# Antimicrobial GH12-fibrin hydrogel for dental pulp regeneration: An in vitro study

Marianne Leveque, Lisa Reiniche, Marjorie Faure, Cédric Orelle, Jean-Christophe Farges, Raphaël Richert, Jean-Daniel Malcor, Edwin-Joffrey Courtial, Jérôme Sohier, Éric Diesis, et al.

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Marianne Leveque, Lisa Reiniche, Marjorie Faure, Cédric Orelle, Jean-Christophe Farges, et al. "Antimicrobial GH12-fibrin hydrogel for dental pulp regeneration: An in vitro study." Dental Materials, In press, 10.1016/j.dental.2025.08.010. hal-05343288

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# Antimicrobial GH12-fibrin hydrogel for dental pulp regeneration: An *in vitro* study

Marianne Leveque<sup>a</sup>, Lisa Reiniche<sup>a</sup>, Marjorie Faure<sup>a,b,c</sup>, Cédric Orelle<sup>d,e</sup>,
Jean-Christophe Farges<sup>a,b,c</sup>, Raphaël Richert<sup>b,c,e</sup>, Jean-Daniel Malcor<sup>a,d</sup>,
Edwin-Joffrey Courtial<sup>f</sup>, Jérôme Sohier<sup>a,d</sup>, Éric Diesis<sup>g</sup>, Mourad Bekhouche<sup>a,d</sup>,
Maxime Ducret<sup>a,b,c,*</sup><sub>d</sub>

## ARTICLE INFO

**Keywords:**

Biomaterial

Antimicrobial peptides

Dental pulp

Dentistry

Regeneration

Antibacterial

Hydrogel

## ABSTRACT

**Aim:** Fibrin hydrogels hold great promise as drug delivery systems in tissue engineering, due to their biocompatibility, degradability, and injectability. However, their inability to prevent bacterial growth limits their usefulness in infection-prone environments such as the dental root canal. GH12 was described as a potent antimicrobial peptide for oral applications. In the present study, we aimed to evaluate the preclinical relevance of an innovative fibrin hydrogel incorporating antimicrobial peptide GH12 to prevent bacterial contamination of the material during the regeneration process.

**Methodology:** Hydrogel's mechanical properties were characterised by rheology and Scanning Electron Microscopy (SEM). GH12 antibacterial effectiveness against *Enterococcus faecalis*, *Porphyromonas gingivalis*, and *Streptococcus gordonii* was determined using Minimum Inhibitory Concentration (MIC) assays as well as inhibition circle assays. Cytocompatibility was assessed through a Live and Dead assay using Dental Pulp Mesenchymal Stem Cells (DP-MSCs) embedded in GH12-fibrin hydrogels.

**Results:** SEM showed no significant difference in fibre and pore average sizes. MICs were 75 µg/mL for *E. faecalis*, 4.25 µg/mL for *P. gingivalis* and 28.15 µg/mL for *S. gordonii*. GH12 effectively inhibited bacterial growth in both liquid and solid media. DP-MSCs embedded in GH12-fibrin hydrogels (GH12 150 µg/mL) showed a 90% survival rate at 48 h, compared to fibrin-alone hydrogels.

**Conclusions:** These results suggest that GH12-fibrin hydrogels might control endodontic infection, as it shows significant antibacterial activity without compromising the hydrogel's structure or cytocompatibility with DP-MSCs. Further studies *in vitro* and *in vivo*, are required to optimise GH12 release from the fibrin hydrogel and confirm its safety and effectiveness.

## 1. Introduction

Regenerative Endodontic Procedures (REPs) aim to recreate a functional dental pulp tissue, allowing the tooth to regain its functionality [1]. To achieve full regeneration, recent studies suggest that a delicate

balance between bacterial load, associated inflammation, cell migration, and extracellular matrix remodelling must be reached [2,3]. The presence of bacteria in the root canal has been linked to a significant increase in failure to regenerate the dental pulp tissue in *in vivo* models [4,5]. Moreover, excessive inflammation due to residual matter from

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

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

<footnote><sup>d</sup> Microbiologie Moléculaire et Biochimie Structurale (MMSB), UMR 5086 CNRS/Université de Lyon, Lyon, France</footnote>

<footnote><sup>e</sup> LaMCoS, CNRS UMR 5259, INSA Lyon, Villeurbanne 69621, France</footnote>

<footnote><sup>f</sup> 3d.FAB, Université Lyon1, CNRS, INSA, CPE-Lyon, ICBMS, UMR 5246, Villeurbanne, France</footnote>

<footnote><sup>g</sup> Université de Lyon, INSERM, ENS Lyon, CNRS, Protein Science Facility, SFR BioSciences, UAR3444/US8, Lyon 69366, France</footnote>

<footnote>* Corresponding author at: 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>E-mail address: maxime.ducret@chu-lyon.fr (M. Ducret).</footnote>

<footnote>https://doi.org/10.1016/j.dental.2025.08.010</footnote>

<footnote>Received 13 May 2025; Received in revised form 17 July 2025; Accepted 15 August 2025</footnote>

<footnote>0109-5641/© 2025 The Author(s). Published by Elsevier Inc. on behalf of The Academy of Dental Materials. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</footnote>
pathogenic microorganisms, such as molecules from bacterial mem-
branes and/or cytoplasm, that are recognised by the immune system
(Pathogen Associated Molecule Pattern, PAMPs), and various associated
antigens has been shown to strongly impact cell differentiation, miner-
alisation, and scaffold modelling through direct and indirect mecha-
nisms [6–8].

Various antibacterial strategies have been investigated to reduce
bacterial load and the associated inflammation in dental root canals
[9–11]. These strategies are to be placed in a single appointment, after
pulp extraction and root canal irrigation. They are built on two main
parameters: the active molecule(s), and the galenic strategy used
[12–14]. The galenic strategy aims to allow the release of the molecule
in the environment in a controlled manner, but also to act as a cellular
scaffold. As such, the selected biomaterial has to remain stable long
enough for cells to colonise it, differentiate, produce their own envi-
ronment etc. Fibrin is a very interesting candidate for this, and is one of
the most studied scaffolds for dental pulp regeneration, due to its cell
compatibility, biodegradability and complete safety for patient use, but
has no intrinsic antibacterial activity [15,16]. The addition of an anti-
microbial agent to endodontic hydrogels is then necessary to provide
antimicrobial protection to the scaffold [11,17]. Various strategies such
as propolis, light-reactive components or peptides have been explored
for this purpose [18–20] but are also associated with challenges such as
the reliability of the component’s quality, cost and quantities delivered,
as well as long-term stability [11,12].

Antimicrobial Peptides (AMPs) were proposed during the last decade for various regenerative applications such as bone or wound healing [21, 22]. One of the most widely used antimicrobial peptides is the human cathelicidin LL37 and its derivatives, mostly because of their cyto-compatibility and large number of biological activities [23,24]. Alpha helicoidal peptides such as LL-37 and its derivatives are thought to be active on the bacterial membrane, by forming pores and/or dissolving the phospholipids of the membrane, thus leading to a leak of cellular contents and eventually to cellular death [25,26]. They are few mech- anisms that could allow bacteria to resist this mechanism, and thus, few to no resistance to antimicrobial peptides are observed [26,27]. More- over, AMPs were reported to have lower toxicity on cells than antibiotics currently used for endodontic disinfection (ciprofloxacin, minocycline, metronidazole, etc) [21,25,28]. However, most of these peptides are quickly degraded in bodily fluids (blood, serum, etc.), which limits po- tential clinical applications and explains the recent search for de- rivatives [29]. Several AMPs have been recently described and proposed for various applications in dentistry, including GH12, a 12 amino acid derivative of LL37, that has shown promising efficacy on endodontic, cariogenic and oral bacteria such as *Streptococcus gordonii*, *Streptococcus mutans* and *Enterococcus faecalis* [30–32]. GH12 has been previously investigated as a free-form for caries [33–37], as a root canal irrigant [31,38] and incorporated in hydrogels for periodontal use [33,37]. However, this candidate has shown moderate stability, and authors in previous studies suggested designing a galenic form for GH12 to enhance stability [30,31,33,39]. GH12 has yet to be considered as a protective antimicrobial agent, included in a dental pulp regeneration scaffold. This study hypothesises that GH12 may prevent bacterial contamination of the material during the dental pulp regeneration process when included in a fibrin hydrogel.

It aimed to evaluate the preclinical relevance of an innovative fibrin
hydrogel incorporating antimicrobial peptide GH12 for REPs. Firstly,
the rheological and physicochemical properties of the hydrogel were
characterised. Then, the effectiveness of the peptide on three aerobic
gram-positive and one anaerobic gram-negative bacteria was studied.
GH12 effectiveness was assessed in aqueous and solid media, upon
release from a fibrin hydrogel cast in a recently developed Endodontic
Release Insert (ERI) [13]. Finally, the cytocompatibility of this hydrogel
was estimated using human dental pulp mesenchymal stem/stromal
cells (DP-MSCs) embedded in the hydrogel.

**2. Material and method**

2.1. GH12 synthesis

The 12-amino acids peptide GH12 (GLLWHLLHHLLH) was syn-
thesised as described by Wang et al. [40]. A fluorescent derivative of
GH12 was also produced, onto which, the fluorophore 5(6)-Carboxy-
Fluorescein (5(6)-CF) was grafted in the N-terminal part. Both pep-
tides were synthesised using the Fmoc strategy by the Protein Science
Facility (PSF) platform (SFR Bioscience, Lyon, France), using Tri-
Fluoroacetic Acid (TFA) as the counter ion.

GH12 peptides were produced with a reported purity of at least 95 %,
and further purified using High-Performance Liquid Chromatography
(HPLC). Purification spectres are in supplementary data 1.

Tertiary conformation prediction was performed using the PepFold
4.2 server using the following GH12 sequence: GLLWHLLHHLLH.

2.2. GH12 incorporation into the fibrin hydrogel

Fibrin hydrogels were synthesised as previously described [41]. Human fibrinogen (ref. F3879) and thrombin (ref. T6884; Sigma-Aldrich, Saint-Louis, MO, USA) were used. Fibrin–GH12 formulations were prepared at Room Temperature (RT) by mixing solutions of 50 mg/mL fibrinogen, 3 M sodium chloride, 0.4 M calcium chloride, and 4 U/mL thrombin with GH12 diluted in PBS at various concentrations, in appropriate proportions to obtain final fibrin concentrations into the hydrogel ranging from 1.5 mg/mL to 6 mg/mL. These concentrations were derived from previous work as optimal for early remodelling and colonization by cells [41–44]. Final GH12 concentration ranged from 37.5 to 150 µg/mL, which are the minimal bactericidal concentrations extracted from previous literature [30,31,33,34–38]. Hydrogels were left at RT to polymerise for 15 min.

2.3. *GH12 release assessment*

GH12 release from the hydrogel was assessed as previously described
in Leveque et al. [13], using the 5(6)-CF labelled GH12. Briefly, End-
odontic Release Inserts (ERIs) were 3D printed using a Formlabs 3 3D
printer (Formlabs Sommerville, MA, USA) in a photosensitive auto-
clavable resin. Hydrogels were then moulded in ERIs. The ERI-hydrogel
device was placed on top of a bottom solution (PBS) in a 96 well plate.
The 100 % fluorescence was defined as the maximal fluorescence that
could be released from the hydrogel; its value was established based on
the fluorescence obtained from the maximal amount of fluorescent
peptide (15 µL at 150 µg/mL), suspended in wells without hydrogel.
Fluorescence release from the hydrogels was measured by excitation at
492 nm and emission at 515 nm at 1, 3, 5, and 24 h (early release), using
a Tecan Spark ® wellplate reader (Tecan, Männedorf, Switzerland), and
then every 24 h. The percentage of release was the ratio between fluo-
rescence measured in the medium and the 100 % fluorescence. The
negative controls were 110 µL PBS. Release assessment was carried out 3
times (N = 3).

2.4. Hydrogel viscoelastic properties

A stress-controlled rheometer (DHR2, TA Instruments, New Castle, DE, USA) equipped with a 25-mm plate geometry was used to characterise the changes in hydrogels viscoelastic properties at 21°C. A time oscillation procedure was applied to the hydrogels at 0.5 % shear strain at 1 Hz during 1200 s after 300 s at rest. The linear viscoelastic region was validated before time tests. Time oscillation procedure was carried out 3 times (N = 3).

**2.5. Hydrogel ultrastructure**

Discs of fibrin were prepared using 30 µL of hydrogel formulation,
left to polymerise for 15 min at RT and frozen at -70°C. They were then lyophilised at -52°C under 10 mbar pressure for 6 h. Samples were placed on their edges and image acquisition was carried out using a scanning electron microscope (SEM; TM4000, Hitachi, Tokyo, Japan) at 10 kV in backscattered electron (BSE) mode at 500-2500 × magnification.

Three images of each condition were analysed using the Fiji software. Each image was scaled, and pores as well as fibres were measured 10 times each, manually, until 60-70 data points were gathered on each image for fibre and pore size. Data were gathered, averaged, and treated with the Excel software (2018, 16.0). SEM analysis was carried out 3 times (N = 3).

## 2.6. Growth inhibition in liquid

Bacteria were cultured in a closed sterile 15 mL Falcon tube (Corning, NY, USA), at 37°C, 10% CO2, until they reached the stationary growth phase. Each strain was individually diluted to obtain an optical density at 600 nm (OD600nm) of 0.08. Then, 110 µL of the diluted solution were placed in each well of a 96-well plate (Greiner, Kremsmünster, Austria). The molecule of interest was weighted and diluted in PBS at 600 µg/mL, added to the plate, and serially diluted 12 times. Optical density was read at 5 and 24 h using a Tecan Spark® well plate reader (OD600nm; Tecan, Lyon, France). The Minimum Inhibitory Concentration (MIC), minimum concentration for which no bacterial growth is observed, and Minimum Bactericidal Concentration (MBC), minimum concentration for which no bacterial survival is observed, were determined. All results were then confirmed by seeding and counting the Colony Forming Units (CFUs) after 24 h in a 10% CO2 incubator at 37°C. A volume of 10 µL from of each well was placed on a 9 cm circular plate either coated with BHI-agar 2.5% (W/V) for *Streptococcus gordonii* (ATCC 35105), and *Enterococcus faecalis* (ATCC 19433), or coated with BHI-agar 2.5% (W/V) containing 10% (V/V) defibrinated sheep blood for *Porphyromonas gingivalis* (ATCC 33277). CFUs were counted manually using a Galaxy 330 Colony counter (Rocker, New Taipei City, Taiwan). All MIC assays were carried out 4 times (N = 4).

## 2.7. Inhibition circle

Bacteria were cultured in a closed sterile 15 mL Falcon tube, at 37°C, 10% CO2, until they reached the stationary growth phase. Each strain was diluted individually to obtain an OD600nm of 0.6. Then, 50 µL of each broth was seeded on a 9-cm plate BHI-agar 2.5% (W/V) for *S. gordonii*, and *E. faecalis*; or BHI-agar 2.5% (W/V), 10% (V/V) defibrinated sheep blood for *P. gingivalis*. Immediately after, 30 µL of a pre-polymerised fibrin hydrogel was placed on the plate.

The fibrin hydrogel was made as previously described. The molecule of interest was either clindamycin (final concentration 75 µg/mL) or GH12 (final concentrations 75 or 150 µg/mL). Plates were then cultured 24 h at 37°C, 10% CO2. Pictures were then taken on a standard setup, and the inhibition circles were measured using the Fiji software. All inhibition circle assays were carried out 4 times (N = 4).

## 2.8. Apical release from ERI and effectiveness

Hydrogels were made as previously described, and 30 µL were cast in Endodontic Release Inserts (ERIs) as previously described in Leveque et al. [13]. Each of the three endodontic bacterial strains (*S. gordonii*, *P. gingivalis*, and *E. faecalis*) were diluted to obtain OD600nm of 0.08 and then, 110 µL were placed in each well of the 96-well plate. Hydrogel filled ERIs were placed on the well. After 5 and 24 h of culture at 37°C, 10% CO2, 10 µL of the bottom solutions were sampled.

Samples were then diluted to 1/50, 1/500 and 1/5000, and 10 µL of each dilution was seeded on a 9-cm plate BHI-agar 2.5% (W/V; for *S. gordonii*, and *E. faecalis*) or a BHI-agar 2.5% containing 10% defibrinated sheep blood (for *P. gingivalis*). After 24 h, the CFUs were

counted. All apical release disinfection assays were carried out 4 times (N = 4).

## 2.9. Cell culture

Isolation and expansion of dental pulp mesenchymal stem cells (DP-MSCs) were performed as previously described [43]. Briefly, healthy impacted human third molars were collected from 13-17 years old donors who provided informed consent, in accordance with the World Medical Association's Declaration of Helsinki and the Public Health Code (Article R1211-49), and following a protocol approved by the Ministry of Higher Education and Research (CODECOH: DC-2014-2325). DPs from teeth between Nolla developmental stages 5 (crown almost completed) and 7 (one third root completed) were gently extirpated from pulp cavities and cut into fragments of approximately 0.5-2 mm³. Pulp fragments were seeded as explants on dishes pre-coated with a mixture of human placental collagens I and III (0.5 mg/cm²) and cultured in Optistem serum-free medium (Univercell Biosolutions, Toulouse, France) supplemented with 100 IU/mL penicillin and 100 mg/mL streptomycin (Thermo Fischer Scientific, Waltham, MA, USA).

After 2 weeks, cells outgrowing from the explants were harvested using TrypLEs (Thermo Fischer Scientific) and aliquoted and stored at -196°C.

At the time of the experiment, cell aliquots were thawed and mixed with 7 mL of serum-free, GMP culture media [41] before being seeded in 75 cm² flasks previously coated with a mixture of collagen I and III. Cells were allowed to expend and were then harvested and incorporated in the hydrogel formulation as previously described, at a concentration of 5000 cells/µL. Hydrogels were then slowly injected into a 1-mm diameter transparent plastic tubes and allowed to polymerise for 15 min at RT. Tubes were then placed into wells of 48-well plates, and covered with 500 µL of culture medium. In these experiments the final fibrin concentration of the hydrogel was 6 mg/mL.

## 2.10. Cytotoxicity test

The cytotoxicity of GH12 on DP-MSCs (0 or 150 µg/mL of GH12) was studied during 48 h, using a Live and Dead assay (04511-1KT-F, Sigma-Aldrich). Three conditions were tested: DP-MSCs embedded in fibrin hydrogel were the positive control), and DP-MSCs embedded in a fibrin-Triton X100 hydrogel as the negative control. Test was carried out on DP-MSCs embedded in a GH12-fibrin (6 mg/mL fibrin, 150 µg/mL GH12). Hydrogel filled plastic tubes were collected and cut in half lengthways. The medium was removed and 300 µL of the prepared Live and Dead assay solution was added in each well. After 15 min of incubation at 37°C, photographs were taken using an eclipse TE300 microscope (Nikon, Japan) in the PRIMATISS facility of the lab using Fluorescein IsoThioCyanate (FITC) and TetramethylRhodamine-5-(and 6)-IsoThioCyanate (TRITC) filters. A 660-µm² square area was selected, ensuring it was blur free. Red fluorescent cells were counted as dead cells, and green fluorescent cells were counted as living cells. Images were analysed using the Fiji software. Viability and mortality were calculated using the following formulas for quantification:

* Total Cell Number = Living Cells + Dead Cells

* Percentage of Living Cells = (Living Cells/Total Cell Number) *100

* Percentage of Dead Cells = (Dead Cells/Total Cell Number) *100

The Live and Dead assays were carried out 3 times (N = 3).

## 2.11. Statistics

Data are expressed as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, CA, USA) using ANOVA tests followed by multiple comparisons Bonferroni correction (A two-way ANOVA statistical test was performed along with a TUKEY multiple comparison test).
All tests were run in triplicates (n = 3), either 3 or 4 independent times (N = 3 or N = 4). Rheology and SEM samples were carried out 3 times (N = 3). For microbiology samples: MICs were carried out 4 times (N = 4), inhibition circles assays were carried out 4 times (N = 4) and ERI model apical release assays were carried out 4 times (N = 4). All samples were checked for normal distribution and variance equality using the Shapiro Wilks test. A P-value < 0.05 was considered statistically significant.

## 3. Results

### 3.1. GH12 synthesis and representation

The GH12 peptide composed of 12 amino acids based on the physicochemical characteristics of LL37 was produced with a reported purity of at least 95%, and was further purified using HPLC. The MS Spectra displayed a peak at 1487 m/z, characteristic of the formed peptide (supplementary data 1). The structure and the associated physicochemical characteristics were assessed by predictive computing. The Pepfold software predicted the 3D structure of GH12 as an amphipathic right α-helix. This helix displayed a hydrophobic side coloured in orange and red colours (Fig. 1A).

### 3.2. GH12-fibrin hydrogel synthesis

The hydrogels were analysed using SEM imaging. The hydrogels with or without GH12 displayed a mean pore size of 6.56 µm, and fibre mean diameters of 0.38 µm. The addition of 150 µg/mL GH12 did not lead to significant variation in hydrogels pore size or fibre diameters (Figs. 1B and 1C). All pores and fibre sizes results are gathered in Table 1.

Storage modulus (G') and loss modulus (G'') of a 6 mg/mL fibrin hydrogel were 105.00 ± 41 Pa and 10.00 ± 3 Pa, respectively, after 11 min of reading (16 min of total polymerisation). Stiffness reached a plateau of 295.00 ± 90 Pa after 25 min of total polymerisation. Storage modulus (G') and loss modulus (G'') of a 6 mg/mL fibrin hydrogel loaded with 150 µg/mL GH12 were 100.12 ± 12 Pa and 9.35 ± 2 Pa,

<caption>**Table 1**</caption>

Mean fibre and pore sizes measured in SEM images of 1.5 mg/mL fibrin hydrogel containing 0 or 150 µg/mL GH12. N = 3.

<table><thead><tr><td>Hydrogel</td><td>Mean fibre width (µm)</td><td>Average pore diameter (µm)</td></tr></thead><tbody><tr><td>Fibrin 1.5 mg/mL</td><td>0.378 ± 0.141</td><td>6.560 ± 1.764</td></tr><tr><td>Fibrin 1.5 mg/mL - GH12 150 µg/mL</td><td>0.324 ± 0.091</td><td>6.354 ± 3.217</td></tr></tbody></table>

respectively, after 16 min and reached a stiffness plateau of 140.12 Pa after 23 min of polymerisation. (Fig. 1D).

Release of GH12 from the hydrogels was studied using Endodontic Release Inserts (ERIs). Fibrin hydrogels at concentrations of 1.5, 3, and 6 mg/mL were tested. At 24 h, 72.80% of the total amount of fluorescent GH12 was released in PBS from the 1.5 mg/mL fibrin hydrogels, 61.20% was released from the 3 mg/mL hydrogels, and 24.50% was released from the 6 mg/mL hydrogels (Fig. 1D, Supplementary data 2).

### 3.3. Growth inhibition in liquid

After 24 h of incubation at 37°C, MBCs and MICs were measured using OD₆₀₀ nm. The growth of oral gram-positive S. gordonii was inhibited in BHI at GH12 concentration of 27.37 µg/mL. E. faecalis was inhibited from 75.00 µg/mL onwards. P. gingivalis, oral gram-negative bacteria, was more sensitive than the tested gram-positive, the MIC being 4.00 µg/mL. All values are gathered in Table 2.

The bacterial growth was also measured at 5 h and 48 h using OD₆₀₀ nm. No growth was observed when at least 18.75 µg/mL of GH12 was added for S. gordonii, 37.75 µg/mL for E. faecalis, and 1.85 µg/mL for P. gingivalis. After 48 h, OD₆₀₀ nm readings no longer allowed to read MICs, as bacterial growth reached the stationary state (Supplementary data 3).

### 3.4. Growth inhibition in solid media

GH12 was rapidly released from fibrin hydrogels (Fig. 1F), we tested whether it retained its antibacterial effect. A total of 6 mg/mL of fibrin

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Integration of GH12 into the fibrin hydrogel. 3D GH12 forming an α-helix as proposed by PepFold 4.0. (A). Effect of GH12 incorporation on SEM aspect of the hydrogel without GH12 (B), or charged with GH12 (150 µg/mL) (C). Effect of GH12 on the hydrogel's rheological properties (G', D). Release of fluorescent GH12 from fibrin hydrogels at various concentrations (E). N = 3.</figcaption></figure>
<caption>**Table 2**</caption>

The deduced MIB and MBCs from the optical density (OD) in 96-well plate at 24 h incubation on gram-positive oral bacteria *Streptococcus gordonii*, *Enterococcus faecalis*, and gram-negative *Porphyromonas gingivalis*.

<table><thead><tr><th>Bacterial strain</th><th>Minimum Inhibitory Concentration (MIC) (µg/mL)</th><th>Minimum Bactericidal Concentration (MBC) (µg/mL)</th></tr></thead><tbody><tr><td><i>Streptococcus gordonii</i> (ATCC35105)</td><td>28.15</td><td>37.50</td></tr><tr><td><i>Enterococcus faecalis</i> (ATCC19433)</td><td>75.00</td><td>150.00</td></tr><tr><td><i>Porphyromonas gingivalis</i> (ATCC49417)</td><td>4.25</td><td>9.75</td></tr></tbody></table>

hydrogels were placed on seeded agar plates. Both the 75 and 150 µg/mL concentration of GH12 led to an inhibition circle ranging from 1 to 6 mm in diameter, depending on the bacterial strain. The diameter was proportional to GH12 concentration; a twice greater GH12 concentration in the hydrogels led to twice greater diameter of the inhibition circle. Compared to the other strains, inhibition circle diameters were greater for *P. gingivalis* (4 and 6 mm for GH12 µ and 150 µg/mL, respectively). Clindamycin yielded circles 2–10 times greater than those of GH12, depending on the strain. All inhibition circle values (measured and plotted at 24 h) were maintained during 72 h (Fig. 2).

### 3.5. Apical release and effectiveness

During the apical release assays, CFUs were counted 5 and 24 h after contact with GH12 in hydrogels placed in ERIs on top of the bacterial cultures (Fig. 3A). A reduction ranging from 70 % to 99.99 % of the CFU count was found, depending on the bacterial strain and the hydrogel concentration, as well as the exposure duration. All results are gathered in Supp data 4.

The effect of GH12 on the CFU count of S gordonii, E faecalis and P gingivalis was plotted compared to a hydrogel containing no active molecule (Fibrin) and a hydrogel containing a positive control (clindamycin 75 µg/mL) after 5 h (B). After 24 h incubation, bacterial regrowth was assessed on S. gordonii. ("Fibrin": Fibrin 1.5 mg/mL, "GH12": Fibrin 1.5 mg/mL - GH12 150 µg/mL, "Clindamycin": Fibrin 1.5 mg/mL - Clindamycin 75 µg/mL) (C) The effect of fibrin concentration in

presence of GH12 was assessed at 5 h on S. gordonii (D). N = 4, p value < 0,01 = *, p value < 0,001 = **, non significant = ns.

The addition of GH12 in fibrin hydrogels led to a complete eradication of *P. gingivalis* cultures at 150 µg/mL, while the 75 µg/mL led to a 99.96 % reduction of the total CFU count. GH12 appears less potent on gram-positive *S. gordonii* and *E. faecalis*, the reduction ranging from 99.00 % to 90.00 %, after 5 h of contact (Fig. 3B). After 24 h of contact, an increase in CFU was observed in all bacterial strains (including *S. gordonii* Fig. 3C)

An increase in the hydrogel density (fibrin concentration from 1.5 mg/mL to 6 mg/mL) led to a decrease in effectiveness of the GH12-fibrin hydrogel on *S. gordonii*, (CFU count was higher when GH12-fibrin hydrogel was more concentrated (6 mg/mL)). CFU count was reduced with both GH12-fibrin hydrogels (1.5 mg/mL and 6 mg/mL) compared to fibrin alone (Fig. 3D). Significant reduction of CFU counts (p values < 0.05) are shown in Fig. 3.

### 3.6. Cellular assays

GH12 cytotoxicity on DP-MSCs in a 3D cellular environment was assessed at GH12 concentration 150 µg/mL. The survival rate of DP-MSCs in a fibrin hydrogel without GH12 is considered to be a negative control. In hydrogels with 150 µg/mL GH12, the survival rate was 90 % (Fig. 4). Addition of Triton X100 as a positive control led to 0 % survival.

## 4. Discussion

This study hypothesised that GH12 could be included in a fibrin hydrogel to prevent bacterial contamination of the material during the dental pulp regeneration process. It aimed to evaluate the rheological, morphological and antibacterial impact of GH12 inclusion in a cyto-compatible fibrin scaffold. GH12 has demonstrated good effectiveness against endodontic bacteria and outstanding cytocompatibility with DP-MSCs, making it a very promising candidate for dental pulp regeneration. The first objective of this study was to integrate GH12 in a fibrin hydrogel without impacting its structural conformation and properties, thereby maintaining GH12's antibacterial activity. The second objective was to determine GH12 effectiveness against several endodontic bacteria. Lastly, the study aimed to assess the applicability of this hydrogel in the specific endodontic anatomical and galenic context, by determining whether GH12 was able to both protect the hydrogel from

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Antibacterial activity of the GH12-fibrin hydrogel in a semi solid environment. *Streptococcus gordonii*, *Enterococcus faecalis* and *Porphyromonas gingivalis* were incubated on agar plates with 30 µL fibrin hydrogel (6 mg/mL) containing GH12 (0, 75 or 150 µg/mL), or Clindamycin (75 µg/mL). Inhibition circles were photographed after 24 h (E. faecalis photos, A). The average inhibition circle diameters were gathered in a table and compared to a negative (fibrin alone) and positive control (75 µg/mL clindamycin fibrin gel) (B), N = 4. [* = < 0.05; ** = < 0.005; *** = < 0.001; **** = < 0.0001].</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** GH12 release and antibacterial effectiveness. Fibrin hydrogels at concentration 1.5 or 6 mg/mL, containing GH12 (75 or 150 µg/mL) were cast in ERI models [13] and GH12 antibacterial effect was assessed on Streptococcus gordonii, Enterococcus faecalis or Porphyromonas gingivalis. At 5 and 24 h, a fraction (10 µL) of the bottom solution was sampled and seeded on agar plates. Colony forming units (CFUs) were counted after 24 h (A).</figcaption></figure>

bacterial infection and decrease the bacterial load in the periapical region, while being non-toxic to Dental Pulp (DP) cells.

Fibrin has been extensively used in studies as a potential injected vector for long lasting release of active molecules in the fields of tissue engineering and RNA delivery, as well as for cell delivery [45,46]. However, fibrin has a limited antibacterial activity and can even promote bacterial growth by providing a scaffold onto which various strain can attach [47], which was also observed herein. Of note, the inhibition circle assay carried out showed an absence of bacterial growth on and around the GH12-loaded fibrin hydrogel, and fast-growing colonies on and around fibrin hydrogels alone. When included in the hydrogel, GH12 impacted the hydrogel polymerisation kinetic; it took 4–5 min longer to reach a 100 Pa stiffness, which was the average stiffness observed for fibrin hydrogels at similar concentrations overall in other tissue engineering studies [48–51]. However, this delay did not impact significantly the final structure of the hydrogel, which reaches a plateau stiffness of approximately 180 Pa, similar to that observed for the fibrin hydrogel without GH12. The hypothesis of a lack of interaction between fibrin fibres and GH12 is further supported by the rapid release rate of GH12, (45 % is released from a 2000 µm apex ERI during the first 5 h), which suggests that the peptide is able to diffuse freely through the gel.

MIC assays were first carried out in liquid media without release. This model is easy and cheap, and was therefore used to confirm GH12

effectiveness as described in the literature on endodontic bacteria, *E. faecalis*, *P. gingivalis*, and *S. gordonii* [31,33,34]. As this study aims at using GH12 as a protective agent for dental pulp regeneration, its efficacy in a semi solid environment was tested. Inhibition circles were carried out on agar plates to study the ability of GH12 to protect the hydrogel from bacterial infection coming from outside the hydrogel in a semi-solid environment. Studies have shown that more than 600 bacterial strains can be found in a necrotic pulp, depending on patients and teeth [52]. To remain close to a reliable model of endodontic infection, it is necessary to use strains from both gram groups. Therefore, two gram-positive and one gram-negative bacteria were studied. Of note, *E faecalis* presence has been associated with higher risks of reinfections as well as biofilm formation and resistance, making it a severity factor [53]. *S. gordonii* and *P. gingivalis* were selected due to their frequent presence in infected pulps [52,54]. All strains showed reduced or stopped growth, and were killed by GH12 at concentrations greater than 100 µg/mL in both endodontic and non-endodontic models. These results are accordance with those of Jiang et al., Li et al., and Wang et al. on *S. mutans*, *E. faecalis*, and *S. gordonii*, in liquid and dental models [31, 36,40].

GH12 has a very rapid action on membranes (within 10 min as demonstrated by Wang, [40] that was herein associated with a rapid release from a fibrin hydrogel. It was hypothesised that this very fast
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Cytocompatibility assessment in GH12-containing 6 mg/mL fibrin hydrogels. Fibrin hydrogels with 5000 cells per µL, containing GH12 (0, 75 or 150 µg/mL) were cast in transparent cylindrical tubules. After 48 h, the tubules were cut in half and the samples were treated with a Live and Dead staining kit. Green: live cell; red: dead cell (A). The number of living cells was counted and compared to the number of living cells in a hydrogel treated with triton. Results are gathered in a graph (D). N = 3, p value < 0,001 = *, ns = non-significant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

release within the first few hours allows GH12 to reduce the apical bacterial load, by allowing a high concentration of the fast-acting peptide to be released in the apical space [30,34]. In the present study, a low concentration of fibrinogen (1.5 mg/mL) was found optimal to obtain rapid and effective action against bacteria outside the endodontic space. However, GH12 release suggests that a rapid depletion of the hydrogel will occur and lead to reduced *in situ* antibacterial action as well as lack of long-term hydrogel protection. It would be interesting to explore strategies aiming to optimize GH12 release from the hydrogel, as performed on other peptides such as LL-37 or IDR002 for tissue engineering purposes [55,56].

Current models used in studies investigating endodontic regeneration often fail to convey the full complexity of endodontic features. The inhibition circles model, although reliable and easy to read, does not consider the very particular anatomy of the endodontic environment, including its small volume (a few mm³) and the intricate shape of the root canal, which only allows for minor contact and a low volume of hydrogel [10,57,58]. These anatomical parameters make it challenging to achieve a thorough endodontic disinfection; the antibacterial molecule might not reach bacteria deeply located into the canal or at the apex. To overcome these challenges, various models were developed to test diffusion, apical release, and activity of molecules. The most used are pipette cones cut and put into agar and individually prepared extracted human tooth. However, both these models are time consuming, difficult to use, and prone to variability [12,59–61]. To better mimic the *in vivo* conditions, release from the hydrogel as well as apical antibacterial effectiveness tests were performed using the Endodontic Release Insert (ERI) model [13]. This highly reproducible model, based on the shape and volume of a single root human tooth, has previously been used to study apical release of several model molecules [13]. It demonstrated very low variability, was easy to manage, and could be cleaned and reused. This model was used to study the ability of the peptide to diffuse in the periapical region and retain its antibacterial

properties. Furthermore, from the ability of the molecule to circulate and diffuse in the gel and in the periapical region, it was possible to anticipate the total amount of molecule that will be released into the bloodstream, and thus, its potential toxicity [13,60].

Of note, clinical use and systemic toxicity must be taken into account to design clinically usable and safe products; this includes products that are injectable, with a long shelf life, which can be manufactured on an industrial scale, while being non-toxic to patients and professionals [12, 62]. Regarding the peptide itself, its short half-life, estimated around 4–5 h in body fluids such as human serum, is a double-edged knife suggesting lower systemic toxicity for the patient; it reduces risks of long-term toxicity associated with molecule accumulation in other organs. However, it implies only short-term bactericidal activity. Regarding peptide production, GH12 was synthesised using the Fmoc technique, a cheap, quick, and overall scale-upable method. Being able to synthesise the peptide through this technique is an important advantage over antibiotics, which require long, sometimes patented synthesis reactions, or complex purification process, as for metronidazole or ciprofloxacin [63,64]. Altogether these features make GH12-based medical devices cheap, accessible, and marketable options. However, further studies are required to determine the ability of the GH12-fibrin composite to make contact with the root dentin wall and diffuse deeply in dentin tubules to disrupt biofilm formation.

Several limitations can be raised from this study. Regeneration was not studied in a thorough manner in this study, and further work is necessary to ensure GH12 does not impact regenerative mechanisms in cells. Mainly, mineralisation processes are to be studied, as they are vital to tooth impermeability [65]. Biofilm models were not investigated in this study, even though endodontic infection is mostly biofilm mediated [66]. However, GH12 was first designed as an antibiofilm peptide, and has shown extensive antibiofilm action in the literature [31,35,37,38, 40]. Furthermore, previous work by Li et al. has demonstrated the effect of GH12 as a canal irrigant, and shown the ability of GH12 in liquid
solution to eradicate *E. faecalis* within 20 min from dentin slabs previ-
ously infected with an *E. faecalis* biofilm [31]. This work by Li et al.
suggests that dentin does not limit the access of GH12 to bacteria inside
tubules, nor interact with it in a relevant manner to antimicrobial ac-
tivity. Further investigations are necessary to study the behaviour of a
GH12-fibrin hydrogel, in the context of a biofilm and dentin complex,
and ensure inclusion in the hydrogel does not hinder the ability of GH12
to enter tubules, and thus the clinical relevance of the final medical
device [67]. The 3D model used does not currently allow to model the
specific root canal with its secondary canals and tens of thousands of
micrometre-sized tubules. Similarly, it should be noted that GH12
release was investigated herein in a "static" way, therefore the overall
release of GH12 may be underestimated (not considering the presence of
various enzymes, proteins, ions, and blood flow). Regarding the selected
bacteria, a greater number of anaerobic strains and biofilms should be
further investigated. However, the required material to grow and study
them is much more complex and expensive, as it includes anaerobic
chambers and incubators, as well as specific microbiological training for
the operator. This common issue [11,68,69], limits the frequency of
successful transitions from bench to bedside as the effect of the tested
molecules cannot be anticipated on an important part of the bacterial
population [70].

5. Conclusion

This study describes the antimicrobial effects of GH12, a LL37-
derived synthetic peptide, upon release from a fibrin hydrogel previ-
ously developed for endodontic regeneration. GH12 in fibrin hydrogel
showed some impact on hydrogel rheological properties, but no signif-
icant changes were observed in fibre and pore size. An up to 24 h anti-
microbial activity was evidenced on both gram-positive and gram-
negative bacteria often found in infected root canals. After 24 h, the
activity decreases, which, from a clinical point of view, limits risks of
systemic toxicity by reducing exposition duration and overall dose.
Further *in vitro* and *in vivo* investigations are necessary to evaluate the
biological effects of GH12 on Dental Pulp Mesenchymal Stem Cells, as
well as to ensure safety and effectiveness of GH12 in animal models of
infection.

Funding

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), the Fondation de
l'Avenir (AP-RM-21-030), the French National Centre for Scientific
Research (CNRS) and the French Institute for Odontological Research
(IFRO). ML is a recipient of a Ph.D. grant from the French National
Association of Research (ANR JCJC ENDONANOBIOTIC).

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 are grateful to the Protein Science Facility of the SFR Biosciences (Université Claude Bernard Lyon 1, CNRS UAR3444, Inserm US8, ENS de Lyon) for their support in GH12 peptide synthesis.

We are grateful to the ImpressiOdont plateform (Université Claude Bernard Lyon 1, Faculté d'Odontologie) for their help designing and producing the ERI model.

**Appendix A. Supporting information**

Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.dental.2025.08.010.

**References**

[1] Atala A, Irvine DJ, Moses M, Shaunak S. Wound healing versus regeneration: role of the tissue environment in regenerative medicine. MRS Bull 2010;35:597-606. https://doi.org/10.1557/mrs2010.528.

[2] Nakashima M, Iohara K, Zayed M. Pulp regeneration: current approaches, challenges, and novel rejuvenating strategies for an aging population. J Endod 2020;46:S135-42. https://doi.org/10.1016/j.joen.2020.06.028.

[3] Schmalz G, Widbiller M, Galler KM. Clinical perspectives of pulp regeneration. J Endod 2020;46:S161-74. https://doi.org/10.1016/j.joen.2020.06.037.

[4] Hsiao WWL, Li KL, Liu Z, Jones C, Fraser-Liggett CM, Fouad AF. Microbial transformation from normal oral microbiota to acute endodontic infections. BMC Genom 2012;13:345. https://doi.org/10.1186/1471-2164-13-345.

[5] Endo MS, Ferraz CCR, Zaia AA, Almeida JFA, Gomes BPFA. Quantitative and qualitative analysis of microorganisms in root-filled teeth with persistent infection: monitoring of the endodontic retreatment. Eur J Dent 2013;07:302-9. https://doi.org/10.4103/1305-7456.115414.

[6] Verma P, Nosrat A, Kim JR, Price JB, Wang P, Bair E, et al. Effect of residual bacteria on the outcome of pulp regeneration in vivo. J Dent Res 2017;96:100-6. https://doi.org/10.1177/0022034516671499.

[7] Vishwanat L, Duong R, Takimoto K, Phillips L, Espitia CO, Diogenes A, et al. Effect of bacterial biofilm on the osteogenic differentiation of stem cells of apical papilla. J Endod 2017;43:916-22. https://doi.org/10.1016/j.joen.2017.01.023.

[8] Liu C, Xiong H, Chen K, Huang Y, Huang Y, Yin X. Long-term exposure to pro-inflammatory cytokines inhibits the osteogenic/dentinogenic differentiation of stem cells from the apical papilla. Int Endod J 2016;49:950-9. https://doi.org/10.1111/iej.12551.

[9] El-Fiqi A, Mandakhbayar N, Jo SBin, Knowles JC, Lee J-H, Kim H-W. Nanotherapeutics for regeneration of degenerated tissue infected by bacteria through the multiple delivery of bioactive ions and growth factor with Antibacterial/Angiogenic and osteogenic/odontogenic capacity. Bioact Mater 2021;6:123-36. https://doi.org/10.1016/j.bioactmat.2020.07.010.

[10] Mehdawi I, Neel EAA, Valappil SP, Palmer G, Salih V, Pratten J, et al. Development of remineralizing, antibacterial dental materials. Acta Biomater. 2009;5:2525-39. https://doi.org/10.1016/j.actbio.2009.03.030.

[11] Leveque M, Guittat M, Thivichon-Prince B, Reuzeau A, Eveillard M, Faure M, et al. Next generation antibacterial strategies for regenerative endodontic procedures: a scoping review. Int Endod J 2023. https://doi.org/10.1111/iej.13958.

[12] Leveque M, Bekhouche M, Farges J-C, Aussel A, Sy K, Richert R, et al. Bioactive endodontic hydrogels: from parameters to personalized medicine. Preprints 2023.

[13] Leveque M, Bekhouche M, Farges J, Richert R, Ducret M. Investigation of the early apical cell response from endodontic hydrogels: a 3D printed model. Int Endod J 2024. https://doi.org/10.1111/iej.14049.

[14] Kim JY, Xin X, Moioli EK, Chung J, Lee CH, Chen M, et al. Regeneration of dental-pulp-like tissue by chemotaxis-induced cell homing. Tissue Eng Part A 2010;16:3023-31. https://doi.org/10.1089/ten.tea.2010.0181.

[15] Allotey JK, King AH, Kumins NH, Wong VL, Harth KC, Cho JS, et al. Systematic review of hemostatic agents used in vascular surgery. J Vasc Surg 2021;73:2189-97. https://doi.org/10.1016/j.jvs.2020.10.081.

[16] Campos F, Bonhome-Espinosa AB, Chato-Astrain J, Sánchez-Porras D, García-García ÓD, Carmona R, et al. Evaluation of fibrin-agarose tissue-like hydrogels biocompatibility for tissue engineering applications. Front Bioeng Biotechnol 2020; 8. https://doi.org/10.3389/fbioe.2020.00596.

[17] Jacobs JC, Troxel A, Ehrlich Y, Spolnik K, Bringas JS, Gregory RL, et al. Antibacterial effects of antimicrobials used in regenerative endodontics against biofilm bacteria obtained from mature and immature teeth with necrotic pulps. J Endod 2017;43:575-9. https://doi.org/10.1016/j.joen.2016.12.014.

[18] El-Tayeb MM, Abu-Seida AM, El Ashry SH, El-Hady SA. Evaluation of antibacterial activity of propolis on regenerative potential of necrotic immature permanent teeth in dogs. BMC Oral Health 2019;19. https://doi.org/10.1186/s12903-019-0835-0.

[19] da Cunha Neto MA, de Almeida Coelho J, Pinto KP, Cuellar MRC, Marcucci MC, Silva EJNL, et al. Antibacterial efficacy of triple antibiotic medication with macrogol (3Mix-MP), traditional triple antibiotic paste, calcium hydroxide, and ethanol extract of propolis: an intratubular dentin ex vivo confocal laser scanning microscopic study. J Endod 2021;47:1609-16. https://doi.org/10.1016/j.joen.2021.07.014.

[20] Siddiqui Z, Acevedo-Jake AM, Griffith A, Kadincesne M, Dabek K, Hindi D, et al. Cells and material-based strategies for regenerative endodontics. Bioact Mater 2022;14:234-49. https://doi.org/10.1016/j.bioactmat.2021.11.015.

[21] Hanna CC, Hermant YO, Harris PWR, Brimble MA. Discovery, synthesis, and optimization of Peptide-Based antibiotics. Acc Chem Res 2021;54:1878-90. https://doi.org/10.1021/acs.accounts.0c00841.

[22] Di Somma A, Moretta A, Canè C, Cirillo A, Duilio A. Antimicrobial and antibiofilm peptides. Biomolecules 2020;10:652. https://doi.org/10.3390/biom10040652.

[23] Nagant C, Pitts B, Nazmi K, Vandenbranden M, Bolscher JG, Stewart PS, et al. Identification of peptides derived from the human antimicrobial peptide LL-37 active against biofilms formed by pseudomonas aeruginosa using a library of truncated fragments. Antimicrob Agents Chemother 2012;56:5698-708. https://doi.org/10.1128/AAC.00918-12.
[24] Lima SMF, Freire MS, Gomes ALO, Cantuária APC, Dutra FRP, Magalhães BS, et al. Antimicrobial and immunomodulatory activity of host defense peptides, clavanins and LL-37, in vitro: an endodontic perspective. Peptides 2017;95:16-24. https://doi.org/10.1016/j.peptides.2017.07.005.

[25] Talapko J, Městrovíč T, Juzbašíč M, Tomas M, Eríč S, Horvat Aleksijevič L, et al. Antimicrobial Peptides-Mechanisms of action, antimicrobial effects and clinical applications. Antibiotics 2022;11. https://doi.org/10.3390/ANTIBIOTICS11101417.

[26] Bechinger B, Gorr SU. Antimicrobial peptides: mechanisms of action and resistance. J Dent Res 2017;96:254-60. https://doi.org/10.1177/0022034516679973.

[27] Peschel A. How do bacteria resist human antimicrobial peptides? Trends Microbiol 2002;10:179-86. https://doi.org/10.1016/S0966-842X(02)02333-8.

[28] Li Y, Xiang Q, Zhang Q, Huang Y, Su Z. Overview on the recent study of antimicrobial peptides: origins, functions, relative mechanisms and application. Peptides 2012;37:207-15. https://doi.org/10.1016/j.peptides.2012.07.001.

[29] Kim EY, Rajasekaran G, Shin SY. LL-37-Derived short antimicrobial peptide KR-12-A5 and its D-amino acid substituted analogs with cell selectivity, anti-biofilm activity, synergistic effect with conventional antibiotics, and anti-inflammatory activity. Eur J Med Chem 2017;136:428-41. https://doi.org/10.1016/j.ejmech.2017.05.028.

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

[31] Li Y, Wang Y, Chen X, Jiang W, Jiang X, Zeng Y, et al. Antimicrobial peptide GH12 as root canal irritant inhibits biofilm and virulence of enterococcus faecalis. Int Endod J 2020;53:948-61. https://doi.org/10.1111/iej.13296.

[32] Kong X, Vishwanath V, Neelakantan P, Ye Z. Harnessing antimicrobial peptides in endodontics. Int Endod J 2024;57:815-40. https://doi.org/10.1111/iej.14043.

[33] Jiang W, Wang Y, Luo J, Chen X, Zeng Y, Li X, et al. Antimicrobial peptide GH12 prevents dental caries by regulating dental plaque microbiota. Appl Environ Microbiol 2020;86. https://doi.org/10.1128/AEM.00527-20.

[34] Li X, Wang Y, Jiang X, Zeng Y, Zhao X, Washio J, et al. Investigation of drug resistance of Caries-Related streptococci to antimicrobial peptide GH12. Front Cell Infect Microbiol 2022;12. https://doi.org/10.3389/fcimb.2022.991938.

[35] Jiang W, Wang Y, Luo J, Li X, Zhou X, Li W, et al. Effects of antimicrobial peptide GH12 on the cariogenic properties and composition of a cariogenic multispecies biofilm. Appl Environ Microbiol 2018;84. https://doi.org/10.1128/AEM.01423-18.

[36] Jiang W, Luo J, Wang Y, Chen X, Jiang X, Feng Z, et al. The PH-Responsive property of antimicrobial peptide GH12 enhances its anticaries effects at acidic PH. Caries Res 2021;55:21-31. https://doi.org/10.1159/000508458.

[37] Zeng Y, Chen Y, Duan C, Jiang X, Wang Y, Zhang L. A transcriptional analysis showing the effects of GH12 combined with fluoride for suppressing the acidogenicity of streptococcus mutans biofilms. Microorganisms 2023;11. https://doi.org/10.3390/microorganisms11071796.

[38] Sinha A, Taneja S, Shetty DC, Bhalla VK. Efficacy of antimicrobial peptide GH12 on a multispecies endodontic biofilm model: an in-vitro study. Eur Endod J 2024;9: 411. https://doi.org/10.14744/EEJ.2024.75983.

[39] Xie S-X, Song L, Yuca E, Boone K, Sarikaya R, VanOosten SK, et al. Antimicrobial Peptide-Polymer conjugates for dentistry. ACS Appl Polym Mater 2020;2:1134-44. https://doi.org/10.1021/acsapm.9b00921.

[40] Wang Y, Fan Y, Zhou Z, Tu H, Ren Q, Wang X, et al. De novo synthetic short antimicrobial peptides against cariogenic bacteria. Arch Oral Biol 2017;80:41-50. https://doi.org/10.1016/j.archoralbio.2017.03.017.

[41] Ducret M, Montembault A, Josse J, Pasdeloup M, Celle A, Benchrih R, et al. Design and characterization of a Chitosan-Enriched fibrin hydrogel for human dental pulp regeneration. Dent Mater 2019;35:523-33. https://doi.org/10.1016/j.dental.2019.01.018.

[42] Ducret M, Costantini A, Gobert S, Farges JC, Bekhouche M. Fibrin-based scaffolds for dental pulp regeneration: from biology to nanotherapeutics. Eur Cell Mater 2021;41:1-14. https://doi.org/10.22203/ecm.v041a01.

[43] Bekhouche M, Bolon M, Charriaud F, Lamrayah M, Da Costa D, Primard C, et al. Development of an antibacterial nanocomposite hydrogel for human dental pulp engineering. J Mater Chem B 2020;8:8422-32. https://doi.org/10.1039/ DOTB00989J.

[44] Piglionico SS, Varga B, Pall O, Romieu O, Gergely C, Cuisinier F, et al. Biomechanical characterization of a Fibrinogen-Blood hydrogel for human dental pulp regeneration. Biomater Sci 2023;11:6919-30. https://doi.org/10.1039/ D3BM00515A.

[45] Janmey PA, Winer JP, Weisel JW. Fibrin gels and their clinical and bioengineering applications. J R Soc Interface 2009;6:1-10. https://doi.org/10.1098/ rsif.2008.0327.

[46] Ngarande E, Doubell E, Tamgue O, Mano M, Human P, Giacca M, et al. Modified fibrin hydrogel for sustained delivery of RNAi lipopolyplexes in skeletal muscle. Regen Biomater 2023;10. https://doi.org/10.1093/rb/rbac101.

[47] Kearney KJ, Ariëns RAS, Macrae FL. The role of fibrin(ogen) in wound healing and infection control. Semin Thromb Hemost 2022;48:174-87. https://doi.org/ 10.1055/s-0041-1732467.

[48] Brinkmann J, Malyaran H, Enezy-Ulbrich MAI, Jung S, Radermacher C, Buhl EM, et al. Assessment of Fibrin-Based hydrogels containing a Fibrin-Binding peptide to tune mechanical properties and cell responses. Macromol Mater Endo 2023;308: 2200678. https://doi.org/10.1002/MAME.202200678.

[49] Jung SA, Malyaran H, Demco DE, Manukanc A, Häser LS, Kućikas V, et al. Fibrin-Dextran hydrogels with tunable porosity and mechanical properties. Biomacromolecules 2023;24:3972-84. https://doi.org/10.1021/ACS. BIOMAC.3C00269.

[50] Gao Y, Cho HJ. Quantifying the Trade-off between stiffness and permeability in hydrogels. Soft Matter 2022;18:7735-40. https://doi.org/10.1039/D2SM01215D.

[51] Moreno-Arotzena O, Meier J, Del Amo C, García-Aznar J. Characterization of fibrin and collagen gels for engineering wound healing models. Materials 2015;8: 1636-51. https://doi.org/10.3390/ma8041636.

[52] Siqueira JF, Róças IN. Diversity of endodontic microbiota revisited. J Dent Res 2009;88:969-81. https://doi.org/10.1177/0022034509346549.

[53] Alghami D, Shakir M. The influence of enterococcus faecalis as a dental root canal pathogen on endodontic treatment: a systematic review. Cureus 2020. https://doi.org/10.7759/cureus.7257.

[54] Ricucci D, Siqueira JF, Loghin S, Lin LM. Pulp and apical tissue response to deep caries in immature teeth: a histologic and histobacteriologic study. J Dent 2017;56: 19-32. https://doi.org/10.1016/j.jdent.2016.10.005.

[55] Galler KM, Hartgerink JD, Cavender AC, Schmalz G, D'Souza RN. A customized Self-Assembling peptide hydrogel for dental pulp tissue engineering. Tissue Eng Part A 2012;18:176-84. https://doi.org/10.1089/ten.tea.2011.0222.

[56] Comune M, Rai A, Palma P, TondaTuro C, Ferreira L. Antimicrobial and pro-angiogenic properties of soluble and nanoparticle-immobilized LL37 peptides. Biomater Sci 2021;9:8153-9. https://doi.org/10.1039/d1bm01034d.

[57] O'Shea R, Moser HE. Physicochemical properties of antibacterial compounds: implications for drug discovery. J Med Chem 2008;51:2871-8. https://doi.org/ 10.1021/jm700967e.

[58] Dai J, Han R, Xu Y, Li N, Wang J, Dan W. Recent progress of antibacterial natural products: future antibiotics candidates. Bioorg Chem 2020;101:103922. https:// doi.org/10.1016/j.bioorg.2020.103922.

[59] Abbott PV. Systemic release of corticosteroids following intra-dental use. Int Endod J 1992;25:189-91. https://doi.org/10.1111/j.1365-2591.1992.tb00748.x.

[60] Robert G, Liewfer F, Buxton T, McPherson J. Apical diffusion of calcium hydroxide in an in vitro model. J Endod 2005;31:57-60. https://doi.org/10.1097/ 01.DON.0000134211.85578.38.

[61] Aubeux D, Valot-Salengro A, Gautier G, Malet A, Pérez F. Transforaminal and systemic diffusion of an active agent from a zinc oxide Eugenol-Based endodontic sealer containing Hydrocortisone—in an in vivo model. Clin Oral Investig 2020;24: 4395-402. https://doi.org/10.1007/s00784-020-03305-5.

[62] Foud AF, Verma P. Healing after regenerative procedures with and without pulpal infection. J Endod 2014;40:S58-64. https://doi.org/10.1016/j.joen.2014.01.022.

[63] Chen J, Zhao ZY, Mu XF, Li XL, Tang J, Bi QQ. Mechanochemical synthesis of novel metronidazole cocrystal: structure characterization and pharmaceutical properties study. Biochem Biophys Res Commun 2024;738. https://doi.org/10.1016/J. BBRC.2024.150546.

[64] Zhang GF, Liu X, Zhang S, Pan B, Liu ML. Ciprofloxacin derivatives and their antibacterial activities. Eur J Med Chem 2018;146:599-612. https://doi.org/ 10.1016/J.EJMECH.2018.01.078.

[65] Wang Y, Zhang X, Zheng H, Zhou Z, Li S, Jiang J, et al. Remineralization of dentin with cerium oxide and its potential use for root canal disinfection. Int J Nanomod 2023;19:1-17. https://doi.org/10.2147/IJN.S441060.

[66] Jhajharia K, Parolia A, Kv Shetty, Mehta L. Biofilm in endodontics: a review. J Int Soc Prev Community Dent 2015;5(1). https://doi.org/10.4103/2231- 0762.151956.

[67] Neelakantan P, Romero M, Vera J, Daood U, Khan AU, Yan A, et al. Biofilms in endodontics—current status and future directions. Int J Mol Sci 2017;18:1748. https://doi.org/10.3390/LJMS18081748.

[68] Diogenes A, Ruparel NB, Shiloah Y, Hargreaves KM. Regenerative endodontics: a way forward. J Am Dent Assoc 2016;147:372-80. https://doi.org/10.1016/j. adaj.2016.01.009.

[69] Ribeiro JS, Münchow EA, Ferreira Bordini EA, de Oliveira da Rosa WL, Bottino MC. Antimicrobial therapeutics in regenerative endodontics: a scoping review. J Endod 2020;46:S115-27. https://doi.org/10.1016/j.joen.2020.06.032.

[70] Almutairi W, Yassen GH, Aminoshaaria E, Williams KA, Mickel A. Regenerative endodontics: a systematic analysis of the failed cases. J Endod 2019;45:567-77. https://doi.org/10.1016/j.joen.2019.02.004.