GelMA/TCP nanocomposite scaffold for vital pulp therapy

Abstract

Pulp capping is a necessary procedure for preserving the vitality and health of the dental pulp, playing a crucial role in preventing the need for root canal treatment or tooth extraction. Here, we developed an electrospun gelatin methacryloyl (GelMA) fibrous scaffold incorporating beta-tricalcium phosphate (TCP) particles for pulp capping. A comprehensive morphological, physical-chemical, and mechanical characterization of the engineered fibrous scaffolds was performed. In vitro bioactivity, cell compatibility, and odontogenic differentiation potential of the scaffolds in dental pulp stem cells (DPSCs) were also evaluated. A pre-clinical in vivo model was used to determine the therapeutic role of the GelMA/TCP scaffolds in promoting hard tissue formation. Morphological, chemical, and thermal analyses confirmed effective TCP incorporation in the GelMA nanofibers. The GelMA+20%TCP nanofibrous scaffold exhibited bead-free morphology and suitable mechanical and degradation properties. In vitro, GelMA+20%TCP scaffolds supported apatite-like formation, improved cell spreading, and increased deposition of mineralization nodules. Gene expression analysis revealed upregulation of ALPL, RUNX2, COL1A1, and DMP1 in the presence of TCP-laden scaffolds. In vivo, analyses showed mild inflammatory reaction upon scaffolds’ contact while supporting mineralized tissue formation. Although the levels of Nestin and DMP1 proteins did not exceed those associated with the clinical reference treatment (i.e., mineral trioxide aggregate), the GelMA+20%TCP scaffold exhibited comparable levels, thus suggesting the emergence of differentiated odontoblast-like cells capable of dentin matrix secretion. Our innovative GelMA/TCP scaffold represents a simplified and efficient alternative to conventional pulp-capping biomaterials.

1. Introduction

Nestled at the tooth’s core, the dental pulp plays a central role in responding to a myriad of injuries and insults [1]. In minor injuries or bacterial intrusion, the pulp activates a series of defensive mechanisms that elicit the odontoblasts in proximity to produce reactionary dentin. Conversely, in more severe scenarios that result in odontoblast death, specialized cells, commonly referred to as odontoblast-like cells or pulp fibroblasts, generate reparative dentin from a stem cell recruitment and differentiation process [2-4]. These dentin formations represent an extraordinary adaptive response, protecting the pulp from further harm. Therefore, it is crucial to establish a suitable microenvironment when treating pulp injuries. While root canal therapy (RCT) is widely regarded as the standard of care treatment for pulpitis, it has drawbacks. Issues such as the possibility of reinfection, tooth discoloration, and brittleness can arise [5]. However, these concerns can be effectively addressed by opting for a direct vital pulp procedure, like pulp capping, to maintain the pulp vitality and facilitate reparative dentin production [6,7]. This approach helps retain the tooth’s natural strength, responsiveness, and protective mechanisms, eliminating the need for extensive structure removal.

Tissue engineering strategies have made significant strides in utilizing cells, particularly dental pulp stem cells (DPSCs), to regenerate and repair dental tissues. DPSCs possess remarkable differentiating capabilities, enabling them to develop into odontoblasts and other specialized cells for dental tissue formation. Electrospinning, a widely employed technique, allows for the creation of fibrous scaffolds, offering a promising microenvironment to interact with these cells and enhance the regenerative process [8]. The fiber scaffolds produced with this method have suitable pores and offer a significant surface area for cells to adhere to. Gelatin Methacryloyl (GelMA) is a well-established biomaterial obtained by modifying amine-containing side groups of naturally derived gelatin, i.e., denatured collagen, with a methacrylate group. GelMA is usually cured using light and can be tailored to exhibit specific physical and mechanical characteristics [9-11].

Recent research successfully employed electrospinning to engineer GelMA fibers that are photocrosslinkable and capable of carrying azithromycin for localized treatment of endodontic infection [12]. Moreover, a GelMA-based drug carrier has been developed, incorporating antibiotic-loaded fibrous microparticles into GelMA, enabling controlled and sustained release of antimicrobial drugs within the root canal system [10,13]. Additionally, the integration of beta-tricalcium phosphate (TCP) into a GelMA/PCL membrane led to increased osteogenic gene expression in alveolar bone-derived mesenchymal stem cells and promoted robust bone regeneration in rat calvarial critical-size defects [14].

GelMA is an ideal biomaterial for facilitating cellular interactions, and it degrades when exposed to matrix metalloproteinases. However, its pure form lacks substantial mineralization potential, making it unsuitable as a standalone material for pulp capping. Biologically active ceramic additives, such as bioglass and calcium phosphates (e.g., TCP), are commonly added to GelMA to overcome these limitations [14]. One of the most widely used ceramic additives is beta-tricalcium phosphate ( Ca₃(PO₄)₂), which is an osteoconductive, osteoinductive, and cell-mediated resorbed synthetic bone graft substitute that elicits a remarkable tissue response [15]. However, combining different polymers and ceramics to optimize their mechanical and biocompatible features for dental tissue applications still requires improvement.

The current optimal materials for VPT, such as mineral trioxide aggregate (MTA) and Biodentine^™, also present certain disadvantages and difficulties [16]. One of the primary drawbacks is their cost, posing a financial challenge for dental practitioners and patients, potentially limiting their accessibility. Furthermore, the handling characteristics of MTA can be challenging for some clinicians. It has a gritty texture and can be difficult to manipulate effectively, making it less user-friendly for less experienced clinicians working with this material. Another limitation is the setting time of MTA and Biodentine^™. It can take several hours to fully set, which can be impractical when quick treatment is required in urgent situations. This delay can lead to patient discomfort and inconvenience.

To effectively address these challenges, our study aimed to develop an innovative GelMA-based fibrous scaffold incorporated with TCP nanoparticles as a potential substitute for conventional pulp capping materials. To test this hypothesis and assess the potential of our fibrous scaffold, we conducted a thorough assessment characterizing its morphological, physical-chemical, and mechanical characterization properties. Additionally, we performed detailed cell-based in vitro assays and in vivo studies using a pulp capping model in rats to determine its biological properties and assess its capacity to promote hard tissue formation.

2. Materials and methods

2.1. Gelatin methacryloyl (GelMA) synthesis and preparation

GelMA was synthesized using a previously reported method [17]. The process involved dissolving 10% w/v of Gelatin Type A (Sigma-Aldrich, St. Louis, MO, USA) from porcine skin in DPBS and heating the solution to 50 °C. Methacrylic anhydride purchased from Sigma-Aldrich was then introduced into the solution in a 0.8 mL/g ratio, which initiated a methacrylation reaction kept under stirring conditions for 2 h. An equal amount of warm DPBS was added to the solution to halt the reaction. The solution was then dialyzed using a 12-14 kDa dialysis membrane (Spectrum^™ Spectra/Por^™, Fisher Scientific International Inc., Hampton, NH, USA) in deionized water at 45 ± 5 °C for seven days to remove any impurities. Following the synthesis process, the obtained solution was subjected to freezing overnight at a temperature of −80 °C. Subsequently, freeze-drying was performed for seven days (Labconco FreeZone 2.5 L lyophilizer, Labconco Corporation, Kansas City, MO, USA), resulting in a porous white foam stored at −20 °C until subsequent use.

2.2. Preparation of electrospun scaffolds

To prepare a 150 mg/mL GelMA solution, GelMA was dissolved in acetic acid (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Next, β-TCP particles (~ particle size of 100 nm) (Berkeley Advanced Biomaterials Inc., Berkeley, CA, USA) were added to the GelMA solution at 5%, 10%, or 20% (w/v). To ensure adequate particle dispersion, the solution was sonicated for 90 minutes. For crosslinking, Lithium Phenyl (2,4,6-trimethyl benzoyl) phosphinate (LAP) solution (TCI America Inc., Portland OR, USA) was added at a concentration of 0.075% (w/v). The solutions were loaded into a plastic syringe (Becton, Dickson and Company, Franklin Lakes, NJ, USA) connected to stainless steel needles (CML Supply LLC, Lexington, KY, USA.) with gauges ranging from 21-27G. The syringe was then connected to a high-voltage source (ES50P-10W/DAM, Gamma High-Voltage Research, Inc., Ormond Beach, FL, USA), and the solutions were pumped (with a flow rate ranging from 1.8-2 mL/h) over a grounded stainless steel collecting drum with a diameter of 4 cm. The scaffolds were electrospun using a high-voltage source (with voltage ranging from 20-22 KV) and collected onto a rotating mandrel (18 cm-distant) at room temperature and 18% relative humidity. The resulting scaffolds were named GelMA, GelMA+5%TCP, GelMA+10%TCP, and GelMA+20%TCP. The electrospun scaffolds were then vacuum-dried for two days, cut to the desired size, wetted with 85% Isopropyl Alcohol (v/v), gently dried using Kimwipes^™ (Kimberly-Clark Corporation, Irving, TX, USA), and crosslinked using Light Zone II (BesQual-E300N, Meta Dental Corp, Glendale, NY, USA) with 410 nm wavelength and 65W power for five minutes on each side.

2.3. Morphological, chemical, and thermal characterizations

The processed electrospun scaffolds, including uncrosslinked and crosslinked fibrous scaffolds, were morphologically assessed using scanning electron microscopy (SEM) (MIRA3, FEG-SEM, TES-CAN Brno, Kohoutovice, Czech Republic). Before imaging, the scaffolds were mounted on AI stubs and coated with Au using an SPI-Module Carbon/Sputter coater (Thermo Fisher Scientific Inc). The average fiber diameter (AFD) was determined from three images captured at the same magnification and analyzed using imageJ software (National Institutes of Health in Bethesda, MD, USA). The results are presented as mean ± standard deviation.

The attenuated total reflection mode of Fourier transform infrared spectroscopy (ATR-FTIR) was employed using a Nicolet iS50 instrument (Thermo Fisher Scientific, Inc.) to analyze the presence of specific chemical groups in GelMA and its interaction with TCP particles. 16 scans were taken between 4000 cm⁻¹ and 600 cm⁻¹ at a resolution of 4 cm⁻¹. The elemental composition of TCP-laden scaffolds was evaluated using energy-dispersive X-ray spectroscopy (EDS) with a Kratos Axis Ultra XPS instrument. The structure and phase content of GelMA, GelMA+5%TCP, GelMA+15%TCP, GelMA+20%TCP, and TCP were examined via X-ray diffraction (XRD) using Cu K (λ = 1.5406 Angstrom) in Bragg-Brentano geometry on a Rigaku Ultima IV diffractometer (Rigaku Americas Corporation, Woodlands, TX, USA). The 2-theta (2) scan ranged from 5° to 45° with 0.05° steps at a scan speed of 2 degrees min⁻¹. The thermal characteristics of GelMA, GelMA+5%TCP, GelMA+10%TCP, and GelMA+20%TCP scaffolds were examined by thermogravimetric analysis (TGA, Perkin-Elmer TGA-7, Perkin-Elmer Inc.). The samples (n=3) were heated to 900 °C at 40 °C min⁻¹ and in a nitrogen atmosphere.

2.4. Biomechanical properties

To assess the mechanical properties, the tear resistance of both uncrosslinked and crosslinked scaffolds was evaluated using uniaxial tensile testing (Expert 5601, ADMET, Inc., Norwood, MA, USA). The samples were cut into rectangular shapes measuring 25 mm × 3 mm, and the thickness was measured at three distinct positions using a Mitutoyo Digimatic Caliper (Mitutoyo Corporation, Tokyo, Japan) to obtain the average thickness for each sample analyzed. A total of four samples were analyzed per group. Testing was performed at a crosshead speed of 1 mm min⁻¹ to obtain three properties: Young’s Modulus, Elongation at break, and Tensile Strength. Load-position curves were analyzed to obtain the values reported in MPa. The tests were carried out according to standard procedures [18].

2.5. In vitro degradation and swelling capacity

The scaffolds (n=3/group) were cut into 1×1 cm² samples, and their initial weights were recorded. Subsequently, the samples were incubated in 2 mL DPBS containing collagenase type A (1U/mL - Roche Holding AG, Basel, Switzerland) at 37 °C for 28 days. At predetermined intervals, the samples were washed twice with DI water and allowed to air-dry at RT for 24 h before being weighed to obtain their dried weight. The degradation ratio of the scaffolds was calculated using the following equation (Eq. 1):

$$\text{The remaining mass}(\%)=\frac{W_{\mathrm{t}}}{W_{\mathrm{o}}}\times 100$$ (1)

Where $W_{\mathrm{t}}$ is the residual wet weight at different time points, and $W_{\mathrm{o}}$ is the initial wet weight.

Meanwhile, after soaking the scaffolds in PBS solution for 24 h, the swelling capacity was measured at different time intervals of 1, 3, 6, and 24 h using the following formula:

$$\text{Swelling Capacity}(\%)=\frac{W_{\mathrm{t}}-W_{\mathrm{o}}}{W_{\mathrm{o}}}\times 100$$ (2)

Where $W_{\mathrm{t}}$ is the wet weight after soaking, and $W_{\mathrm{o}}$ is the original weight.

2.6. Cell culture

For the in vitro experiments, we utilized human dental pulp stem cells (DPSCs) obtained from adult third molars (PT-5025, Lonza, Walkersville, MD, USA). These cells were generously provided to us by Dr. Jacques Nör at the University of Michigan and have been thoroughly characterized for their phenotypic properties by his research team [19,20]. Low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) obtained from Gibco (Grand Island, NY, USA) was utilized. The culture medium was supplemented with 10% fetal bovine serum (FBS) from Hyclone (Logan, UT, USA) and 1% penicillin-streptomycin from Sigma-Aldrich. The cells were incubated in a humidified incubator at 37 °C with 5% CO₂ . Regularly, every 2-3 days, the culture medium was replaced. For our experiments, cells from passages 4-6 were used.

2.7. Cell-scaffold interaction

Following crosslinking, scaffolds measuring 1 cm × 1 cm (n=3 per group) were disinfected under UV light for 30 minutes and placed into a 24-well plate. DPSCs at passage 6 were seeded on the scaffolds at a density of 3×10³ cells/well and cultured for 1 and 7 days to observe the cell-scaffold interaction. The scaffolds were washed with PBS and fixed with 4% Paraformaldehyde (PFA) for 30 minutes at room temperature. Samples at day 1 were stained with F-action (Invitrogen^™ ActinGreen^™ 488 ReadyProbes^™ Reagent) and DAPI (5 min), followed by imaging using a fluorescence microscope (Echo Revolve, BICO Company, San Diego, CA, USA). For samples at day 7, the scaffolds were dehydrated in ascending ethanol (70%, 80%, 90%, and 100%, 15 minutes each), incubated overnight in hexamethyldisilazane (HMDS), sputtered with gold, and imaged using SEM (MIRA3, FEG-SEM, TESCAN). Crosslinked scaffolds measuring 1.5 cm × 1.5 cm (n=4 per group) were affixed to sterile cell crowns (CellCrown^™, Scaffdex Ltd., Tampere, Finland) to evaluate cell proliferation. DPSCs at passage 6 were harvested and seeded at a density of 3 × 10³ cells/scaffold. Cell proliferation was assessed at 1, 4, and 7 days using the AlarmarBlue^™ Cell Viability Assay (Invitrogen Corporation, Carlsbad, CA, USA). In brief, 500 μL of the reagent was added to each well and incubated at 37 °C for 3 h at each time point. Subsequently, 100 μL of the component from each well was transferred in triplicate to a 96-well plate, and the fluorescence was measured using Spectra-Max iD3 microplate readers (Molecular Devices LLC, San Jose, CA, USA) with excitation at 530 nm and emission at 590 nm. After measurement, the wells were washed with PBS, and fresh media was added.

2.8. In vitro mineralization nodule quantification

DPSCs at a density of 5×10⁴ cells/well were seeded on individual scaffolds (GelMA, GelMA+5%TCP, GelMA+10%TCP, and GelMA+20%TCP) and cultured for 21 days in an osteogenic induction medium consisting of α-MEM supplemented with 15% FBS, 1% antibiotic, 10 mM β-glycerol phosphate, 100 nM dexamethasone, and 50mg/mL ascorbic acid. After that, the scaffolds were imaged using micro-CT with specific parameters (45 kV, 133 μA, and 10 μm voxel sizes) to quantify mineralization nodules [14]. Cell-free scaffolds were imaged in air, while cell-seeded scaffolds were imaged in phosphate-buffered saline (PBS). The imaging process involved collecting images at regular intervals over 360° rotation, with three images per step averaged to reduce noise. The reconstructed images were analyzed using the Scanco micro-CT software (SCANCO Medical AG, Brüttisellen, Switzerland). A global threshold was applied to detect hard tissue formation and segment the scaffolds and mineralization nodules. The quantification of mineralization nodules was obtained by subtracting the value of scaffolds without cells (as background) from the value of scaffolds with cells [14,21].

2.9. In vitro bioactivity test

The in vitro bioactivity of the fibers was assessed using simulated body fluid (SBF), prepared according to the protocol of Kokubo et al. [22]. Briefly, the SBF was made by dissolving reagent grade NaCl, NaHCO₃, KCl, K₂HPO₄·3H₂O, MgCl₂·6H₂O, CaCl₂, and Na₂SO₄ (all from Sigma Aldrich) in ultra-pure water and buffered to a pH of 7.40 with tris(hydroxymethyl)aminomethane and 1 M hydrochloric acid (Fisher Scientific Inc.) at 37 °C. Four sample groups were tested: GelMA, GelMA+5%TCP, GelMA+10%TCP, and GelMA+20%TCP; n=3 per group. Each sample was immersed in SBF for 7, 14, and 21 days without shaking in an incubator at 37 °C. After immersion, the fibers were washed with deionized water to prevent further reaction and dried in an oven at 60 °C for 24 h. The bioactivity and calcium phosphate deposition were analyzed using SEM and EDS, while HA forming ability was characterized using FTIR and XRD.

2.10. Dentinogenic gene expression RT-PCR

Quantitative polymerase chain reaction (qPCR) was employed to evaluate odontogenic gene expression at 7, 14, and 21 days. Total RNA was extracted from the collected cells using TRIzol^™ Reagent, followed by the TRIzol^™ Plus RNA Purification Kit and Phasemaker^™ Tubes Complete System (all from Invitrogen Corporation). The extracted RNA was reverse transcribed into complementary DNA (cDNA) using SuperScript^™ II Reverse Transcriptase (Invitrogen Corporation). Real-time polymerase chain reaction (RT-PCR) was performed using TaqMan^™ Gene Expression Master Mix (Thermo Fisher Scientific, Inc.) to quantify the expression levels of crucial odontogenic genes, specifically Alkaline phosphatase (ALPL, Hs01029144_m1), Runt-related transcription factor 2 (Runx2, Hs01047973_m1), Collagen alpha 1 (COL1A1, Hs0 01640 04_m1), and Dentin matrix protein1 (DMP1, Hs01009391_g1). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs02758991_g1) was used as the housekeeping gene for normalization. The 2^−ΔΔCT method was employed to determine the relative gene expression levels.

2.11. In vivo pulp capping model

Thirty-two upper first molars were used from sixteen Fischer 344 rats (Envigo RMS, USA) with similar weights (250-300 g) and age ranges. The rats were kept in a controlled environment with a regulated temperature. The study complied with the guidelines and protocols approved by the Institutional Animal Care and Use Committee (PRO00010911). Under general anesthesia achieved by using 50 mg/kg of ketamine (Hospira, USA) and 5 mg/kg of xylazine (Akorn, USA) intraperitoneally, the pulp exposure was conducted on the central portion of the occlusal surface in the upper first molars of the rats. A dental microscope (Evolution XR6, Seiler, St. Louis, Missouri, USA) was used during the dental pulp exposure with a ½ dental round burr (Kavo Kerr Group, USA). A sterile #25 endodontic file (Dentsply Maillefer, Switzerland) was used to confirm the exposure. The bleeding from exposures were controlled with sterile paper points before placing the tested materials. The rats were allocated into four groups based on the treatment applied: SHAM (pulp exposure and no treatment), GelMA, GelMA+20%TCP, and MTA (Angelus, Londrina, Brazil). Glass ionomer (Fuji II, GC, Tokyo, Japan) and temporary sealing material Coltosol (Coltene, Cuyahoga Falls, OH, USA) were used to seal the teeth of all groups. Four weeks after treatment, the rats were euthanized by inhalation of carbon dioxide, and the right and left sides of the maxilla were removed and preserved in a 10% buffered formaldehyde solution.

2.12. In vivo micro-computed tomography (micro-CT)

Scanco CT 100 (Scanco Medical AG) equipment was used to examine the hard tissue formation inside the pulp chamber, employing the following scanning parameters: 70 kV, 114 μA, and 8 μm voxel sizes. The exposure period was kept to an average of 500 ms per frame. For 3D image reconstruction, the Scanco Medical System software was employed. The 3D image was then used to trace the pulp chamber area, defined as the region of interest afterward (ROI). Each sample’s ROI was examined using the same hard tissue volume deposition threshold.

2.13. Immuno-histology analysis

Following microCT, the maxillae samples were decalcified in 10% EDTA for four weeks. Subsequently, the samples were histologically processed to be paraffin-embedded. Histological sections (5-μm thick) were stained with hematoxylin and eosin (HE) to examine soft and mineralized tissue neoformation inside the pulp chamber, using a High-Capacity Digital Pathology Slide Scanner (Aperio GT 450, Leica Biosystems, Deer Park, IL, USA) at 20×. The histologic sections of dental pulp were evaluated for inflammation and mineralization using previously established criteria and a grading system [23] as follows: Score 0 indicates no presence of inflammatory infiltrate; Score 1 indicates mild inflammation with enlarged blood vessels and infiltrates next to a dentine bridge; Score 2 indicates moderate inflammation occupying part of the coronal dental pulp; Score 3 indicates severe inflammation with infiltrate occupying the entire coronal pulp or necrosis of pulp tissue near the exposure site. The scoring system for pulp mineralization is as follows: Score 0 indicates no formation of mineralized tissue; Score 1 indicates the partial or complete formation of a dentine bridge beneath the pulp capping material at the exposure site; Score 2 indicates extensive mineralized tissue formation within the coronal pulp tissue.

The sections were dewaxed at 60 °C for 15 minutes for immunostaining and rehydrated using established ethanol gradients. To decrease endogenous peroxidase activity, the sections were incubated for 20 minutes in a 3% hydrogen peroxides solution at room temperature (RT). The slides were treated with a background snipper solution for 20 minutes at RT to block nonspecific binding. The primary antibodies used to evaluate hard tissue formation, DMP1 (PA5-47621, Invitrogen Corporation) and Nestin (19483-1-AP, Proteintech, Rosemont, IL, USA), were applied overnight at 4 °C at a dilution of 1:100. Subsequently, the sections were incubated with the corresponding secondary antibodies: Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488, Abcam) for Nestin and Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 568, Abcam) for DMP1, at a dilution of 1:200 for 2 h at RT. Cell nuclei were stained with DAPI using Vectashield antifade mounting media (Vector Laboratories, Newark, CA, USA). The images were captured at 20× magnification using an Echo Revolve microscope (BICO Company, San Diego, CA, USA). The positive immunofluorescence staining areas were quantified using ImageJ software (National Institutes of Health) from six randomly selected images per group [24].

2.14. Statistical analysis

One-way ANOVA or two-way ANOVA in multiple comparisons for parametric data and Kruskal-Wallis with Dunn’s multiple comparisons test for non-parametric data were used to determine statistical significance between the different groups using Prism 9.4 software (GraphPad Software, San Diego, CA, USA). Statistical significance was considered at p<0.05 level.

3. Results

3.1. Fiber morphology, diameter, and chemical analysis

The diameter of fibers processed via electrospinning plays a crucial factor in influencing the biological activity of cells and scaffold physiomechanical properties [25]. Smaller fiber diameters result in a larger scaffold surface area, leading to enhanced biological responses such as improved cell attachment and proliferation. Before crosslinking, we observed uniform-sized fibers and a microstructure with interconnected pores and bead-free (Fig. 1). Upon the integration of TCP, the fibers exhibited evidence of TCP particles. However, post-crosslinking, the fibers tended to merge, and their diameter increased, except for the GelMA+20%TCP group. Notably, in the uncrosslinked samples, the incorporation of 20%TCP caused an increase in fiber diameter from 0.31 ±0.09 μm to 0.64 ±0.23 μm. Conversely, after crosslinking, the incorporation of higher concentrations of TCP resulted in smaller fiber diameters.

Fig. 1.

Images showing the morphological characterization through scanning electron microscopy (SEM) and fiber diameter histogram from uncrosslinked and crosslinked GelMA, GelMA+5%TCP, GelMA+10%TCP, and GelMA+20%TCP electrospun scaffolds; average fiber diameter (AFD) = mean ± SD. Note that the fiber thickness increases after crosslinking and TCP addition for GelMA, GelMA+5%TCP, and GelMA+10%TCP, compared to uncrosslinked counterparts.

Chemical composition analysis of the engineered scaffolds was conducted using FTIR and XRD (Fig. 2). FTIR confirmed the presence of TCP in GelMA+20%TCP, indicated by an enhanced peak attributed to PO₄⁻³ (1041 cm⁻¹). Crosslinking of GelMA is indicated by the emergence of sharp peaks around 1600 cm⁻¹, corresponding to the C═O vibrations in the crosslinked amide groups. XRD spectra showed that pure GelMA had a broad hump centered around 22° in the 2θ range of 10 to 35°, suggesting an amorphous structure. However, increasing TCP concentration to 10% and 20% led to distinct sharp peaks at 2θ=30-35°, indicating the crystalline nature of TCP. Additionally, EDS analysis of GelMA+20%TCP confirmed the successful incorporation of TCP particles into the electrospun scaffold, as evidenced by peaks corresponding to calcium and phosphate.

Fig. 2.

Chemical characterization by (A) Fourier transform infrared spectroscopy (FTIR) analysis, presenting peaks around 1600 cm⁻¹ and 1041 cm⁻¹, corresponding to the crosslinked amide groups in GelMA and TCP, respectively; and (B) X-ray diffraction (XRD) pattern of TCP particles and crosslinked electrospun scaffolds (pure GelMA, GelMA+5%TCP, GelMA+10%TCP, and GelMA+20%TCP); C) Elemental energy-dispersive X-ray spectroscopy (EDS) of crosslinked GelMA+20%TCP scaffold showing 16.35% and 6.52% of calcium and phosphorus present in the sample, respectively.

Thermogravimetric analysis (Fig. 3D) was conducted to assess the thermal properties of GelMA, GelMA+5%TCP, GelMA+10%TCP, and GelMA+20%TCP. As observed in the thermograms, all scaffolds demonstrate a degradation phase around 100 °C, likely attributed to water loss. Consistent with earlier findings [26], the curves for GelMA and GelMA/TCP groups exhibited a loss of integrity and mass decrease, particularly between 350 °C and 450 °C. At 900 °C, the remaining weights for GelMA, GelMA+5%TCP, GelMA+10%TCP, and GelMA+20%TCP were approximately 15%, 19%, 21%, and 23%, respectively.

Fig. 3.

(A) Mechanical characterizations (elongation at break, modulus of elasticity, and tensile strength), (B) Swelling capacity, (C) Degradation profiles, and (D) Weight loss of the crosslinked assembled scaffolds measured by thermogravimetric analysis (TGA). Data are shown as mean ± SD. Observe the increased mechanical properties by adding βTCP and decreased swelling capacity and mass stability over 28 days compared to pure GelMA.

3.2. Scaffold mechanical properties

The mechanical properties of the fabricated scaffolds, specifically elongation at break, modulus of elasticity, and tensile strength, were evaluated under uniaxial tensile loading (Fig. 3A). In general, incorporating TCP up to 10% led to a decrease in mechanical properties. However, an increase in TCP concentration to 20% resulted in an improvement in stiffness compared to the pure GelMA scaffold. For instance, GelMA+20%TCP displayed an elastic modulus of 1.58 ± 0.16 MPa, whereas pure GelMA exhibited a modulus of 1.31 ± 0.29 MPa. Additionally, the incorporation of TCP at 20% (w/v) successfully restored the elongation at break (approximately 68%) and tensile strength (about 1.06 MPa) values to those exhibited by the pure GelMA.

3.3. Swelling capacity and biodegradability

Swelling and biodegradability are crucial for bone scaffolds, as they play a key role in promoting proper integration, enabling cell infiltration, and supporting tissue regeneration [27]. These properties ensure the scaffold can adapt to the dynamic healing process and gradually degrade as new bone tissue forms. During the initial hour, all scaffolds exhibited rapid water sorption, reaching a saturated state (Fig. 3B). Notably, GelMA+10%TCP and GelMA+20%TCP demonstrated similar swelling capacities, approximately 150%, while GelMA+5%TCP and pure GelMA showed 180% and 205%, respectively.

Regarding degradation, all electrospun scaffolds exhibited continuous mass loss over 28 days (Fig. 3C). However, the rate of biodegradation increased with the addition of TCP. For instance, on day 14, GelMA+20%TCP retained approximately 68% of its ini tial mass, while pure GelMA retained around 75%. Incorporating 5% and 10% TCP led to higher degradation after 28 days, with only 49% and 48% of the original mass remaining, respectively. Although GelMA+20%TCP slightly improved these values to 53%, it still deviated significantly from the degradation observed in pure GelMA, which retained 64% of its original mass.

3.4. In vitro bioactivity and mineralization

Osteoconductivity is another crucial characteristic of scaffolds designed to facilitate the formation of new bone [28]. To access the bioactivity and mineralization of the electrospun scaffolds, in vitro experiments were conducted using simulated bodily fluid (SBF) under static conditions (Fig. 4). FTIR spectroscopy of the SBF-soaked TCP-laden fibers revealed the presence of calcium phosphate wavelengths at 1041 cm⁻¹ and 561 cm⁻¹ (Fig. 4 A). After 14 and 21 days in SBF, a crystalline Ca-P layer was formed on the GelMA+TCP scaffolds, evident by a split in the P-O bending vibration band between 500 cm⁻¹ and 600 cm⁻¹. These peaks were also observed after 7 days of immersion, and their intensity increased over time, with GelMA+20%TCP showing a higher intensity in the P–O bending peaks after 14 days, thus indicating a faster rate of hydroxyapatite formation and enhanced bioactivity compared to the other scaffolds. The XRD pattern revealed that all TCP-incorporated scaffolds exhibited a calcium phosphate peak (~32°) even after 7 days of SBF immersion, indicating hydroxyapatite formation. The crystalline peak became more distinct after 21 days of immersion. In contrast, pure GelMA did not exhibit any peak at 32 °, suggesting the absence of calcium phosphate. The distinctive peaks also observed in GelMA included peaks near ~1600 cm⁻¹ and 1550 cm⁻¹, primarily arising from the N─H bonds, C═O bonds, and N─H bond wagging, respectively. Overall, the results indicate that the GelMA+TCP scaffolds demonstrated enhanced bioactivity and mineralization ability in SBF, as evidenced by the presence of calcium phosphate wavelengths, the development of a crystalline Ca-P layer, and the appearance of a calcium phosphate peak in the XRD pattern.

Fig. 4.

Chemical characterization of the electrospun scaffolds (pure GelMA, GelMA+5%TCP, GelMA+10%TCP, and GelMA+20%TCP) after immersion in SBF for 7, 14, and 21 days by (A) Fourier transform infrared spectroscopy (FTIR) analysis (dashed lines show peaks corresponded to the apatite formation) and (B) X-ray diffraction (XRD) pattern of crosslinked, the peak around 32° is indicating hydroxyapatite formation (mean).

SEM and EDS analysis of the SBF-immersed scaffolds revealed a significant deposition of calcium phosphate particles (Fig. 5), particularly noticeable after 14 days, with the GelMA+20%TCP scaffold exhibiting the most pronounced deposition. After 21 days of immersion in SBF, the calcium phosphate ratio (Ca/P) increased from 0.45 to 1.62 (Fig. 5B), indicating the formation of calcium-deficient hydroxyapatite [29]. The SEM images of the TCP-laden scaffolds also demonstrated the presence of hydroxyapatite-like minerals on the surface, while the pure GelMA scaffold displayed a smooth surface (data not shown). Furthermore, after 21 days of immersion in SBF, the GelMA+20%TCP scaffold formed numerous precipitated globules, which were more prominent than the other scaffolds, suggesting a tendency for the GelMA+20%TCP scaffold to form calcium-deficient hydroxyapatite. Overall, the SEM and EDS analysis provides evidence of calcium phosphate deposition, and the propensity for calcium-deficient hydroxyapatite formation on the GelMA+20%TCP scaffold. In contrast, the pure GelMA scaffold maintained a smooth surface.

Fig. 5.

Elemental energy-dispersive X-ray spectroscopy (EDS) and scanning electron microscopy (SEM) images of crosslinked electrospun scaffolds (pure GelMA, GelMA+5%TCP, GelMA+10%TCP, and GelMA+20%TCP) after immersion in SBF for 7, 14, and 21 days.

3.5. Cell-scaffold interaction

Fluorescence images showed improved cell spreading after one day on GelMA/TCP scaffolds compared to pure GelMA (Fig. 6A). In contrast to the round-shaped structures observed on the pure GelMA scaffold, the GelMA/TCP scaffolds exhibited more actin filaments and an increase in the number of nuclei, indicating enhanced cell spreading and attachment. SEM images further illustrated the interaction of DPSCs with the fibers on all electrospun scaffolds (Fig. 6B). The cells were attached to the scaffold’s fibrous structure and spread throughout its surface, indicating favorable cell-material interactions and cell adhesion. Importantly, all assembled scaffolds demonstrated a non-cytotoxic nature, as evidenced by the fact that cell proliferation increased from day 4 to day 7 (Fig. 6C). These combined findings highlight the potential of the TCP-laden electrospun scaffolds in supporting cell adhesion and proliferation.

Fig. 6.

Cell viability of DPSCs seeded on the GelMA, GelMA+5%TCP, GelMA+10%TCP, and GelMA+20%TCP electrospun scaffolds: (A) Fluorescence images of DPSCs stained using F-Actin and DAPI (nuclei) after one day; (B) SEM micrographs of DPSCs on the electrospun scaffolds after seven days, showing cell attachment and spreading; (C) Quantitative analysis of cell proliferation; and (D) in vitro bone mineralization nodules formation after twenty-one days. Data presented as mean ± SD.

3.6. In vitro mineralization nodule quantification

To investigate the mineralization ability of the proposed GelMA/TCP scaffold, we evaluated the deposition of mineralization nodules induced by DPSCs when cultured on the scaffolds. By subtracting the background (GelMA/TCP scaffolds cultured without cells), we observed a significantly greater formation of mineralization nodules in groups containing TCP than in TCP-free GelMA. Among the TCP concentrations tested, the highest values were observed in the 5% TCP (3.68 ± 0.33mm³), followed by the 10% TCP (3.12 ± 0.74mm³) and the 20% TCP (2.75 ± 0.77mm³) groups. However, no statistically significant differences were found when comparing the three TCP concentrations (Fig. 6D). These results demonstrate the potential of the GelMA/TCP scaffold in promoting DPSCs’ mineralization.

3.7. Dentinogenic gene expression RT-PCR

The expression of odontogenic genes ALPL, RUNX2, COL1A1, and DMP1 was investigated (Fig. 7). The activity of ALPL was significantly higher on days 7, 14, and 21 for the GelMA+20%TCP scaffold compared to pure GelMA, indicating enhanced osteoblast activity and suggesting an increase in mineralized tissue formation. qPCR analysis was also performed to assess the presence of RUNX2 and COL1A1. DPSCs seeded on the GelMA+20%TCP scaffold exhibited significantly higher RUNX2 and COL1A1 mRNA expression levels than those on pure GelMA scaffolds. After 21 days of culture, RUNX2 and COL1A1 expression levels were upregulated approximately two-fold in DPSCs seeded on GelMA+20%TCP scaffolds compared to pure GelMA.

Fig. 7.

ALPL, Runx2, COL1A1, and DMP1 expression of DPSCs seeded on crosslinked electrospun scaffolds (pure GelMA, GelMA+5%TCP, GelMA+10%TCP, and GelMA+20%TCP) measured by RT-qPCR after 7, 14, and 21 days. Data presented as mean ± SD.

Furthermore, the impact of GelMA/TCP scaffolds on odontogenic differentiation was examined by analyzing DMP1 expression. The expression of DMP1 significantly increased with the addition of TCP, with approximately a six-fold increase on day 14 and a twelve-fold increase on day 21 for the GelMA+20%TCP scaffold. These results show that the GelMA/TCP scaffolds are suitable for stimulating DPSCs’ odontogenic differentiation.

3.8. In vivo micro-computed tomography (micro-CT)

Based on the in vitro results described earlier, these findings underscore the importance of transitioning from in vitro assessments to in vivo methods for evaluating the potential applications and effectiveness of the GelMA/TCP scaffold. After four weeks post-implantation of the scaffolds within the pulp chamber, we evaluated the extent of hard tissue formation in the pulp using micro-CT analysis (Fig. 8A). In the sham group (negative control), where only the pulp tissue was exposed, followed by coronal sealing, minimal mineralization was observed (0.011 ± 0.007 mm³). Similarly, the pure GelMA group led to limited mineralization (0.024 ± 0.017 mm³). However, when GelMA was combined with 20% TCP (GelMA+20%TCP), a significant increase in hard tissue deposition was observed, reaching 0.057 ± 0.024 mm³. Although this value was lower than that of the clinical reference (MTA), the GelMA+20%TCP scaffold displayed promising potential for hard tissue formation.

Fig. 8.

In vivo assessment of GelMA+20%TCP membranes using a pulp capping rat model by (A) Micro-computed tomography (microCT). (B) Pulp inflammation and mineralization through hematoxylin and eosin (H&E) staining. Floating bar graphs depict the score distribution for pulp mineralization and inflammation; data are presented as minimum and maximum values, with the blue line corresponding to the median. p: pulp tissue; d: dentin; nt: necrotic tissue; sc: scaffold. Representative images to classify the degree of mineralization are H&E panels SHAM (score 0), GelMA+20%TCP-figure(2) (score 1), and MTA (score 2). Representative images for inflammation scores 0, 1, 2, and 3 are figures GelMA, GelMA+20%TCP figure (4), GelMA+20%TCP figure (1), and SHAM, respectively. (C) Immunolabelling of DMP1 and Nestin as newly differentiated odontoblast-like cell markers capable of dentin matrix secretion. The blue arrow points to new mineralized areas in microCT and histology. Bar graphs for (A) and (C) show mean ± SD of hard tissue formation in mm³ and DMP1/Nestin staining area in %.

3.9. Immuno-histology analysis

Evaluating inflammation and mineralization through hematoxylin and eosin staining is paramount in understanding dental tissue’s responses to biomaterials. Through visualizing and quantifying inflammatory cell infiltration, our observations revealed distinct patterns among the different experimental groups (Fig. 8B).

In the SHAM group, most of the samples exhibited a high inflammatory infiltrate or the presence of disorganized necrotic tissue within the pulp chamber, scoring a median of 3. Conversely, the pure GelMA and GelMA+20%TCP groups displayed similar cellular and structural tissue organization. These groups exhibited a mild inflammatory infiltrate near the coronal sealing with no signs of necrosis, resulting in a median score of 1.5. Notably, the positive control group treated with MTA, the clinical reference, demonstrated the most favorable outcomes, scoring a median of 0, indicating minimal to no inflammation after four weeks. This finding was statistically significant when compared to the SHAM group. Although our innovative GelMA+20%TCP scaffold did not elicit a lower inflammatory response than MTA, they proved suitable as pulp capping materials as they yielded similar inflammation scores. Regarding the ability of the employed treatments to induce mineralization, only GelMA+20%TCP and MTA groups were able to significantly induce regions of hard tissue formation, with the latter showing the best results, but not different from our GelMA+20%TCP scaffold.

Immunolabeling for DMP1 and Nestin was employed to evaluate mineral deposition during pulp capping procedures (Fig. 8C). We observed a significant increase in DMP1 expression in the GelMA+20%TCP group compared to the SHAM group, although not as high as the expression levels seen for MTA. These findings suggest that the scaffold effectively induces DMP1 expression. However, it is essential to note that MTA, considered the clinical reference, showed a substantial increase in protein expression compared to all other groups, indicating its superior performance. Regarding Nestin expression, we observed a mild increase in the GelMA+20%TCP group compared to the SHAM group; however, statistical analysis did not reveal any significant differences between the two groups. In contrast, MTA demonstrated a marked increase in Nestin expression compared to all other groups, further emphasizing its effectiveness as the gold standard.

4. Discussion

The current study has successfully demonstrated that our innovative GelMA nanocomposite scaffold incorporating TCP is a promising alternative to traditional pulp capping materials, substantiated by its superior handling characteristics linked to similar in vivo responses. In detail, the study investigated the morphology and nanofibrous structure of electrospun scaffolds, revealing distinct differences in fiber morphology influenced by crosslinking and the concentration of TCP. The crosslinking process significantly affected the diameter of GelMA fibers due to water absorption from isopropyl alcohol embedding, causing the filaments to merge and lose their fine morphology. As a result, the fiber diameter increased compared to the uncrosslinked groups, highlighting the crosslinking process effect in determining the diameter of the GelMA fibers [14]. Overall, incorporating TCP particles in scaffolds directly affects the conductive properties of the polymer solution, serving synergistically toward the enhancement in charge density on the surface of the electrospinning jet, which decreases self-repulsion tension and enhances elongation forces and, consequently, the formation of thinner and finer fibers [30,31]. However, the impact of viscosity on fiber diameter should be considered. For example, the uncrosslinked GelMA+20%TCP nanofibrous observed a larger diameter not only compared to its crosslinked counterpart, also in comparison with other groups, potentially due to differences in solution viscosity since it has been reported that an increase in viscosity results in a greater degree of chain entanglement and, consequently, an increase in fiber diameter [25,32].

The presence of TCP within the nanofibrous scaffold was confirmed through FTIR and XRD analyses, supporting previous findings [33]. As the fundamental form of calcium phosphates, TCP exhibited multiple peaks indicative of a crystal structure with an orthorhombic phase, as determined by indexing analysis. The XRD pattern confirms that the fabricated fibers exhibited chemical properties conducive to TCP acting as a nucleant for crystallization. Regarding the TGA, our results align with the previous one [34], indicating a slight enhancement in thermal stability attributed to incorporating TCP. Our findings collectively validate the scaffolds’ chemical composition and the successful integration of TCP particles into each TCP-laden electrospun scaffold.

As previously mentioned, scaffolds for dental pulp capping must possess suitable mechanical properties to withstand compressive forces from the surrounding soft tissues and chewing [35]. Recent research has indicated that the viability and proliferation of fibroblasts cultured on rigid scaffolds surpassed those cultured on softer ones [36]. Moreover, studies have highlighted the importance of scaffold stiffness in influencing stem cell differentiation. It has been found that a scaffold with a stiffness of at least 30 kPa can stimulate stem cell differentiation, leading to the secretion of osteocalcin and the formation of an extracellular matrix resembling bone [37,38]. In our present study, we conducted uniaxial tensile testing to assess the mechanical characteristics of the electrospun scaffolds, including tensile strength, modulus of elasticity, and elongation at break. The results of this investigation provided further confirmation that incorporating 20% TCP enhanced the mechanical properties of the scaffolds. Previous research has shown that the incorporation of TCP into scaffolds can positively influence mechanical properties due to its inherent characteristics. TCP’s interaction with physiological fluids, such as simulated body fluid, leads to the formation of calcium-deficient hydroxyapatite, which can impart improved mechanical properties to the scaffolds. Calcium-deficient hydroxyapatite contains impurities that create a composition analogous to natural bone minerals, thus contributing to the mechanical integrity and strength of the scaffolds [39].

Another crucial characteristic of a scaffold for tissue regeneration is its swelling behavior, which directly affects the absorption of body fluids, the transport of nutrients and metabolites, and the mechanical stress exerted on the surrounding tissues [40]. The GelMA scaffolds used in this study demonstrated a progressive increase in swelling during the first hour, reaching a steady state afterward. This swelling behavior is mainly attributed to the hydrophilicity of the GelMA material, further enhanced by incorporating hydrophilic TCP ceramic particles. Although the developed scaffolds exhibited similar degradation, the TCP-laden samples degraded faster. This particular degradation pattern observed in the TCP-laden scaffolds is highly beneficial, as it facilitates the gradual release of essential minerals such as Ca²⁺ and PO₄³⁻ into the surrounding environment. These minerals play a vital role in bone regeneration by providing the necessary elements for effective bone remodeling [41].

Osteoconductivity is crucial in developing bioactive scaffolds to promote new bone growth [42]. One material that stands out in this regard is TCP, which is known for its propensity to readily undergo a reaction with physiological fluids such as simulated body fluid and phosphate solution, leading to the creation of calcium-deficient hydroxyapatite, finds common application in the bone tissue engineering. Calcium-deficient hydroxyapatite contains impurities encompassing hydrogen phosphate, carbonate, chloride, and sodium ions. These layers possess a composition similar to that of natural bone minerals and facilitate adhesion to the surrounding tissues [42]. The outcomes of FTIR analyses align with earlier investigations [43,44], corroborating the presence of specific characteristic peaks. These peaks are also discernible in SEM images of scaffolds embedded with TCP, manifesting as particles of precipitated calcium carbonate. These findings correspond well with the results obtained from XRD and EDS, collectively reinforcing the confirmation of the formation of calcium-deficient hydroxyapatite.

The dental pulp is a diverse and versatile tissue known for its multipotent and heterogeneous nature [45]. A noteworthy lineage of mesenchymal-like stem cells called DPSCs exists naturally within the dental pulp. These DPSCs play a crucial role as the primary source of stem cells for regenerating pulp tissue and have the inherent ability to repair damaged tissue [46]. Consequently, for this study, DPSCs were specifically selected based on their remarkable capacity for both osteogenic and odontogenic differentiations, making them ideal candidates for tissue regeneration purposes. We observed that TCP-laden scaffolds exhibited enhanced cell spreading but similar cell proliferation compared to pure GelMA, agreeing with previous research showing no cytotoxicity when incorporating TCP into GelMA in human adipose-derived stem cells (hASCs) [47]. These findings are particularly relevant for scaffolds intended for pulp tissue applications, as they should possess the ability to facilitate mineralization and promote odontogenic differentiation of host cells upon implantation, thereby supporting healing and pulp regeneration [48]. To evaluate the odonto/osteogenic differentiation of DPSCs, we measured the expression of several genes. ALPL gene expression was assessed since alkaline phosphatase is an early marker for osteoblastic differentiation [49]. Additionally, Runx2 and Col1A1 are considered positive regulators that play a vital role in promoting the expression of bone matrix genes, such as collagen type I, osteopontin, osteocalcin, bone sialoprotein, and matrix metalloproteinase 13 [49].

Furthermore, the expression of the DMP1 gene was evaluated due to its close association with hard tissue-forming cells, particularly undifferentiated osteoblasts, since this gene regulates the mineralization process, dentin tubule formation, and the response to mechanical stress [49]. The expression of DMP1 was notably elevated in the GelMA+20%TCP group at both the 14 and 21-day time points when compared to the other concentration groups, denoting enhanced odontogenic differentiation of DPSCs, probably attributed to TCP hydrolysis, which releases Ca²⁺ and HPO₄⁻ ions [50]. Interestingly, despite observing a lower level of mineralized nodule formation in vitro within this group, no statistically significant discrepancy was observed between the three tested TCP concentrations. When we consider these findings alongside the comprehensive assessment of various in vitro gene expressions and mechanical properties, particularly elongation at break and tensile strength, it becomes evident that the 20% concentration emerges as the most promising candidate for further exploration in an in vivo model.

Based on that, the GelMA+20%TCP scaffold was successfully implanted in vivo, utilizing a model that effectively mimics the pulp capping dental procedure performed in humans [51-53]. After four weeks, comprehensive analysis through micro-CT scans and pulp mineralization histology provided compelling evidence that the GelMA+20%TCP scaffold prompted the generation of hard tissue close to the scaffold, positioned adjacent to the exposure site. Regarding the inflammatory response triggered by scaffold placement, the TCP-free GelMA and GelMA+20%TCP exhibited mild reactions with no necrosis. This observation further reinforces the adequate biocompatibility of these materials. Although the volume of deposited tissue and the inflammatory response did not surpass those of the positive control (MTA), our scaffolds demonstrated statistical comparability. Furthermore, the physical characteristics of the scaffolds present a pioneering approach for manipulating and utilizing the material in a clinical setting since the current standard of care (benchmark material), namely (MTA), necessitates laborious procedures involving multiple steps and specialized instruments for the application. Although other materials, such as Biodentine^™, have been introduced to facilitate manipulation, they also possess undesirable setting times [54].

In recent years, Nestin, a class VI intermediate filament protein, has received significant attention in the context of mineral deposition during pulp capping procedures [55]. This protein has emerged as a notable marker for undifferentiated mesenchymal and progenitor cells, indicating its crucial role in the regenerative and mineralization processes within the dental pulp [56,57]. Several studies have consistently demonstrated the participation of nestin-positive cells in forming dentin-like structures and synthesizing mineralized tissue in response to various pulp-capping materials [58]. Therefore, examining nestin expression and localization holds remarkable potential for providing valuable insights into the dental pulp’s cellular dynamics and regenerative capacity. Although our observations revealed that both GelMA+20%TCP and MTA induced noticeable nestin expression between odontoblasts within the pulp tissue, only the latter exhibited a significant difference compared to the other groups. However, it is worth noting that the visual enhancement of nestin expression associated with the GelMA+20%TCP scaffold, compared to the TCP-free, aligns with the findings from microtomography and histology analyses. These findings strongly suggest the emergence of differentiated odontoblast-like cells capable of dentin matrix secretion in response to the GelMA+20%TCP scaffold. Together, these results indicate the potential involvement of Nestin in reparative processes, particularly in the secretory activity of odontoblastic cells following dental injury and scaffold placement.

DMP1 is a vital protein associated with mineral deposition in pulp capping procedures [59]. Primarily expressed by odontoblasts, this protein plays a critical role in dentin formation and mineralization, acting as a regulator of calcium and phosphate metabolism, promoting odontoblast differentiation, and facilitating the deposition of hydroxyapatite crystals, the primary mineral constituent of dentin [60]. Experimental studies using DMP1-deficient mice have consistently demonstrated impaired dentin mineralization, highlighting the pivotal role of DMP1 in the mineralization process [61]. In this way, understanding the involvement of DMP1 in mineral deposition holds superior potential for guiding the development of innovative therapeutic strategies to enhance dentin regeneration and improve the long-term success of pulp capping procedures. Our immunolabeling analysis revealed a marked increase in DMP1 expression for GelMA, GelMA+20%TCP, and MTA treatments compared to the SHAM group. The favorable outcomes observed with GelMA alone can be attributed to the separation of pulp tissue from direct contact with the restorative sealing material, as the pure GelMA scaffold acted as a barrier. While MTA exhibited a higher overall expression, it did not display a statistically significant difference compared to the GelMA+20%TCP composite, indicating a positive correlation between DMP1 expression and new hard tissue formation. These findings highlight the potential of GelMA+20%TCP as an effective material for promoting DMP1-mediated dentin regeneration.

5. Conclusion

This study represents a significant advancement in developing GelMA scaffolds incorporating TCP for pulp capping procedures. The electrospun GelMA/TCP scaffolds demonstrated favorable mechanical properties essential for their intended application. Additionally, these scaffolds exhibited adequate biocompatibility, as evidenced by their cytocompatibility and ability to promote the formation of apatite-like structures, suggesting their potential for supporting mineralization. Furthermore, our in vivo data solidly supports the effectiveness and biocompatibility of GelMA+20%TCP scaffolds as an alternative to conventional MTA for simplified and efficient pulp capping in dental practice. These findings highlight the promising potential of GelMA/TCP scaffolds, combining desired mechanical properties with favorable biological characteristics, making them a viable option for pulp capping applications.

Statement of significance.

Vital pulp therapy (VPT) aims to preserve dental pulp vitality and avoid root canal treatment. Biomaterials that bolster mineralized tissue regeneration with ease of use are still lacking. We successfully engineered gelatin methacryloyl (GelMA) electrospun scaffolds incorporated with beta-tricalcium phosphate (TCP) for VPT. Notably, electrospun GelMA-based scaffolds containing 20% (w/v) of TCP exhibited favorable mechanical properties and degradation, cytocompatibility, and mineralization potential indicated by apatite-like structures in vitro and mineralized tissue deposition in vivo, although not surpassing those associated with the standard of care. Collectively, our innovative GelMA/TCP scaffold represents a simplified alternative to conventional pulp capping materials such as MTA and Biodentine^™ since it is a ready-to-use biomaterial, requires no setting time, and is therapeutically effective.