Synergistic proanthocyanidin-copper oxygen-generating microneedle enhances anti-inflammatory activity in dental pulp stem cells and macrophage efferocytosis

Abstract

Background

Dental pulp inflammation and impaired tissue repair remain critical challenges in endodontic therapy, often exacerbated by hypoxia and dysregulated immune responses.

Methods

We developed a procyanidin-copper-loaded oxygen-generating microneedle hydrogel (CA-PCC-CAT) releasing active components into deeply infected pulp tissue in a pH-responsive manner to exert potent antioxidative and anti-inflammatory effects.

Results

In vitro experiments demonstrated that the hydrogel effectively protected hDPSCs through its multifunctions, including antibacterial activity, antioxidative stress modulation, anti-inflammatory action, and hypoxia alleviation. Simultaneously, it augmented macrophage efferocytosis by scavenging reactive oxygen species (ROS), thereby facilitating the clearance of apoptotic cells and enhancing tissue repair.

Conclusions

This study represents a promising strategy for developing multifunctional biomaterials to amplify the anti-inflammatory capacity of hDPSCs and advance the field of pulp preservation and regenerative therapy.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12903-026-07870-1.

Introduction

Oral diseases continue to seriously affect people’s health and quality of life all over the world. In the fourth national oral epidemiological survey, caries prevalence was 89% among 35–44 years old people [1]. Caries as well as mechanical and chemical stimulation usually accompany dental pulp-dentin complex injury due to the inflammation and immune reaction. These inflammatory responses lead to vascular injury, edema, and lactic acid accumulation, creating a hostile microenvironment that impairs hDPSCs-mediated regeneration, which is not conducive to the alleviation of inflammation and even ultimately lead to pulp necrosis. It is crucial to maintain vital pulp and preserve the function of pulp-dentin complex (defense, nutrition and regeneration) by taking effective measures [2]. Recently, more and more researches focus on how to promote the physiological regeneration of pulp-dentin complex such as simulating extracellular matrix, regulating physical cues [3, 4], regulating growth factors [5, 6], transplanting stem cells [7], removing ROS, inhibiting inflammatory response, at etc. Cerium oxide nanoparticles with diverse morphologies have showed significant potential in dental pulp regeneration [8]. It can be internalized into hDPSCs to exert antioxidant effects and improve cell viability under inflammatory conditions, mainly by alleviating DNA damage and lipid peroxidation and protein carbonylation. However, single inorganic nanoparticles face limitations, such as low efficiency, unstable activity and cytotoxicity [9].

Procyanidins (PCs) are natural polyhydroxyl compounds and have excellent antioxidant, antibacterial, anti-inflammatory properties. PCs can effectively scavenge excessive ROS in the inflammatory microenvironment to prevent ROS-induced DNA damage and continuously inhibit the activation of nuclear factor-κb (NF-κb) pathways through multi-target inhibition of this pathway [10, 11]. Also it plays an anti-inflammatory role by inhibiting the activity of lipoxygenase and enzymes involved in enzyme peroxidation, inhibiting ROS production and reducing mitochondrial membrane potential [12]. At present, the supramolecular amorphous network formed by the coordination interaction between polyphenols and multivalent metal ions has been used to prepare a class of polyphenol nanoparticles [13]. Because of its excellent physical chemistry properties, such as pH responsiveness and good thermal stability, it has attracted extensive attention in drug delivery and disease treatment applications. The polyphenol-metal nanoparticles can dissociate and release therapeutic drugs in the acidic environment of inflammation, which makes it a promising drug release platform [14].

Hydrogel microneedles, a cutting-edge technology in the field of drug delivery and transdermal therapy, are tiny, needle-like structures made from biocompatible hydrogel materials. As minimally invasive systems, they allow for the targeted delivery of diverse therapeutics. Their tunable physicochemical properties ensure controlled release to enhance efficacy [15]. Until now it has not been applied in the treatment of pulp diseases [16]. In the narrow pulp cavity, microneedles may have a promising application, releasing the active drugs into the deeply infected pulp tissue to adjust cell’s biology and homeostasis, and increasing the contact area to improve the efficiency of treatment.

Therefore, in this study procyanidins and copper ions were used to form a stable metal-polyphenol nanocomplex to remove ROS in the pulpitis microenvironment and inhibit inflammation. In addition, to maintain stem cell viability by addressing hypoxia in inflammatory conditions, catalase (CAT) was added to break down H₂O₂, thereby producing oxygen and alleviating hypoxic injury [17]. We hypothesized that the newly designed active substances bridge the gap between oxidative stress modulation and regenerative endodontics, offering a novel therapeutic platform for pulpitis management (scheme 1).

Scheme 1.

Mechanistic Illustration of CA-PCC-CAT MNs in Modulating Oxidative Stress and Inflammation

Materials and methods

Materials

Proanthocyanidins(PCs) were obtained from Jiuding Chemistry (Shanghai, China), copper sulfate pentahydrate (CuSO₄·5H₂O), collagen, calcium chloride, and F-127 were obtained from Energy Chemical (Shanghai, China). Sodium alginate and Tris buffer were purchased from Sigma (America). CAT, Fetal bovine serum (FBS), Dulbecco’s modified eagle medium (DMEM) and α-MEM were obtained from Gibco (America).

Synthesis of PCC nanoparticles

10 mg procyanidins were dissolved in 1 ml Tris buffer (10 mmol/L) with 0.1 g F-127, stirred for 30 min, followed by adding a 5 mg CuSO₄(pH = 8.0)and stirred for 10 h at room temperature. The excess copper ions were removed by dialysis bag (MWCO = 10 kDa) for 2 days and then freeze-dried.

Fabrication of CA-PCC-CAT microneedle hydrogels

Sodium alginate (30 mg) and cinnamaldehyde modified collagen (30 mg) were crosslinked with CaCl₂ in microneedle molds to form blank microneedle hydrogel (CA). PCC (3 mg/mL) and CAT (1 mg/mL) were incorporated for functionalization (Scheme 1).

Physicochemical characterization

Morphology analysis

Nanoparticles were characterized using Transmission Electron Microscopy (TEM, HT7800, Talos F200X). Morphology and elemental mapping of hydrogels were assessed via Scanning Electron Microscopy (SEM, GeminiSEM 500) with Energy-Dispersive Spectrometer (EDS).

Chemical composition

Fourier Transform Infrared Spectroscopy (FTIR, Nicolet iS50, China) was performed on PCC and hydrogels before and after doping with PCC and CAT, covering a range of 4000 to 600 cm⁻¹.

Antioxidant activity

To assess the radical scavenging ability of PCC nanoparticles in vitro, various concentrations of PCC nanoparticles were mixed with 3 mL of 100 µM DPPH solution and incubated at 37 °C for 30 min. Ascorbic acid served as the positive control. The absorbance at 517 nm was measured using a UV spectrophotometer.

Mechanical properties

Rheological tests conducted using a rheometer (Gemini HRnuno, UK) to examine hydrogel behavior.

Swelling test

To evaluate the swelling ratio of CA-PCC-CAT hydrogel, the hydrogel was freeze-dried, and its dry weight (WD) was recorded. The hydrogel was then immersed in PBS at 37 °C for 1, 10, 30, 60, and 120 min. After removing excess liquid, the wet weight (WW) was measured. The swelling ratio was calculated as [(WW-WD)/WD ] × 100% .

Degradation experiments

CA and CA-PCC-CAT hydrogel samples (30 mg) were placed in 50 mL centrifuge tubes with 20 mL of PBS (pH 7.4). At each time point, three hydrogel groups were collected, dried, and weighed to determine the remaining mass after degradation.

In vitro procyanidins release

CA-PCC-CAT hydrogel samples (30 mg) were suspended in 20 mL of PBS (pH 5.5 or 7.4) in a 50 mL centrifuge tube. At each time point, 2 mL of the solution was collected for procyanidins detection and replaced with fresh PBS to maintain constant conditions. Procyanidins concentrations were measured using a spectrophotometric method.

Oxygen release experiment

In order to analyze the responsiveness and oxygen production capacity of CA-PCC-CAT hydrogel to H₂O₂, the hydrogel was placed in a 50 ml centrifugal tube, suspended in 20 ml H₂O₂ solutions with different concentrations, and the dissolved oxygen concentration was determined at each time point.

Cytotoxicity

Human dental pulp stem cells (hDPSCs) was incubated in α-MEM medium supplemented with 10% FBS and 1% streptomycin/penicillin at 37 °C in a CO₂ incubator. The cytotoxicity of the samples was assessed by the CCK-8 assay (Solabol, China). Cells were seeded into 96-well plates at a proper density of 2 × 10³ cells per well and cultured for 24 h. After that, the original medium was replaced by 100µL of impregnation solution of the samples with different concentrations. Then the cell viability was quantified by the OD values at 450 nm using a microplate reader on days 1, 3 and 5, respectively. Cell viability was calculated as the following formula:

where OD is the absorbance of CA、CA-PCC and CA-PCC-CAT groups, and OD₀ is the absorbance of the control group.

Antibacterial activity

Streptococcus mutans(S.mutans)and lactobacillus casei (L.casei) as the typical bacteria of dental caries and pulpitis, were selected to characterize the antibacterial activities of CA-PCC by the plate colony-counting method. CA-PCC were cocultured with 10 µL of a bacterial solution (10⁶ CFUs/mL) for 2 h. After the coculture, the suspension was further diluted, uniformly plated, and incubated. The antibacterial rate was calculated by the following formula:

where N₀ represents the number of bacteria without any treatment, and N₁ represents the number of bacteria in the experimental group.

Detection of the biological effects of CA-PCC-CAT on hDPSCs

Intracellular ROS scavenging activity

hDPSCs cells were seeded and cocultured with 100 µL of α-MEM containing 20 µg/mL LPS for 24 h to induce the endogenous oxidative stresses. Afterward, the culture medium was replaced by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), which could be oxidized by ROS to fluorescent 2,7-dichlorofluorescein (DCF). The cells were observed under the inverted fluorescence microscope, and the fluorescence of DCF was measured by fluorescence analysis for the quantification (Ex: 488 nm, Em: 525 nm). In additionally, according to the steps of lipid peroxide (MDA) detection kit (Solabol, China) and Reduced/Oxidation Glutathione detection kit (Beytotime, China), Lipid Peroxidation and the contents of reduced/oxidized Glutathione in blank, LPS, CA, CA-PCC, CA-PCC-CAT groups were detected respectively.

Anti-DNA damage property

Induce endogenous and exogenous oxidative stress in hDPSCs. hDPSCs cells were seeded at a density of 5 × 10⁵ cells per well in confocal dishes, incubated for 24 h, then added 1 mL of α-MEM containing 20 µg/mL LPS, 200 µmol H₂O₂ and α-MEM containing samples of different concentrations for 24 h. The samples were fixed with 4% paraformaldehyde for 20 min, treated with 0.25% TritonX-100 for 15 min after PBS washing, blocked with goat serum for 20 min after PBS washing, incubated with γH₂AX antibody at 4 °C overnight, and incubated with corresponding fluorescent secondary antibody for 30 min after PBS washing, the fluorescence intensity was observed by confocal microscope (FV3000, China) after DAPI anti-fluorescence quenching tablets were used.

Scratch assay

Scratch assay was conducted to evaluate the migration ability of dental pulp stem cells in vitro. As above mentioned, hDPSCs were pretreated with different medium for 48 h, respectively. A fresh 200 µl pipette tip was employed to create a straight-line scratch across the center of each well. At different time intervals (0 h, 12 h, 24 h), the gap distance was observed under the microscope and quantitatively evaluated using Image J.

Anti-inflammatory experiment

The levels of inflammatory factors in each group after LPS induction were detected by RT-qPCR. Total RNA from hDPSCs and macrophages was extracted and reverse transcribed into cDNA following the manufacturer’s protocol. Real-time quantitative PCR was performed with SYBR Green PCR kit according to standard protocol. The denaturation, annealing and extension conditions of each polymerase chain reaction cycle were 95 ° C for 30s, 95 ° C for 5s and 60 ° C for 30s.

Western Blot (WB) analysis of P65/p-P65 levels

The protein of hDPSCs was cleaved and the concentration was measured by BCA protein assay kit (Bosterbio, China). Protein samples were collected, boiled, separated by polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (PVDF). PVDF membrane was blocked with rapid blocking solution and incubated with primary antibody (P65 and P-P65, Proteintech, China) at 4 °C overnight. TBST washed the membrane and incubated with secondary antibody (SparkJade, China) for 1 h at room temperature. Finally, the images were acquired by ECL kit (EpiZyme, China) and exposed to film.

Early odontogenic differentiation potential of hDPSCs

The osteogenic induction medium was prepared using α-MEM supplemented with 10 mM β-glycerophosphate, 50 µg/mL vitamin C, 0.1 µM dexamethasone, 10% fetal bovine serum (FBS), and 1% double antibiotics. The hDPSCs were cultured in a 6-well plate for 7 days to induce mineralization. ALP staining was performed according to the manufacturer’s instructions provided in the ALP staining kit (Beytotime, China). The stained cells were then observed and photographed under a microscope.

Detection of the biological effects of CA-PCC-CAT on macrophages

Intracellular ROS scavenging activity

The procedure follows the same protocol as previously described.

Anti-inflammatory experiment

The levels of inflammatory factors in each group after LPS induction were detected by RT-qPCR. The procedure follows the same protocol as previously described.

Immunofluorescence staining of macrophages efferocytosis

After 6 h of inducing hDPSCs apoptosis by 1 mM H₂O₂, apoptotic hDPSCs cells were stained with CellTracker Red CMTPX dye (SparkJade, China). Macrophages (RAW264.7) were incubated for 24 h, and 1 mL of DMEM containing 200 ng/mL LPS was added to induce inflammation for 24 h, then 5 × 10⁴ stained apoptotic cells and samples of different groups were added and co-cultured for 24 h. Then, it was fixed with 4% paraformaldehyde for 20 min, washed with PBS, blocked with goat serum for 20 min at room temperature, and incubated with F4/80 antibody (Bosterbio, China) overnight at 4°C. The corresponding fluorescent secondary antibody (Bosterbio, China) was added for 30 min, and the fluorescence was observed by confocal microscope after sealing the tablets with DAPI anti-fluorescence quenching blocking agent. Statistical analysis of the efferocytosis index.

Flow cytometry analysis of macrophage efferocytosis

Macrophages were incubated for 24 h, and 1 mL of DMEM containing 200 ng/mL LPS was added to induce inflammation for 24 h, then 5 × 10⁴ stained apoptotic cells and samples of different groups were added and co-cultured for 24 h. Cells were collected in 1.5 mL centrifuge tubes, centrifuged, and added to 100 µl of PBS containing 0.25 µg TruStain FcXTM PLUS (anti-mouse CD16/32) for 5–10 min to block Fc receptors. After discarding the supernatant, 100 ΜL of 0.25 µg FITC-F4/80 (anti-mouse) (Biolegend, United States) staining buffer was added, and it was incubated on ice for 1 h and washed twice in PBS. Testing on the flow cytometry.

Statistical analysis

All data were presented as mean ± standard deviation and analyzed using GraphPad Prism 8.0 for Windows. Normality of data within each group was assessed using the Shapiro-Wilk test (applied when sample size n < 50); data were considered normally distributed when p > 0.05. Homogeneity of variances across groups was evaluated with Levene’s test; variances were considered homogeneous when p > 0.05. For comparisons among multiple independent groups that met both normality and variance homogeneity assumptions, one-way analysis of variance (ANOVA) was conducted. Post hoc multiple comparisons were then selected based on the outcome of Levene’s test: if p > 0.05, Tukey’s HSD test was applied; if p < 0.05, the Games-Howell test was used. P < 0.05 was considered statistically significant.

Results

Synthesis and characterization of CA-PCC-CAT

The morphology of PCC nanoparticles were spherical, with a particle size about 224 nm. The hydrated particle size of PCC was about 164 nm and the zeta potential value was approximately − 9.03 mV (Fig. 1A, B,C, D). EDS microelement analysis revealed that copper ions and oxygen were uniformly distributed within the PCC nanoparticles (Fig. 1F). FTIR spectrometer analysis indicated the coordination of copper ions with phenolic hydroxyl groups and further confirmed the successful synthesis of PCC nanoparticles. This was evidenced by the shift of the benzene ring skeleton at 1520 cm⁻¹ and the disappearance of 1,2 - Dihydroxybenzene substitution peaks at 770 cm⁻¹.(Fig. 1E). DPPH scavenging experiments demonstrated that 3 mg/mL PCC nanoparticles completely scavenged free radicals in vitro (Fig. 1G), and this concentration was subsequently used in further experiments.

Fig. 1.

A: TEM images; (B): Particle size distribution; (C): Zeta potential; (D): Hydrodynamic size; (E): FTIR analysis of PCC nanoparticles and proanthocyanidins; (F): Elemental mapping; (G):DPPH scavenging results

The analysis of the infrared (IR) spectra confirmed the characteristic peaks of CA, CAT, and the carboxyl group in ALG has shifted, indicating the successful synthesis of the hydrogel (Fig. 2B). CA-PCC-CAT microneedles were uniformly distributed, with an average length of about 500 μm, as observed under optical microscopy and scanning electron microscopy (SEM) (Fig. 2A, D). The elements within the hydrogel were evenly distributed (Fig. 2D). The hydrogel exhibited a swelling rate of about 7–8 times, with the swelling process slowing down after loading (Fig. 2C). The rheological test indicated that the hydrogel was stable for its storage modulus was larger than the loss modulus(Fig. 2E). The force-displacement curve showed that the maximum force generated by the microneedle was approximately 2 N, sufficient to penetrate the soft tissue of the dental pulp (Fig. 2F). The oxygen release rate of CA-PCC-CAT varied in response to different concentrations of hydrogen peroxide solution (Fig. 2H). The release rate of proanthocyanidin from the hydrogel was higher at pH 5.5 than at pH 7.4, with faster release within the first 6 h, followed by a slower release rate (Fig. 2G). The cumulative proanthocyanidin release reached approximately 80% over one week (Fig. 2I). None of the hydrogel groups showed cytotoxicity toward hDPSCs on days 1, 3, and 5 (Fig. 2J).The hydrogel demonstrated strong antibacterial activity, with inhibition rates of 99.9% against Streptococcus mutans and 87.9% against Lactobacillus casei (Fig. 2K, L,M).

Fig. 2.

A: Bright-field image of CA-PCC-CAT; (B): Infrared analysis spectra of each component of the hydrogel; (C): Swelling curve; (D): SEM images and mapping images of each hydrogel group; (E): Rheological test results of CA-PCC-CAT; (F): Force-displacement curve analysis of CA-PCC-CAT; (G): Release curves of proanthocyanidins at pH 5.5 and 7.4; (H): Oxygen release curves of CA-PCC-CAT under different hydrogen peroxide concentrations; (I): Degradation curve of the hydrogel; (J): Cytotoxicity assay of hDPSCs in the leachate of each hydrogel group; (K): Antibacterial experiment of the hydrogel against Streptococcus mutans and Lactobacillus casei; (L): Statistical analysis of the antibacterial rate against Streptococcus mutans; (M): Statistical analysis of the antibacterial rate against Lactobacillus casei

Detection of the biological effects of CA-PCC-CAT on hDPSCs

CA-PCC and CA-PCC-CAT significantly reduced ROS production in hDPSCs after LPS induction (Fig. 3A, B), and CAT combined PCC nanoparticles could produce better antioxidant activity in vitro. MDA detection showed that both groups significantly lowered lipid peroxidation levels in LPS-induced hDPSCs (Fig. 3C). The ratio of reduced/oxidized glutathione in CA-PCC and CA-PCC-CAT group was higher compare to the stimulation group (Fig. 3D). Similarly, the immunofluorescence staining showed that the γH₂AX (a marker of DNA damage) red fluorescence intensity was statistically decreased in the CA-PCC and CA-PCC-CAT groups(Fig. 3E, F), indicating hydrogel could protect the cells from free radical oxidation.

Fig. 3.

A: Fluorescence staining to detect ROS levels in hDPSCs cultured in the leachate of each hydrogel group; (B): Semi-quantitative statistical analysis of ROS fluorescence staining; (C): Detection of malondialdehyde (MDA) content in each group; (D): Detection of reduced glutathione (GSH) percentage in each group; (E): Semi-quantitative statistical analysis of γH₂AX immunofluorescence staining; (F): Immunofluorescence staining to detect γH₂AX levels in hDPSCs cultured in the leachate of each hydrogel group

CA, CA-PCC and CA-PCC-CAT groups all significantly enhanced hDPSCs migration at 12 h and 24 h, with no obvious differences among the three groups(Fig. 4A, B). This suggests that the hydrogel matrix itself may play a crucial role in promoting cell migration, regardless of the additional functional components.

Fig. 4.

A: Scratch assay of hDPSCs in the leachate of each hydrogel group; (B): Semi-quantitative statistical analysis of the scratch assay; C: Detection of HIF-1α expression levels in each group; (D, E, F): Detection of IL-6, TNF-α, and IL-1β expression levels in each group; (G): Western blot (WB) analysis of P65 and P-P65 protein levels; (H): Semi-quantitative analysis of WB results; (I): Semi-quantitative analysis of ALP staining assay; (J): ALP staining assay

The expressions of HIF-1α, IL-1β, IL-6 and TNF-α induced by LPS significantly decreased in CA-PCC and CA-PCC-CAT groups (Fig. 4C, D,E, F), indicating that PCC nanoparticles could exert anti-inflammatory activity in vitro and alleviate cell hypoxia.

Following inflammatory stimulation, the CA-PCC and CA-PCC-CAT groups showed a significant reduction in P-P65/P65 levels compared to the LPS alone stimulation group (Fig. 4G, H), indicating that PCC nanoparticles may inhibit NF-κB pathway activation and subsequent inflammatory cytokine expression. In addition, the effect of CA-PCC-CAT on the odontogenic differentiation of hDPSCs after 7 days mineralization induction were detected. The elevated ALP activity observed in the CA-PCC and CA-PCC-CAT groups (Fig. 4I, J) suggests that these hydrogels promote early odontogenic differentiation of hDPSCs. This implies that the hydrogel may create a microenvironment conducive to the subsequent odontogenic differentiation of hDPSCs.

Detection of the biological effects of CA-PCC-CAT on macrophages

After LPS induction, macrophages in the CA-PCC and CA-PCC-CAT groups showed a decrease in ROS production (Fig. 5A, B) and lower expression of IL-1β, IL-6, and TNF-α (Fig. 5C, D, E). It gave a further support to the antioxidant and anti-inflammatory effects of PCC and CAT. Notably, only the CA-PCC-CAT group showed a modest but statistically significant increase in the efferocytosis index, as evidenced by enhanced co-localization of red and green fluorescent signals upon phagocytosis of apoptotic cells. (Fig. 5H, F). This enhancement was further validated by flow cytometry (Fig. 5G, I). No statistically significant difference in macrophage efferocytosis was observed among the CA, CA-PCC, and LPS groups (p > 0.05). These results indicate that although PCC nanoparticles alone exhibited notable anti-inflammatory activity, they did not directly enhance efferocytic function. This highlights the essential synergistic inclusion of catalase (CAT) to simultaneously suppress inflammation and promote the efferocytic activity of macrophages.

Fig. 5.

A: Fluorescence staining of ROS levels in macrophages cultured in the leachate of each hydrogel group;　(B): Semi-quantitative analysis of ROS fluorescence staining;　(C, D, E): Expression levels of IL-6, IL-1β, and TNF-α in macrophages cultured in the leachate of each hydrogel group;　(F): Statistical analysis of macrophage efferocytosis index by immunofluorescence staining;　G: Statistical analysis of macrophage efferocytosis index by flow cytometry;　(H): Immunofluorescence staining to detect macrophage efferocytosis levels (blue: nuclei of　macrophages; green: cell membrane of macrophages; red: apoptotic hDPSCs);　(I): Flow cytometry detection of macrophage efferocytosis levels (FITC-A: F4/80-labeled macrophages; PE-A: CellTracker-labeled apoptotic cells)

Discussion

Inflammation is a critical prerequisite for tissue healing and regeneration; however, persistent inflammation can cause irreversible tissue damage. This is particularly relevant in the dental pulp, where the rigid pulp cavity restricts swelling, inevitably leading to pulp necrosis [18]. Current clinical gold standard material for vital pulp therapy such as Mineral Trioxide Aggregate (MTA) excel in biocompatibility and sealing but offer limited active immunomodulation [19]. Our CA-PCC-CAT system is designed as a complementary strategy to address this gap. It aims to actively intervene in the early inflammatory phase by scavenging reactive oxygen species (ROS), alleviating hypoxia, and modulating the immune response, thereby creating a conducive microenvironment for subsequent reparative therapies.

Transition metal-based smart nanosystems (TMSNs) have emerged as a transformative platform in biomedical applications. They exhibit unique electronic structures, high surface area-to-volume ratios, efficient photothermal conversion, X-ray absorbency, and multifunctional catalytic activities [20]. By leveraging metal-ligand coordination chemistry, TMSNs achieve high drug-loading capacities with minimal metal ion usage, thereby reducing cytotoxicity risks while enhancing therapeutic specificity [21]. Studies indicate that the cytotoxic threshold of free Cu²⁺ to mammalian cells generally exceeds 10³ µM (>63.5 µg/mL) [22], whereas its antibacterial concentration typically ranges from 0.64 to 6.4 µg/mL [23]. To bridge this gap, we successfully synthesized proanthocyanidin-copper coordination nanoparticles (PCC NPs). In our system, copper ions are stably chelated within the PCC nanoparticles, significantly minimizing the release of free, toxic Cu²⁺ and enabling a more controlled, pH-responsive release, thus effectively reducing inherent metal ion toxicity. The design capitalizes on the negatively charged surface of PCC NPs, allowing targeted adsorption to positively charged inflamed tissues. The localized administration and stimulus-responsive release strategy employed here further minimize systemic toxicity risks. Our in vitro cytotoxicity assays (CCK-8) confirmed that human dental pulp stem cells (hDPSCs) remained viable even at high PCC concentrations (Supplementary Fig. 1). Therefore, our localized microneedle-based delivery system is expected to maintain a favorable biosafety profile at therapeutic doses. A key focus of subsequent investigations will be the detailed characterization of its precise release kinetics.

Actrurally, the design of the PCC NPs incorporates several key features for optimal function. First, their spherical morphology and diameter, which fall within the 10 to 1000 nm range reported for efficient cellular uptake—particularly by cells involved in inflammatory responses—ensure effective internalization [23]. Second, the coordination bonds, exhibiting stability between weak intermolecular forces and strong covalent bonds, provide good dynamic stability in complex biological environments. This stability, coupled with a pH-responsive release mechanism, significantly improves the bioavailability and therapeutic specificity of proanthocyanidin. Moreover, for effective clinical delivery, we integrated the PCC NPs into an alginate-based microneedle hydrogel system. This approach addresses key challenges: it avoids the generation of free radicals associated with photocrosslinking methods and facilitates penetration into soft pulp tissue. Calcium alginate was selected for its high biological safety, rapid cross-linking speed, and controllable gelation properties [24]. Future technical optimization will focus on developing specialized microneedle molds for dental pulp therapy. Specific engineering strategies include minimizing needle diameter while increasing needle length and mechanical strength to enable precise, minimally invasive, and deep drug delivery into the pulp.

Studies have shown that oxidative stress can activate the NF-κB pathway through multiple mechanisms. First, reactive oxygen species (ROS) can directly or indirectly activate the IKK complex, leading to the phosphorylation and degradation of IκB. Second, ROS may indirectly promote NF-κB activation by stimulating upstream kinases such as AKT and MAPKs. Additionally, ROS can enhance the acetylation of p65 by inhibiting the deacetylase SIRT1, thereby increasing its transcriptional activity. The activation of the NF-κB pathway further induces the expression of pro-inflammatory factors and oxidative stress-related genes, forming a positive feedback loop that exacerbates both oxidative stress and inflammatory responses [25]. Proanthocyanidins are known to exert their antioxidant and anti-inflammatory effects by modulating the NF-κB pathway. On one hand, they inhibit IKK activation, reducing IκB degradation and thereby blocking NF-κB nuclear translocation. On the other hand, proanthocyanidins suppress the phosphorylation and acetylation of p65, diminishing its transcriptional activity [12]. Our preliminary findings showed that the p-p65/p65 ratio was significantly decreased in both the CA-PCC and CA-PCC-CAT groups, suggesting that PCC nanoparticles and CAT may inhibit p65 phosphorylation, thereby suppressing NF-κB pathway activation and reducing inflammatory factor expression. Future studies will systematically investigate upstream events, including p65 nuclear translocation, IκBα degradation, and IKKα/β phosphorylation levels to further elucidate the precise regulatory effects of PCC nanoparticles on the NF-κB signaling axis [26].

In addition, pulpitis tissue is usually in a state of relative hypoxia (O₂ < 0.3%), characterized by elevated HIF-1α levels and impaired mitochondrial electron transport chain activity. Inflammatory-induced local vascular damage and tissue edema further exacerbate pulp hypoxia, leading to lactic acid accumulation and inhibition of protein synthesis, thereby establishing a vicious cycle that compensatory mechanisms are often insufficient to counteract [27, 28]. In our study, CAT converted H₂O₂ to O₂, lowering HIF-1α expression, thereby helping restore mitochondrial respiration and break the hypoxia-ROS vicious cycle. However, replicating the hypoxic microenvironment of the dental pulp cavity in vitro and accurately measuring intracellular oxygen levels remain challenging. Consequently, the direct effects of CAT on cellular behavior under hypoxic conditions have not been conclusively demonstrated. Future studies could employ advanced techniques, such as culturing hDPSCs in hypoxia chambers to simulate physiological oxygen levels and utilizing oxygen-sensitive probes to monitor intracellular oxygen dynamics [29, 30]. These approaches may provide deeper insights into CAT’s therapeutic potential and its role in hypoxia-mediated cellular responses, paving the way for innovative strategies in pulp preservation and regeneration.

Recently, the crucial role of macrophages in pulpal inflammation has garnered increasing attention [31]. Macrophages produce large amounts of inflammatory cytokines involved in mediating and modulating inflammatory responses during the initiation and progression of pulpal pathologies [32]. They are not merely passive participants but actively orchestrate inflammatory cascades by modulating immune cell crosstalk—promoting tissue repair via M2 phenotype activation or exacerbating damage through M1-driven pro-inflammatory cytokine release. Modulating macrophage function through pharmacological or immunological interventions could offer new strategies for managing pulpitis, potentially advancing the field of endodontics. Our study showed that the CA-PCC-CAT hydrogel could eliminate excessive ROS in the microenvironment and decrease IL-1β, IL-6, and TNF-α production by macrophages. This immunomodulatory strategy aligns with emerging therapeutic paradigms targeting macrophage plasticity in inflammatory diseases [33].

Emerging evidence indicates that lipopolysaccharide (LPS) enhances macrophage efferocytosis via TLR4-mediated upregulation of Tim4, a process further amplified by TLR agonist [34]. Our results support this mechanism, showing that the CA-PCC-CAT hydrogel promotes efferocytosis potentially by scavenging ROS and modulating the oxidative microenvironment. This is consistent with studies using CeO₂-nanozyme-based systems, where oxidative stress regulation increased efferocytosis efficiency by 40% [35]. Moreover, deficiency in annexin A1 (ANXA1) impairs efferocytosis, leading to the accumulation of apoptotic cells and activation of the cGAS-STING pathway, which promotes chronic inflammation and immune dysregulation [36]. By enhancing efferocytosis, our material may inhibit cGAS–STING signaling, thereby disrupting the cycle of inflammation and apoptosis. The observed reduction in inflammatory cytokines and enhancement of macrophage efferocytosis together indicate a likely phenotypic shift [37]. To precisely elucidate the immunomodulatory mechanism of our intervention, future mechanistic studies should prioritize investigating M1/M2 polarization using canonical markers, including CD86 and iNOS for M1, and CD206 and Arg-1 for M2 phenotypes.

A limitation of this study is the absence of in vivo investigations, such as pulpitis or pulp exposure models, to assess the clinical relevance of our findings. A significant gap exists between our in vitro findings and the intended clinical application in vital pulp therapy and regenerative endodontics. Therefore, future work will involve establishing standardized pulpitis models in rat or canine models to comprehensively validate the therapeutic efficacy of the CA-PCC-CAT microneedles within complex physiological environments. Studies exploring sequential or combined treatment approaches—integrating our immunomodulatory system with established bioactive materials—would be highly valuable for advancing clinical translation.

Conclusion

The designed CA-PCC-CAT hydrogel, through a microneedle-based gel delivery system, responds to the microenvironmental pH to release active substances into deeply infected dental pulp tissues. This system exerts multiple therapeutic effects, including antibacterial activity, hypoxia alleviation, antioxidant stress reduction, and anti-inflammatory action. It effectively protects dental pulp stem cells (hDPSCs). Additionally, the hydrogel enhances macrophage efferocytosis, facilitating tissue repair. This innovative approach provides a promising strategy and methodology for vital pulp therapy and pulp regeneration.

Authors’ contributions

MYT, LRR, LB and WYD contributed to the study conception and design. Material preparation and data collection were performed by MYT, WYD and LZP. Analysis was performed by MYT, YY and WYK. The first draft of the manuscript was written by MYT and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by key project of Shaanxi Provincial Natural Science Found [2022JZ-56] and Shaanxi Natural Science Foundation of China [2023-JC-YB-811].

Data availability

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

Declarations

Ethics approval and consent to participate

Ethical approval was not required for this study because the research did not involve human or animal participants.

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.