Pulpitis is a common infectious dental disease that impairs masticatory function and may contribute to systemic complications such as endocarditis, posing a significant public health burden (Khijmatgar et al., 2024). Pulp capping is a crucial therapeutic modality for vital pulp preservation (Duncan, 2022). The procedure aims to cover exposed or caries-adjacent pulp with bioactive materials, effectively eliminating infection, isolating irritants, and inducing the underlying pulp to generate a reparative dentin barrier (Islam et al., 2023). This procedure applies bioactive materials to exposed or caries-adjacent pulp to eliminate infection, isolate irritants, and stimulate the formation of a reparative dentin barrier (AAE Position Statement on Vital, 2021).

Human dental pulp stem cells (hDPSCs) are the key progenitor cells mediating the regeneration of the dentin-pulp complex (Sultan et al., 2025), and their differentiation capacity dictates the efficacy of regenerative therapy. Consequently, regulating hDPSC differentiation, regeneration, and immunomodulatory capacity has become a central focus in recent regenerative research (Koh et al., 2021). A key strategy for functional dentin–pulp regeneration is to integrate the essential characteristics of ideal capping materials, such as immunomodulation, enhanced repair capacity, and clinical manageability (Nie et al., 2021; Dal-Fabbro et al., 2023). This necessity has made constructing a microenvironment that is both biologically inductive and structurally stable a key area in regenerative dentistry (Hu et al., 2023).

Although modern capping materials such as bioceramics (e.g., iRoot BP Plus and MTA) can achieve clinical success rates above 80%, they still present fundamental limitations (AAE Position Statement on Vital, 2021). Their high rigidity and lack of porosity after setting critically impede the migration and organized regeneration of DPSCs (Rajasekar et al., 2025). Furthermore, the resulting dentin bridge often exhibits structural anomalies or inadequate interfacial sealing, posing a long-term threat of microleakage (Prasad Kumara et al., 2025). Therefore, the development of new materials is not aimed at simple substitution, but at overcoming these persistent clinical limitations to ensure more predictable, higher-quality regenerative outcomes in complex scenarios.

Enamel matrix derivative (EMD, Emdogain®), which is rich in amelogenin and other bioactive components (Quiroga et al., 2025), has been investigated for pulp capping due to its potent bioactivity in promoting cell differentiation and tissue regeneration during tooth development (Tsuchida and Nakayama, 2023). Ample evidence confirms that EMD effectively promotes the odontogenic differentiation of DPSCs and modulates inflammation (Sordi et al., 2022; Panahipour et al., 2022; Zhang et al., 2022a). However, its direct application is problematic due to the washout and unstable concentration associated with its liquid formulation (Fraser et al., 2022; Miron et al., 2016). Additionally, its high cost has not consistently demonstrated clear biological superiority over existing bioceramics. The major obstacle limiting the clinical translation of EMD is the absence of a delivery system that can maintain an effective local concentration within the pulp cavity and guide organized tissue regeneration (Huang et al., 2024). Although previous studies have attempted to combine EMD with various carriers such as bioceramics (Al‐Hezaimi et al., 2013), PRF (Aydemir Turkal et al., 2016), or statin-modified matrices (Karanxha et al., 2013), these systems are generally limited by the intrinsic rigidity of the materials, insufficient microstructural adaptability, or the inability to maintain a stable local concentration of bioactive proteins within the pulp cavity (Duarte et al., 2025). To date, only limited efforts have addressed the development of a biomimetic, photocrosslinkable, degradable hydrogel platform capable of achieving spatiotemporal controlled release of EMD for pulp regeneration. This work therefore fills a critical translational gap by introducing an EMD-loaded GelMA hydrogel that not only stabilizes EMD in situ but also reconstructs a biologically permissive microenvironment capable of simultaneously modulating inflammation and promoting odontogenic differentiation. These functional advantages cannot be achieved by EMD or GelMA alone.

Based on these challenges, this study proposes constructing an EMD-gelatin methacrylate (GelMA) composite scaffold system. The central rationale is to load EMD onto a biomimetic hydrogel scaffold (Kim et al., 2012) to overcome its limitations as a standalone liquid preparation. GelMA provides a stable microenvironment (Karanxha et al., 2013), which facilitates DPSC adhesion and proliferation while enabling the controlled and sustained release of EMD. We hypothesize that this composite will not only resolve EMD’s application issues but, through the synergy of bioactive signals and the physical scaffold (Yuan et al., 2023), will exhibit superior regenerative potential by inducing a more structurally complete reparative dentin compared to mineralization reliant solely on a physical barrier.

To validate this hypothesis, this study first systematically assessed the physicochemical properties of the EMD-Gel scaffold. We established iRoot BP Plus as the control in both in vitro and in vivo experiments—a comparison crucial for objectively determining the new material’s relative benefits. Using a rat pulp capping model, we monitored the short-term (4-day) inflammatory response and evaluated the long-term (28-day) quality of dentin bridge formation to comprehensively compare the material’s impact on healing against the existing standard. Finally, RNA sequencing (RNA-seq) was employed to deeply investigate the molecular mechanisms by which the scaffold regulates cellular behavior. We anticipate this collective work will provide the theoretical and experimental foundation for developing novel pulp capping strategies based on active factor delivery.

This study innovatively developed a methacrylated gelatin hydrogel scaffold loaded with enamel matrix derivative (EMD-Gel) and comprehensively evaluated its anti-inflammatory and odontogenic activities in the context of dental pulp regeneration. Both in vitro and in vivo experiments confirmed that EMD-Gel effectively regulates the inflammatory microenvironment, induces stem-cell differentiation, and promotes reparative dentin formation. Particularly in a rat pulp capping model (Li et al., 2021), the composite scaffold showed excellent biocompatibility and was able to induce the formation of a continuous and dense reparative dentin bridge, with its quality and thickness being significantly superior to the traditional BP (positive control) group (Yang et al., 2024), thereby demonstrating good potential for clinical translation. The following discussion will delve into three aspects: material design, functional validation, and molecular mechanism.

Since the bioactivity of EMD is significantly affected by its local concentration and stability (Chung and Park, 2017), this study first performed an optimal screening of the effective EMD concentration to prevent cytotoxicity caused by excessively high drug concentration or inefficacy due to low concentration (Stout et al., 2014). The results showed that EMD at 10 μg/mL significantly upregulated the expression of odontogenic differentiation-related genes such as ALP and RUNX2, whereas higher or lower concentrations led to a weakened biological effect. This finding is consistent with the dose-dependent characteristic reported in previous studies (Qu et al., 2010). After determining the effective concentration of EMD, we further screened the physicochemical ratios of GelMA to obtain a composite scaffold that offers both mechanical adaptability and cytocompatibility (Sakr et al., 2022). We uniformly incorporated 10 μg/mL EMD into GelMA solutions to construct 5%, 7.5%, and 10% EMD-Gel composite systems, which were then systematically evaluated for their morphology, mechanical properties, and biological characteristics.

The results indicated that increasing the GelMA concentration raised the compressive modulus of the scaffolds while significantly reducing their porosity and permeability (Celikkin et al., 2018). The 5% EMD-Gel system possessed a porous structure with an average pore size of approximately 86.94 ± 11.30 μm, which balanced the needs for cell adhesion, oxygen, and nutrient diffusion (Luo et al., 2025). Its elastic modulus (approximately 10³ Pa) was sufficient to maintain coronal seal stability (Tapsir et al., 2013), while also exhibiting excellent swelling and photo-crosslinking properties. CCK-8 results further confirmed that the 5% EMD-Gel group was significantly superior to the 7.5% and 10% groups in terms of DPSCs viability (P < 0.01), suggesting that a higher GelMA concentration might restrict cell migration and proliferation due to excessive crosslinking density (Sharifi et al., 2021). Compared to traditional liquid EMD preparations using propylene glycol alginate (PGA) as a vehicle (Jung et al., 2023), the EMD-Gel system not only overcame the drawbacks of insufficient physical strength and easy displacement (De Lauretis et al., 2024) but also enabled rapid curing and precise shaping via photo-crosslinking. Scanning Electron Microscopy (SEM) observation showed that the honeycomb-like porous structure of 5% EMD-Gel provided an appropriate space for DPSC adhesion and migration (Huang et al., 2025). Its degradation period of approximately 4 weeks highly matched the dental pulp repair process, preventing long-term material residue from causing tissue interference (Xiao et al., 2019). Swelling experiments and ELISA results further demonstrated that the release of amelogenin, the main component of EMD (Alp and Ulusoy, 2024), could be sustained in GelMA for 28 days, with a cumulative release rate of approximately 70%. This effectively avoided the loss of activity and local irritation caused by the short-term burst release of EMD (Pramanik et al., 2024; Wang et al., 2018). Based on a comprehensive consideration of mechanical performance, pore structure, and biocompatibility, 5% EMD-Gel was determined as the optimal formulation for subsequent functional studies.

Effective control of inflammation is a crucial prerequisite for successful pulp-capping treatment (Duncan and El‐Karim, 2024). Uncontrolled inflammation can destroy the extracellular matrix (ECM), inhibit the odontogenic differentiation of DPSCs, and even lead to irreversible necrosis (Dai et al., 2024; Shi et al., 2025). In in vitro experiments, EMD-Gel exhibited a significant odontogenic induction effect by synergistically modulating inflammation. Specifically, EMD-Gel significantly downregulated the expression of TNF-α and IL-6 (P < 0.01), while simultaneously upregulating anti-inflammatory factors such as TGF-β1 and IL-10 (P < 0.05). In contrast, the anti-inflammatory effect of EMD or GelMA alone was inferior to their composite form, suggesting a significant synergistic effect in microenvironment regulation (Zhang K. et al., 2022).

Building upon effective inflammation control, EMD-Gel further enhanced the odontogenic induction capability of DPSCs. RT-PCR and Western blot (WB) results further confirmed that the expression levels of odontogenic differentiation-related proteins such as DSPP, BSP, and RUNX2 were significantly higher in the EMD-Gel group compared to the EMD-only group and the BP control group (P < 0.01). After 21 days of mineralization induction, both ALP and Alizarin Red S (ARS) staining showed a significant increase in the area of mineralized nodules, which were 3.2 times and 5.9 times that of the control group, respectively (P < 0.01). This further validated its outstanding performance in promoting odontogenic differentiation and mineralization.

The in vivo experimental results similarly supported this conclusion and further confirmed the restorative advantages of EMD-Gel in vivo. The rat molar pulp-capping model (Yang et al., 2024) showed that in the EMD-Gel group, the inflammatory reaction was confined to the coronal pulp area at 4 days post-operation, while the radicular pulp tissue remained intact. The expression of IL-1β was significantly reduced, and DSPP was significantly upregulated (P < 0.01), indicating that the material is capable of early inflammation control and odontogenic differentiation induction (Singh et al., 2019). During the 28-day observation period, the EMD-Gel group formed a dense, continuous, and morphologically regular reparative dentin bridge (Shinde et al., 2024) with a thickness of approximately 95.3 ± 12.1 μm, which was markedly superior to the diffuse or irregular calcified layer observed in the BP group. Concurrently, inflammatory cell infiltration was significantly reduced (P < 0.05). This advantage is attributed to the dual optimization of EMD-Gel’s structure and function: its fluidity and photo-crosslinking properties ensured in situ shaping and sealing effectiveness (Tan et al., 2022), while the hydrogel microporous scaffold provided ideal biological support for cell migration, nutrient transport, and signal diffusion (Ma et al., 2022), thereby optimizing the outcome of the pulp capping repair.

At the molecular level, RNA-seq and PCR results showed that EMD-Gel regulates DPSC differentiation through coordinated modulation of the Wnt/β-catenin pathway and TNF-α–related inflammatory signaling. The mechanism is demonstrated as follows: the significant downregulation of SFRP1 relieves the inhibition on the Wnt signal, promoting the nuclear translocation of β-catenin and activating the transcription of downstream odontogenic differentiation genes (Zhao et al., 2023). Simultaneously, the upregulation of SOST and APCDD1 forms a negative feedback loop to prevent signal overactivation (Filipovich et al., 2011), thereby maintaining the homeostasis of the Wnt pathway and the orderly progression of the mineralization process. Furthermore, EMD-Gel optimized the microenvironment for stem cell differentiation by suppressing inflammatory signaling pathways (Zhang X. et al., 2022). It significantly reduced the expression of the inflammatory chemokine CCL2 and the matrix metalloproteinase MMP3. The downregulation of CCL2 lessens the recruitment of immune cells and the spread of the inflammatory cascade (Li et al., 2022), while the inhibition of MMP3 mitigates ECM degradation, stabilizing the scaffold structure required for cell adhesion and differentiation (Jabłońska-Trypuć et al., 2016). Thus, EMD-Gel provides an ideal matrix environment for the sustained activation of the Wnt signal while maintaining a state of low inflammation and low degradation.

Notably, anti-inflammatory modulation and mineralization differentiation showed a strong synergistic effect within the EMD-Gel system. Excessive inflammation typically inhibits Wnt signaling (Xu et al., 2025), compromises ECM integrity through TNF-α and IL-1β, thereby blocking stem-cell odontogenic differentiation. EMD-Gel effectively alleviated this inhibitory step by downregulating CCL2 and MMP3, allowing the Wnt signal to remain continuously active and drive DPSCs to differentiate toward the odontoblast lineage (Kornsuthisopon et al., 2024). Subsequently, the expression of Wnt downstream effector genes (such as DSPP, DMP1) promotes matrix mineralization and tissue structure stabilization (Hu et al., 2024), further attenuating inflammatory stimuli, thus achieving a positive feedback loop between anti-inflammation and differentiation. Accordingly, the repair mechanism of EMD-Gel can be summarized as follows: it promotes mineralization differentiation by downregulating SFRP1 to activate the Wnt/β-catenin pathway, while simultaneously suppressing CCL2 and MMP3 to reduce inflammation and matrix degradation. These two pathways form a synergistic regulation at the signaling and microenvironmental levels, collectively constructing a stable regenerative ecosystem favorable for dental pulp regeneration (Brizuela et al., 2022).

Compared with previous studies, this research demonstrates significant expansion and advancement in terms of material design, functional performance, and molecular mechanism elucidation. Early studies on EMD primarily focused on its pro-differentiation effect in periodontal tissues or hDPSCs. For instance, (Zhang et al., 2022a) reported that EMD could promote odontogenic differentiation of DPSCs by activating the MAPK signaling pathways. However, these studies mostly used liquid EMD formulations, which have limited sustained action. In contrast, this study introduced GelMA photo-crosslinkable hydrogel as a three-dimensional carrier (Choi et al., 2019; Zeng et al., 2025), achieving sustained release of EMD, effectively extending its active time window, and maintaining a stable microenvironment in vivo. This strategy is relatively rare in previous EMD applications.

Furthermore, compared with other studies on composite delivery systems, EMD-Gel exhibits unique advantages in balancing anti-inflammatory and odontogenic differentiation effects. For example, the EMD/β-TCP composite material constructed by Shirakata et al. (2013), while enhancing mineralization capacity, suffered from high rigidity and lacked good sealing properties. This aligns with previous findings that scaffolds containing substantial inorganic components can enhance mineralization and stiffness but may reduce flexibility and interfacial sealing performance (Nooeaid et al., 2012). In comparison, the EMD-Gel in this study possesses good plasticity and moderate mechanical strength, and establishes a balance between pro-differentiation and anti-inflammation. Although several recent bioactive hydrogel systems have begun to combine immunomodulatory or anti-inflammatory properties with pro-mineralization cues, these platforms frequently rely on inorganic or nanoparticle-based components and do not consistently outperform conventional capping materials with respect to reparative dentin bridge quality (Zhang X. et al., 2024; Zhang Y. et al., 2024; Islam et al., 2025). By contrast, the EMD-Gel system enables sustained delivery of EMD within a photocrosslinkable and degradable GelMA matrix, achieving coordinated suppression of inflammation and robust odontoblast differentiation under inflammatory conditions, and, importantly, generating a continuous and structurally superior reparative dentin bridge compared with iRoot BP Plus.

At the level of signal mechanisms, the findings of this study also expand the understanding of EMD’s mode of action. Existing research often focuses on a single pathway, such as Mrunal et al. (2024), Nokhbe et al. (2012) proposing that EMD alleviates inflammatory response by inhibiting the NF-κB pathway. However, through systematic RNA-seq analysis, this study found that EMD-Gel can synergistically regulate the Wnt/β-catenin and TNF-α signaling axes, forming a bidirectional feedback loop (Nokhbe et al., 2012). This type of signaling interaction regulation mechanism has been rarely reported, suggesting that EMD-Gel possesses unique biological advantages in the field of pulp repair and regeneration.

In summary, EMD-Gel achieves a synergistic effect of anti-inflammation and odontoblast differentiation induction by activating the Wnt/β-catenin pathway and inhibiting the CCL2-MMP3-mediated inflammatory response, thus creating a functional microenvironment conducive to the homeostatic regeneration of pulp tissue (Gong et al., 2016). In vivo, EMD-Gel demonstrated clear advantages relative to iRoot BP Plus, forming a more continuous and better-organized reparative dentin bridge and achieving more effective suppression of inflammatory infiltration, thereby indicating comparatively enhanced regenerative performance. This composite scaffold demonstrates significant advantages in physical properties, biological activity, and signal regulation, providing new theoretical basis and material foundation for vital pulp treatment and pulp regeneration. We will next investigate the temporal sequence and overall synergy among the signaling pathways. Concurrently, a systematic evaluation of its long-term application potential, spanning biosafety, functional integration, and clinical feasibility, must be performed to solidify the foundation for its clinical use in dental pulp bioregeneration.