMP may effectively target intracellular pathogens that evade from traditional antibiotic treatments [44,47]. Further studies using labelled GH12 are required to confirm these hypotheses.

The rapid cytotoxicity observed in the 2D model was expected, as GH12 was shown to permeabilize liposomes within just a few minutes. However, this toxicity was not observed in the fibrin model. These differences in cytotoxicity can likely be attributed to the distinct micro-environments related to 2D and fibrin-based models. Indeed, 2D culture on plastic is known to be stressful for cells and may increase their fragility whereas the 3D environment has been reported to be less sensitive to cytotoxic molecules [48,49]. Finally, interactions between the fibrin matrix and GH12 are likely to occur, as fibrin is known to bind and partially retain bioactive molecules [50], which may reduce the peptide's bioavailability within the hydrogel. Nevertheless, the peptide still appears able to diffuse from the scaffold, as suggested by the agar model. While its partial retention may also be beneficial by maintaining a local reservoir of GH12 at the site of application, further investigations will be required to quantify and optimize the balance between diffusion and retention under different conditions. Altogether, these results indicate that GH12 could have a good biocompatibility.

The present study has however limitations. The *in silico* results
should be interpreted with caution, as membrane interactions may also
influence the multimerization state, and the pTM and ipTM scores of
AlphaFold are relatively low ( < 0.5), reflecting moderate confidence in

the predicted assemblies [51,52]. However, Boltz predictions supported
the same trend towards trimeric and tetrameric states indicating that
these assemblies may represent plausible configurations. These scores
are not directly comparable between models and should be regarded
only as supportive indications, not as proof of structural accuracy. The
observed differences likely reflect the distinct optimization strategies of
each algorithm, with Boltz-2 being specifically designed to improve
predictions for small peptides [18]. Similarly, while a critical method-
ological consideration is the timing of cytotoxicity evaluation, the rapid
cytotoxicity aligns with the findings from the liposome leakage assay, in
which GH12 induced membrane permeabilization within minutes.
Interestingly, GH12 quenched 6-CF fluorescence, suggesting that
membrane disruption may be followed by peptide-dye interaction in
solution. This two-step effect warrants further investigation to under-
stand the underlying biophysical mechanism. Cytotoxicity assessment
was based on the live/dead assay which provides limited insight into the
mechanisms of cell death (apoptosis, necrosis, or autophagy-related
pathways) [53]. Complementary assays, including caspase activity or
markers of oxidative stress, could provide a more comprehensive un-
derstanding of GH12's effects on cellular integrity.

In addition, this study focused only on DP-MSCs, as they contribute to the production of the pulp extracellular matrix [25,54]. However, other cell types are essential for restoring pulp functionality, such as vascular and neuronal cells, and should be investigated [55,56]. Moreover, while this study focuses on the biocompatibility of GH12 in DP-MSCs, further studies are required to evaluate its potential effects on DP-MSCs migration, differentiation, and mineralization [57].

Similarly, fibrin was used here in a single formulation as a simplified
scaffold model. While it does not replicate all aspects of the clinical
setting, its tunable properties allow modulation of release profiles, as
previously demonstrated [58], which could support the controlled de-
livery of GH12 into the root canal system.

Regarding antibacterial activity, while GH12 demonstrated prom-
ising effects in the inhibition zone assay, these results should be inter-
preted with appropriate caution, as this method provides only a partial
view of clinical relevance. As noted by the Journal of Endodontics
editorial board [59], the agar diffusion technique is not fully standard-
ized for endodontic applications and may be influenced by variables
such as pH, media composition, and peptide diffusion properties. In our
study, this experiment was intended only as a proof of concept to
demonstrate that the synthesized peptide retained antibacterial activity
within a scaffold-like environment, rather than as a definitive validation
of efficacy. They do not constitute a deep evaluation of its antibacterial
potential that will require more clinically relevant models, such as
dentin slice infection assays or biofilm-in-tubule models. Additionally,
although *S. aureus* is the most frequent pathogen in device-related in-
fections [60] and *E. faecalis* among the most prevalent microorganisms
associated with endodontic treatment failure [61], future studies should
focus on additional oral pathogens to validate GH12's antimicrobial ef-
ficacy in the fibrin model, as it has already been demonstrated in 2D [62,
63].

Finally, a limitation of our fibrin-based 3D model is that interactions
between fibrin and GH12 may reduce the free concentration of the
peptide and thereby underestimate potential *in vivo* toxicity. Further *in
vivo* studies are therefore essential to assess not only this aspect but also
the potential local and systemic inflammation caused by GH12 before
considering clinical applications. However, concerns about adverse ef-
fects should be mitigated by the short half-life of AMPs [37,64,65],
which limits its persistence and potential inflammatory effects.

**5. Conclusion**

The present study offers new insight into the mechanism of GH12
and its biocompatibility with pulp cells, supporting its potential for
improving the outcome of REPs. In addition, these findings underscore
the importance of using more complex models to assess the cytotoxicity
of an innovative molecule, providing more accurate predictions on cells response in clinic.

## Funding statement

This work was supported by the French National Association of Research (ANR JCJC ENDONANOBIOTIC (ANR-21-CE19-0001) and ANR JCJC TRIDENTOMIC (ANR-21-CE44-0015) and the French National Centre for Scientific Research (CNRS). The PhD grant of LR was funded by the EDISS doctoral school of Lyon.

## CRediT authorship contribution statement

**Lisa Reiniche:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization.
**Marjorie Faure:** Writing – review & editing, Investigation.
**Marianne Leveque:** Writing – review & editing, Investigation.
**Cédric Orelle:** Writing – review & editing, Investigation.
**Audrey Ziverec:** Writing – review & editing, Software.
**Jean-Christophe Farges:** Writing – review & editing.
**François Virard:** Writing – review & editing, Investigation, Conceptualization.
**Jean-Daniel Malcor:** Writing – review & editing.
**Mourad Bekhouche:** Writing – review & editing, Supervision, Methodology.
**Maxime Ducret:** Writing – review & editing, Supervision, Methodology, Conceptualization.

## Declaration of 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.

## Acknowledgements

We would like to express our gratitude to Eric Diesis and the Protein Science Facility of SFR Biosciences (Université Claude Bernard Lyon 1, CNRS UAR3444, Inserm US8, ENS de Lyon) for their support in the synthesis of the GH12 peptide.

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jdent.2025.106105.

## References

[1] M. Leveque, M. Bekhouche, J.C. Farges, A. Aussel, K. Sy, R. Richert, M. Ducret, Bioactive endodontic hydrogels: from parameters to personalized medicine, Int. J. Mol. Sci. 24 (2023) 14056, https://doi.org/10.3390/ijms241814056.

[2] M. Ducret, H. Fabre, A. Celle, F. Mallein-Gerin, E. Perrier-Groult, B. Alliot-Licht, J. C. Farges, Current challenges in human tooth revitalization, Biomed. Mater. Eng. 28 (2017) S159-S168, https://doi.org/10.3233/BME-171637.

[3] D. Erazo, M. Brizuela, D.R. Whetstone, Dental infections. StatPearls, StatPearls Publishing, Treasure Island (FL), 2025. http://www.ncbi.nlm.nih.gov/books/NBK542165/ (accessed March 28, 2025).

[4] M. Kuzekanani, J.L. Gutmann, Latest concepts in the endodontic management of patients with cardiovascular disorders, Eur. Endod. J. 4 (2019) 86-89, https://doi.org/10.14744/eej.2019.70288.

[5] P.R. Cooper, Y. Takahashi, L.W. Graham, S. Simon, S. Imazato, A.J. Smith, Inflammation-regeneration interplay in the dentine-pulp complex, J. Dent. 38 (2010) 687-697, https://doi.org/10.1016/j.jdent.2010.05.016.

[6] F. Mangione, M. EzEldeen, C. Bardet, J. Lesieur, M. Bonneau, F. Decup, B. Salmon, R. Jacobs, C. Chaussain, S. Opsahl-Vital, Implanted dental pulp cells fail to induce regeneration in partial pulpotomies, J. Dent. Res. 96 (2017) 1406-1413, https://doi.org/10.1177/0022034517725523.

[7] X. Li, Y. Wang, X. Jiang, Y. Zeng, X. Zhao, J. Washio, N. Takahashi, L. Zhang, Investigation of drug resistance of caries-related streptococci to antimicrobial peptide GH12, Front. Cell. Infect. Microbiol. 12 (2022) 991938, https://doi.org/10.3389/fcimb.2022.991938.

[8] Antimicrobial resistance, n.d. https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance, 2024 (accessed July 10.)

[9] Y. Wang, X. Wang, W. Jiang, K. Wang, J. Luo, W. Li, X. Zhou, L. Zhang, Antimicrobial peptide GH12 suppresses cariogenic virulence factors of Streptococcus mutans, J. Oral Microbiol. 10 (2018) 1442089, https://doi.org/10.1080/20002297.2018.1442089.

[10] Y. Li, Y. Wang, X. Chen, W. Jiang, X. Jiang, Y. Zeng, X. Li, Z. Feng, J. Luo, L. Zhang, Antimicrobial peptide GH12 as root canal irrigant inhibits biofilm and virulence of Enterococcus faecalis, Int. Endod. J. 53 (2020) 948-961, https://doi.org/10.1111/iej.13296.

[11] A. Sinha, S. Taneja, D.C. Shetty, V.K. Bhalla, Efficacy of antimicrobial peptide GH12 on a multispecies endodontic biofilm model: an In-vitro study, Eur. Endod. J. 9 (2024) 411-417, https://doi.org/10.14744/eej.2024.75983.

[12] P. Verma, A. Nosrat, J.R. Kim, J.B. Price, P. Wang, E. Bair, H.H. Xu, A.F. Fouad, Effect of residual bacteria on the outcome of pulp regeneration *In vivo*, J. Dent. Res. 96 (2017) 100-106, https://doi.org/10.1177/0022034516671499.

[13] S.G. Kim, Infection and pulp regeneration, Dent. J. 4 (2016) 4, https://doi.org/10.3390/dj4010004.

[14] K.M. Gathani, S.S. Raghavendra, Scaffolds in regenerative endodontics: a review, Dent. Res. J. 13 (2016) 379-386, https://doi.org/10.4103/1735-3327.192266.

[15] Y. Wang, Y. Fan, Z. Zhou, H. Tu, Q. Ren, X. Wang, L. Ding, X. Zhou, L. Zhang, De novo synthetic short antimicrobial peptides against cariogenic bacteria, Arch. Oral Biol. 80 (2017) 41-50, https://doi.org/10.1016/j. anchorbio.2017.03.017.

[16] J. Abramson, J. Adler, J. Dunger, R. Evans, T. Green, A. Pritzel, O. Ronneberger, L. Willmore, A.J. Ballard, J. Bambrick, S.W. Bodenstein, D.A. Evans, C.C. Hung, M. O'Neill, D. Reiman, K. Tunyasunakool, Z. Wu, A. Zemgulyte, E. Arvaniti, C. Beattie, O. Bertolli, A. Bridgland, A. Cherepanov, M. Congreve, A.I. Cowen-Rivers, A. Cowie, M. Figurnov, F.B. Fuchs, H. Gladman, R. Jain, Y.A. Khan, C.M. R. Low, K. Perlin, A. Potapenko, P. Savy, S. Singh, A. Stecula, A. Thillaisundaram, C. Tong, S. Yakneen, E.D. Zhong, M. Zielinski, A. Zídek, V. Bapst, P. Kohli, M. Jaderberg, D. Hassabis, J.M. Jumper, Accurate structure prediction of biomolecular interactions with AlphaFold 3, Nature 630 (2024) 493-500, https://doi.org/10.1038/s41586-024-07487-w.

[17] Boltz - Tamarind Online Tool, n.d. https://app.tamarind.bio/boltz, 2025 (accessed June 23

[18] Boltz-2: Towards accurate and efficient binding prediction - MIT Jameel Clinic, n. d. https://j clinic.mit.edu/research-project/boltz-2-towards-accurate-and-efficient-binding-prediction/, 2025 (accessed June 23

[19] PyMOL | pymol.org, n.d. https://www.pymol.org/, 2024 (accessed August 8

[20] J.N. Weinstein, R. Blumenthal, R.D. Klausner, Carboxyfluorescein leakage assay for lipoprotein-liposome interaction, Methods Enzymol. 128 (1986) 657-668, https://doi.org/10.1016/0076-6879(86)28098-2.

[21] M. Ducret, H. Fabre, O. Degoul, G. Atzeni, C. McGuckin, N. Forraz, B. Alliot-Licht, F. Mallein-Gerin, E. Perrier-Groult, J.C. Farges, Manufacturing of dental pulp cell-based products from human third molars: current strategies and future investigations, Front. Physiol. 6 (2015) 213, https://doi.org/10.3389/fphys.2015.00213.

[22] H. Fabre, M. Ducret, O. Degoul, J. Rodriguez, E. Perrier-Groult, E. Aubert-Foucher, M. Pasdeloup, C. Auxenfans, C. McGuckin, N. Forraz, F. Mallein-Gerin, Characterization of different sources of Human MSCs expanded in serum-free conditions with quantification of chondrogenic induction in 3D, Stem Cells Int 2019 (2019) 2186728, https://doi.org/10.1155/2019/2186728.

[23] M. Ducret, H. Fabre, J.C. Farges, O. Degoul, G. Atzeni, C. McGuckin, N. Forraz, F. Mallein-Gerin, E. Perrier-Groult, Production of Human dental pulp cells with a medicinal manufacturing approach, J. Endod. 41 (2015) 1492-1499, https://doi.org/10.1016/j.joen.2015.05.017.

[24] M. Dominici, K.L. Blanc, I. Mueller, I. Slaper-Cortenbach, F.C. Marini, D.S. Krause, R.J. Deans, A. Keating, D.J. Prockop, E.M. Horwitz, Minimal criteria for defining multipotent mesenchymal stromal cells. The International society for cellular therapy position statement, Cytotherapy 8 (2006) 315-317, https://doi.org/10.1080/14653240600855905.

[25] M. Ducret, A. Montembault, J. Josse, M. Pasdeloup, A. Celle, R. Benchrih, F. Mallein-Gerin, B. Alliot-Licht, L. David, J.C. Farges, Design and characterization of a chitosan-enriched fibrin hydrogel for human dental pulp regeneration, Dent. Mater. Off. Publ. Acad. Dent. Mater. 35 (2019) 523-533, https://doi.org/10.1016/j.dental.2019.01.018.

[26] D. Xhindoli, S. Pacor, M. Benincasa, M. Scocchi, R. Gennaro, A. Tossi, The human cathelicidin LL-37 — A pore-forming antibacterial peptide and host-cell modulator, Biochim. Biophys. Acta BBA - Biomembr. 1858 (2016) 546-566, https://doi.org/10.1016/j.bbamem.2015.11.003.

[27] E. Sancho-Vaello, D. Gil-Carton, P. François, E.J. Bonetti, M. Kreir, K.R. Pothula, U. Kleinekathófer, K. Zeth, The structure of the antimicrobial human cathelicidin LL-37 shows oligomerization and channel formation in the presence of membrane mimics, Sci. Rep. 10 (2020) 17356, https://doi.org/10.1038/s41598-020-74401-5.

[28] C.C. Lee, Y. Sun, S. Qian, H.W. Huang, Transmembrane pores formed by human antimicrobial peptide LL-37, Biophys. J. 100 (2011) 1688-1696, https://doi.org/10.1016/j.bpj.2011.02.018.

[29] Y. Engelberg, M. Landau, The Human LL-37(17-29) antimicrobial peptide reveals a functional supramolecular structure, Nat. Commun. 11 (2020) 3894, https://doi.org/10.1038/s41467-020-17736-x.

[30] M. Shahmiri, M. Enciso, C.G. Adda, B.J. Smith, M.A. Perugini, A. Mechler, Membrane core-specific antimicrobial action of cathelicidin LL-37 peptide switches between pore and nanofibre formation, Sci. Rep. 6 (2016) 38184, https://doi.org/10.1038/srep38184.

[31] A. Pino-Angelos, J.M. Leveritt, T. Lazaridis, Pore structure and synergy in antimicrobial peptides of the Magainin Family, PLoS Comput. Biol. 12 (2016) e1004570, https://doi.org/10.1371/journal.pcbi.1004570.

[32] R. Lipkin, T. Lazaridis, Computational studies of peptide-induced membrane pore formation, Philos. Trans. R. Soc. B Biol. Sci. 372 (2017) 20160219, https://doi.org/10.1098/rstb.2016.0219.
[33] W. Hoppe, B. Grill, Prospects of three-dimensional high resolution electron microscopy of non-periodic structures, Ultramicroscopy 2 (1977) 153-168, https://doi.org/10.1016/s0304-3991(76)90926-8.

[34] C.F. Reboul, K. Mahmood, J.C. Whisstock, M.A. Dunstone, Predicting giant transmembrane β-barrel architecture, Bioinforma. Oxf. Engl. 28 (2012) 1299-1302, https://doi.org/10.1093/bioinformatics/bts152.

[35] M.S. Sansom, I.D. Kerr, Transbilayer pores formed by beta-barrels: molecular modeling of pore structures and properties, Biophys. J. 69 (1995) 1334-1343, https://doi.org/10.1016/S0006-3495(95)80000-7.

[36] W. Jiang, Y. Wang, J. Luo, X. Chen, Y. Zeng, X. Li, Z. Feng, L. Zhang, Antimicrobial peptide GH12 prevents dental caries by regulating dental plaque microbiota, Appl. Environ. Microbiol. 86 (2020), https://doi.org/10.1128/AEM.00527-20 e00527-20.

[37] A.A. Bahar, D. Ren, Antimicrobial peptides, Pharmaceuticals 6 (2013) 1543-1575, https://doi.org/10.3390/ph6121543.

[38] H.I. Zeya, J.K. Spitznagel, Antibacterial and enzymic basic proteins from leukocyte lysosomes: separation and identification, Science 142 (1963) 1085-1087, https://doi.org/10.1126/science.142.3595.1085.

[39] C. Yang, X. Wang, Lysosome biogenesis: regulation and functions, J. Cell Biol. 220 (2021), https://doi.org/10.1083/jcb.202102001 e202102001.

[40] F. Wang, R. Gómez-Sintes, P. Boya, Lysosomal membrane permeabilization and cell death, Traffic Cph. Den. 19 (2018) 918-931, https://doi.org/10.1111/tra.12613.

[41] A. Serrano-Puebla, P. Boya, Lysosomal membrane permeabilization in cell death: new evidence and implications for health and disease, Ann. N. Y. Acad. Sci. 1371 (2016) 30-44, https://doi.org/10.1111/nyas.12966.

[42] X. Zhang, X. Cheng, L. Yu, J. Yang, R. Calvo, S. Patnaik, X. Hu, Q. Gao, M. Yang, M. Lawas, M. Delling, J. Marugan, M. Ferrer, H. Xu, MCOLN1 is a ROS sensor in lysosomes that regulates autophagy, Nat. Commun. 7 (2016) 12109, https://doi.org/10.1038/ncomms12109.

[43] D.F. Buccini, M.H. Cardoso, O.L. Franco, Antimicrobial peptides and cell-penetrating peptides for treating intracellular bacterial infections, Front. Cell. Infect. Microbiol. 10 (2021), https://doi.org/10.3389/fcimb.2020.612931.

[44] X. Kong, V. Vishwanath, P. Neelakantan, Z. Ye, Harnessing antimicrobial peptides in endodontics, Int. Endod. J. 57 (2024) 815-840, https://doi.org/10.1111/iej.14043.

[45] X. Tang, D. Basavarajappa, J.Z. Haeggström, M. Wan, P2×7 Receptor regulates internalization of antimicrobial peptide LL-37 by Human macrophages that promotes intracellular pathogen clearance, J. Immunol. Author Choice 195 (2015) 1191, https://doi.org/10.4049/jimmunol.1402845.

[46] M.J. Bordagaray, A. Fernández, M. Garrido, J. Astorga, A. Hoare, M. Hernández, Systemic and extraradicular bacterial translocation in apical periodontitis, Front. Cell. Infect. Microbiol. 11 (2021) 649925, https://doi.org/10.3389/fcimb.2021.649925.

[47] Y. Chen, X. He, Q. Chen, Y. He, F. Chen, C. Yang, L. Wang, Nanomaterials against intracellular bacterial infection: from drug delivery to intrinsic biofunction, Front. Bioeng. Biotechnol. 11 (2023), https://doi.org/10.3389/fbioe.2023.1197974.

[48] S.M. Leano, W. De Souza, R. De Vecchi, A. Lopes, T. Deliberador, J.M. Granjeiro, A multimodal in vitro approach to assess the safety of oral care products using 2D and 3D cellular models, Front. Toxicol. 6 (2024) 1474583, https://doi.org/10.3389/ftox.2024.1474583.

[49] J. Brinkmann, H. Malyaran, M.A.A. Enezy-Ulbrich, S. Jung, C. Radermacher, E. M. Buhl, A. Pich, S. Neuss, Assessment of fibrin-based hydrogels containing a fibrin-binding peptide to tune mechanical properties and cell responses, Macromol. Mater. Eng. 308 (2023) 2200678, https://doi.org/10.1002/mame.202200678.

[50] M.M. Martino, P.S. Briquez, A. Ranga, M.P. Lutolf, J.A. Hubbell, Heparin-binding domain of fibrin(ogen) binds growth factors and promotes tissue repair when

incorporated within a synthetic matrix, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 4563-4568, https://doi.org/10.1073/pnas.1221602110.

[51] J. Xu, Y. Zhang, How significant is a protein structure similarity with TM-score = 0.5? Bioinformatics 26 (2010) 889-895, https://doi.org/10.1093/bioinformatics/btq066.

[52] EMBL-EBI, Confidence scores in AlphaFold-multimer | AlphaFold, n.d. https://www.ebi.ac.uk/training/online/courses/alphafold/inputs-and-outputs/evaluating-alphafolds-predicted-structures-using-confidence-scores/confidence-scores-in-alphafold-multimer/, 2024 (accessed December 16

[53] M.S. D'Arcy, Cell death: a review of the major forms of apoptosis, necrosis and autophagy, Cell Biol. Int. 43 (2019) 582-592, https://doi.org/10.1002/cbin.11137.

[54] M. Bekhouche, M. Bolon, F. Charriaud, M. Lamrayah, D.Da Costa, C. Primard, A. Costantini, M. Pasdeloup, S. Gobert, F. Mallein-Gerin, B. Verrier, M. Ducret, J. C. Farges, Development of an antibacterial nanocomposite hydrogel for human dental pulp engineering, J. Mater. Chem. B 8 (2020) 8422-8432, https://doi.org/10.1039/d0tb00989j.

[55] M. Ducret, A. Costantini, S. Gobert, J.C. Farges, M. Bekhouche, Fibrin-based scaffolds for dental pulp regeneration: from biology to nanotherapeutics, Eur. Cell. Mater. 41 (2021) 1-14, https://doi.org/10.22203/eCM.v041a01.

[56] A. Tenyi, L. Nemeth, A. Golez, K. Cankar, A. Milutinović, Comparison of the vitality tests used in the dental clinical practice and histological analysis of the dental pulp, Bosn. J. Basic Med. Sci. 22 (2022) 374-381, https://doi.org/10.17305/bjbms.2021.6841.

[57] R. Osorio, F.J. Rodríguez-Lozano, M. Toledano, M. Toledano-Osorio, D. García-Bernal, L. Murcia, S. López-García, Mitigating lipopolysaccharide-induced impairment in human dental pulp stem cells with tideglusib-doped nanoparticles: enhancing osteogenic differentiation and mineralization, Dent. Mater. 40 (2024) 1591-1601, https://doi.org/10.1016/j.dental.2024.07.012.

[58] M. Leveque, M. Bekhouche, J.C. Farges, R. Richert, M. Ducret, Investigation of the early apical release from endodontic hydrogels: a 3D printed model, Int. Endod. J. 57 (2024) 943-950, https://doi.org/10.1111/iej.14049.

[59] Wanted: a base of evidence, J. Endod. 33 (2007) 1401-1402, https://doi.org/10.1016/j.joen.2007.09.004.

[60] M. Otto, Staphylococcal biofilms, Microbiol. Spectr. 6 (2018) 10.1128/microbiolspec.gpp3-0023-2018. https://doi.org/10.1128/microbiolspec.gpp3-0023-2018.

[61] S. Yang, X. Meng, Y. Zhen, Q. Baima, Y. Wang, X. Jiang, Z. Xu, Strategies and mechanisms targeting Enterococcus faecalis biofilms associated with endodontic infections: a comprehensive review, Front. Cell. Infect. Microbiol. 14 (2024) 1433313, https://doi.org/10.3389/fcimb.2024.1433313.

[62] H. Tu, Y. Fan, X. Lv, S. Han, X. Zhou, L. Zhang, Activity of synthetic antimicrobial peptide GH12 against oral streptococci, Caries Res. 50 (2016) 48-61, https://doi.org/10.1159/000442898.

[63] W. Jiang, Y. Wang, J. Luo, X. Chen, Y. Zeng, X. Li, Z. Feng, L. Zhang, Antimicrobial peptide GH12 prevents dental caries by regulating dental plaque microbiota, Appl. Environ. Microbiol. 86 (2020), https://doi.org/10.1128/AEM.00527-20 e00527-20.

[64] S.C. Penchala, M.R. Miller, A. Pal, J. Dong, N.R. Madadi, J. Xie, H. Joo, J. Tsai, P. Batoon, V. Samoshin, A. Franz, T. Cox, J. Miles, W.K. Chan, M.S. Park, M. M. Alhamadsheh, A biomimetic approach for enhancing the *in vivo* half-life of peptides, Nat. Chem. Biol. 11 (2015) 793-798, https://doi.org/10.1038/nchembio.1907.

[65] C. Shao, Y. Zhu, Z. Lai, P. Tan, A. Shan, Antimicrobial peptides with protease stability: progress and perspective, Future Med. Chem. 11 (2019) 2047-2050, https://doi.org/10.4155/fmc.2019-0167.