Abstract Background This study investigates the role of PDGFRβ+ dental pulp stem cells (DPSCs) in dental pulp vascular development by remodeling the extracellular matrix (ECM), with implications for angiogenesis and pulp regeneration using GelMA hydrogels. Methods PDGFRβ+ DPSCs were assessed for ECM remodeling and angiogenesis via secretion of ECM proteins (FN, LAMA4, COL1A2). Immunofluorescence and gene expression analyses were performed to evaluate ECM composition and related signaling pathways. GelMA hydrogels loaded with PDGFRβ+ DPSCs were tested for angiogenic support in vitro (HUVEC tube formation) and in vivo (subcutaneous implantation in mice for 6 weeks). Results PDGFRβ+ DPSCs enhanced ECM deposition and modulated angiogenic signaling, promoting vascular development. Encapsulation in GelMA hydrogels supported HUVEC tube formation and facilitated organized pulp-like tissue with increased ECM and angiogenesis in vivo. Integrin pathway inhibition diminished these effects, highlighting the importance of ECM-integrin signaling in angiogenesis. Conclusion PDGFRβ+ DPSCs regulate dental pulp vascular development through ECM remodeling. Their encapsulation in GelMA hydrogels provides a promising strategy for pulp regeneration by establishing an ECM-mediated angiogenic environment, offering potential for clinical pulp-dentin complex repair. Graphical abstract [42]graphic file with name 13287_2025_4382_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s13287-025-04382-7. Keywords: Dental pulp regeneration, Dental pulp stem cells, Extracellular matrix, Angiogenesis, GelMA hydrogels Introduction Dental pulp regeneration is a crucial area in restorative dentistry, aiming to restore the essential functions of the pulp tissue, including sensory detection, immune defense, and regenerative potential [[43]1, [44]2]. A key factor in successful pulp regeneration is the early establishment of vascular networks, as they are essential for nutrient delivery, waste removal, and cell survival in the regenerated tissue [[45]3]. Without adequate angiogenesis, the newly formed pulp tissue faces high risks of necrosis, undermining the overall success rate of regeneration [[46]4]. This pressing need for effective pulp regeneration solutions has driven extensive research into developing methods that can recreate a functional, vascularized pulp-dentin complex capable of sustaining long-term vitality within the tooth. While dental pulp stem cells (DPSCs) are widely studied for their regenerative potential, there remain notable challenges [[47]5]. DPSCs have shown promise in differentiating into odontoblast-like cells and promoting dentin formation; however, their application in pulp regeneration often faces limitations, such as inconsistent angiogenic support and difficulty in establishing a stable pulp-like tissue structure [[48]6–[49]8]. Advances in the use of DPSCs have underscored their potential for pulp tissue engineering, yet outcomes are often suboptimal due to limited sufficient angiogenesis in the regenerated pulp. To address these limitations, PDGFRβ+ DPSCs have emerged as a promising subpopulation, offering unique advantages in ECM deposition, cell-matrix interactions, and promotion of angiogenesis, making them particularly well-suited for supporting the angiogenesis and structural formation needed in pulp regeneration [[50]9]. The establishment of an effective angiogenesis microenvironment is heavily influenced by the presence of ECM components that interact with cellular integrins to regulate cell adhesion, migration, and survival [[51]10–[52]13]. ECM proteins such as fibronectin, laminin, and collagen provide not only structural support but also biochemical signals that are essential for vascular development [[53]14–[54]18]. ECM-integrin interactions activate intracellular signaling pathways (e.g., FAK/SRC, PI3 K, and RHO) that guide endothelial cell behavior, enabling the formation and stabilization of vascular networks [[55]19–[56]25]. Previous studies have shown that PDGFRβ+ DPSCs are capable of secreting higher levels of ECM proteins, notably fibronectin, which plays a crucial role in modulating angiogenesis [[57]9]. These ECM components create a conducive environment for endothelial cell recruitment and tube formation, highlighting the potential of PDGFRβ+ DPSCs to support the formation of a angiogenesis microenvironment [[58]9]. In this study, we aimed to leverage the regulatory role of PDGFRβ+ DPSCs in pulp vascular development to inspire a novel approach to dental pulp regeneration. By investigating the mechanisms through which PDGFRβ+ DPSCs modulate ECM composition and angiogenesis, we developed a strategy involving the encapsulation of PDGFRβ+ DPSCs within GelMA hydrogels. This approach provides a supportive ECM-rich microenvironment conducive to angiogenesis, laying a theoretical and practical foundation for clinical pulp regeneration applications. Through this investigation, we propose a biomimetic method that addresses both structural and vascular requirements for dental pulp regeneration, advancing the field toward more effective clinical treatments. Materials and methods ScRNA-seq data collection and quality control In this study, 40,266 cells, including 26,459 from mature permanent tooth (Adult Group, n = 5) and 13,807 from Young permanent teeth(Young Group, n = 2), were investigated(GEO: [59]GSE146123). The UMAP results using the same dataset have already been published [[60]16, [61]26]. The"Seurat"R package (V4.1.1) facilitated quality control and bioinformatic analysis. Elimination of doublets employed the R package DoubletFinder (3% doublet rate). Cells failing quality criteria (UMIs < 500 or > 5000, mitochondrial gene count proportion > 15%) were filtered out. Subsequent preprocessing involved global scaling normalization ("Log-Normalize") and log transformation. Merged normalized expression profiles used the “merge” function in R v3.6.3. Harmony (v0.1.0) integrated datasets to mitigate technical effects, identifying shared cell states. Identification of signature genes for cell clusters The DEGs in each subcluster were identified using the “FindAllMarkers” function in Seurat. The significance levels of these signature genes were determined using the Wilcoxon rank-sum test and Bonferroni correction. The signature genes of each cluster were determined using the following criteria: (1) expressed in more than 20% of the cells within either or both two groups; (2) |log[2]FC | > 0.5; and (3) Wilcoxon rank-sum test adjusted P-value < 0.01 Pathway enrichment analysis An analysis of Gene Ontology (GO) was performed using the “clusterProfiler” R package v4.0.2 to investigate the potential functions of different cell types [[62]27]. Pathways with P_adj-values < 0.05 were considered significantly enriched. The mean gene expression of each cell type was included as input data using the gene set variation analysis (GSVA) package v1.34.0 for the GSVA and the pathway enrichment analysis [[63]28]. Cell-cell communication analysis with Cellchat Cell-cell communication was analyzed and visualized using CellChat v1.1.0 (github.com/sqjin/CellChat) [[64]29]. The cell type labels were derived from the Harmony integration results using all scRNA-seq data sources. The default values were used for each step parameterization. Tooth sample The premolars(at Nolla stage 8) of 9 to 10-year-old children were extracted due to orthodontic treatment. The healthy mature premolars(at Nolla stage 10) of 18-year-old patients were extracted due to orthodontic treatment. Tooth mobility was checked clinically. Moreover, the history of drug allergy, systemic disease, and family genetic disease were excluded. After tooth extraction, the teeth were categorized into Adult and Young pulp groups. Finally, the number of teeth at each stage of the study was analyzed histologically as follows: A(at Nolla stage 8): 10, Y(at Nolla stage 10): 10. Histological examination HE staining followed the protocol of the HE staining kit (Solarbio, G1120&G1340). Immunofluorescence staining quantified protein expression in tooth pulp samples using primary antibodies: anti-CD31 (Abcam, ab28364), anti-FN (Abcam, ab2413), anti-LAMA4 (Abcam, ab242198), anti-COL1 A2 (Abcam, ab96723), and anti-PDGFR beta (Abcam, ab313777) at 1:200 for 2 hours. Secondary antibody (Goat Anti-Rabbit IgG H&L, Alexa Fluor® 555, Abcam, USA) was used at 1:200 for 1 hour. Slides were mounted with DAPI (Vector Laboratories, H1200, 10 μg/ml) for imaging. Images were captured and the mean optical densities were analyzed using ImageJ. Cell culture Tooth samples were sterilized, split, and pulp was extracted, washed with PBS, and cut into 1 mm^3 pieces. Tissues were digested with collagenase type I (Sigma) at 37 °C for 60 minutes, centrifuged, and re-suspended in α-MEM medium (Gibco). Cells were seeded in six-well plates and incubated at 37 °C with 5% CO₂. Upon reaching 80% confluence, cells were trypsinized (Sigma) for passage. First-generation cells were diluted to 10–15 cells/mL in α-MEM, pipetted into 96-well plates, and incubated. Single-cell wells were labeled, supplemented with medium, and incubated until cell clones occupied half the well bottom. Monoclonal cells were then expanded in six-well plates. HUVECs were cultured in endothelial cell medium (Procell, CP-H082) under 5% CO₂ at 37 °C. Flow Cytometry-based isolation of PDGFRβ+ DPSCs Single-cell suspensions were obtained from actively growing P1 generation DPSCs through routine trypsin digestion. Firstly, DPSCs were collected from culture dishes and digested using 0.25% trypsin to obtain a single-cell suspension. The cells were then centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. This step was repeated twice with PBS to ensure thorough washing. Next, the cells were divided into three groups: a blank control, an isotype control, and a PDGFRβ staining group. Each group was treated with 1% BSA solution to block non-specific binding and incubated in the dark at 4 °C for 10 minutes. The PDGFRβ staining group received the PDGFRβ antibody (diluted 1:100), while the isotype control group received a corresponding isotype control antibody, both incubated in the dark at 4 °C for 15 minutes. Following incubation, the cells were washed twice with PBS and centrifuged at 1000 rpm for 5 minutes. The final cell suspension was analyzed and sorted using a flow cytometer (Beckman Coulter, USA) set to detect FITC at 488 nm. Transcriptome sequencing data analysis The mRNA from PDGFRβ+ DPSCs and P1-DPSCs was extracted, with six samples in each group. RNA concentration and purity were determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). The integrity of RNA samples was evaluated with an Agilent 2100 Bioanalyzer and 2100 RNA Nano 6000 assay kit (Agilent Technologies). After quality control, poly-A RNA was enriched from eukaryotic total RNA using the TIANSeq mRNA Capture Kit (TIANGEN). Library concentration was initially measured with a Qubit 2.0 fluorometer (Life Technologies) and diluted to 1 ng/µl for insert size checking on an Agilent 2100. Accurate quantification was performed by Q-PCR (library activity >2 nM). For biological replicates, DESeq2 R package (1.16.1) was used, employing a negative binomial distribution model and Benjamini-Hochberg correction for false discovery rate (FDR). Genes with adjusted P-values <0.05 were considered differentially expressed. For datasets without biological replicates, edgeR R package (3.18.1) was used, with P-values adjusted by the Benjamini-Hochberg method. Thresholds for significance were set at a corrected P-value <0.05 and a fold change >2. GO enrichment analysis was performed using the topGO R package, correcting for gene length bias, with terms considered significant at corrected P-values <0.05. KEGG pathway enrichment was tested using the clusterProfiler R package, analyzing the statistical enrichment of differential expression genes in KEGG pathways. Gene Set Enrichment Analysis (GSEA) is used to complement the understanding of how subtle but crucial changes impact biological pathways ([65]www.broad.mit.edu/gsea/). Western blot detection DPSCs were lysed in a buffer (10 mM Tris-HCL, 1 mM EDTA, 1% SDS, 1% Nonidet P-40, 1:100 proteinase inhibitor cocktail, 50 mM β-glycerophosphate, 50 mM sodium fluoride). The lysate was sonicated (4 W, 5 s on ice), ice-bathed for 5 min, centrifuged at 25,000 rpm for 10 min at 4 °C, and the supernatant collected for total protein. Protein concentration was measured with a BCA Protein Assay Kit (Beyotime, China). Equal protein amounts were loaded onto 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore), and incubated overnight at 4 °C with primary antibodies (anti-FN, anti-LAMA4, anti-COL1 A2, anti-GAPDH, all Abcam, 1:500). After washing with TBST, membranes were incubated with a secondary antibody (Goat Anti-Rabbit IgG H&L, Alexa Fluor® 555, Abcam, 1:2000) for 1 hour at room temperature. Bands were detected using ECL and analyzed with ImageJ. The experiment was repeated three times. Tube formation assay CellMask Green (Thermo Fisher, USA) was added to the HUVECs culture medium according to the manufacturer’s instructions, ensuring the cells was effectively labeled, resulting in green fluorescence. After incubating for 24 hours to allow stable labeling, the fluorescence-labeled HUVECs (2×10^4 cells/well) were seeded onto GelMA hydrogels loaded with DPSCs. The GelMA hydrogels served as a 3D matrix, providing structural support for cell growth. After 12 hours of incubation, tube formation was observed under an inverted fluorescence microscope. The total tube length was quantified in five randomly selected fields per well using ImageJ software to assess the impact of DPSCs on HUVEC-mediated angiogenesis. In vivo validation of the role of PDGFRβ+ DPSC in angiogenesis and ECM formation The experimental protocol was approved by the Institutional Review Board (IRB) of the Fourth Military Medical University (IACUC-20241397). PDGFRβ-CreERT2 and ROSA-DTA mice, were bred and housed under SPF conditions with a 12-hour light/dark cycle. Conditional knockout of PDGFRβ+ cells was achieved by creating double-transgenic PDGFRβ-CreERT2 and ROSA-DTA mice. Cre activity was induced using tamoxifen corn oil solution (administered via intraperitoneal injection at 100 μg/g body weight for three consecutive days in 4-week-old mice). The mice were divided into three groups: young permanent teeth control group (4 weeks old, 10 mice), mature permanent teeth control group (8 weeks old, 10 mice), tamoxifen-treated group (8 weeks old, 10 mice). The sample size was decided based on our previous research [[66]9]. A random number table was used to randomly group animals. Mandibular molar samples were collected at different developmental stages for histological analysis. Investigators were not blinded to the animal groups. Mice mice were euthanized by cervical dislocation following isoflurane anesthesia (R510-22-10; RWD), and the oral cavity was flushed with saline. Mandibular molars were carefully dissected using a scalpel. The collected mandibular molar samples were fixed, dehydrated, infiltrated, and embedded in paraffin. Tissue sections were prepared using a rotary microtome. Antigen retrieval was performed using heat induction, followed by serum blocking and antibody incubation. Fluorescence microscopy was used to observe and capture images of the samples. The work has been reported in line with the ARRIVE guidelines 2.0. Scaffold-free 3D culture of DPSCs using rotational suspension To culture DPSC spheroids in a scaffold-free 3D environment, we employed a rotational suspension method optimized for stable spheroid formation over a 40-day period. DPSCs were first prepared as a cell suspension at a concentration of 1×10^6 cells/mL to ensure consistent cell density. The cell suspension was then transferred to 100 mL or 250 mL rotary culture bottles, with the culture medium filling one-third to one-half of the bottle volume to maintain adequate nutrient and gas exchange. Bottles were placed in a 37 °C, 5% CO₂ incubator, and the rotation speed was set to 30–40 rpm to keep cells in suspension, promoting aggregation without imposing excessive shear stress that could damage the cells. The medium was refreshed every 2–3 days to remove metabolic waste and supply fresh nutrients, with careful handling to avoid disruption of the forming spheroids. Spheroid formation and growth were monitored periodically under a microscope to observe changes in size, morphology, and cellular organization. At the end of the 40-day culture period, spheroids were collected for subsequent analyses, such as viability assays, ECM secretion, and gene expression profiling, to evaluate their 3D culture characteristics and functionality. This rotational suspension method supports the formation of well-organized DPSC spheroids, suitable for applications in tissue engineering and regenerative medicine research. Real-time polymerase chain reaction (RT-PCR) Trizol (Invitrogen, USA) was used to extract the total RNA of DDPSCs and DPSCs in each group of the fourth generation, and cDNA was synthesized using PrimeScript™ RT-PCR kit (Takara, China). Furthermore, reverse transcription was conducted according to the instructions. The cDNA template obtained by reverse transcription was added to the detection system according to the RT-PCR reagent operating instructions, with cDNA as a template and GAPDH as an internal reference. The ABI7500 fluorescence RT-qPCR system (Applied Biosystems, Germany) was used to detect FN1. The reaction conditions for OPG expression were as follows: pre-denaturation at 95 ℃ for 30 s, one cycle; 95 ℃ (denaturation) for 5 s; and 60 ℃ (annealing and extension) for 30 s, 40 cycles. The experiment was repeated three times. The primer sequences are listed in Supplementary File1. Encapsulation of DPSCs in GelMA Hydrogels To encapsulate DPSCs in GelMA hydrogels, we prepared the GelMA solution by dissolving the powdered GelMA in a 0.25% (w/v) LAP photoinitiator solution. The GelMA concentration was adjusted to 30% (w/v), depending on the desired mechanical properties. The solution was heated in a 60–70 °C water bath for 20–30 minutes with periodic mixing to ensure full dissolution. After dissolving, the solution was centrifuged at 3000 rpm for 2 minutes to remove air bubbles and then sterilized using a 0.22 μm filter. DPSCs were collected and resuspended in the pre-warmed GelMA solution to create a uniform cell suspension. This suspension was then pipetted into a 96-well plate (50–100 μL per well) or other culture plates as required by the experimental design. The GelMA-cell mixture was crosslinked by exposing it to 405 nm light for 10–30 seconds, adjusting the exposure time to control gel stiffness. Following gelation, warm culture medium was added to each well to cover the hydrogel, and the plate was incubated at 37 °C. The medium was refreshed every 2–3 days, and the cultures were maintained for the designated experimental period. The morphology of the obtained hydrogels was observed using a scanning electron microscopy (SEM, HITACHI) at an operating voltage of 5–10 kV, and the porosity, average pore size and distribution range of the pore sizes were analyzed by ImageJ (National Institutes of Health, USA). Live/dead cell staining of DPSCs encapsulated in GelMA Hydrogels To assess the viability of DPSCs encapsulated in GelMA hydrogels, a live/dead cell staining assay was performed using Calcein-AM/PI dual staining(Solarbio, CA1630). First, the 10× Assay Buffer was diluted to 1× with deionized water. DPSCs encapsulated in GelMA hydrogels were washed twice with the 1× Assay Buffer to remove any residual esterase. For staining, Calcein-AM was added at a final concentration of 1–2 μM, and the samples were incubated at 37 °C in the dark for 20–25 minutes to label live cells with green fluorescence. Following Calcein-AM staining, PI was added to the samples at a final concentration of 5 μM to label dead cells with red fluorescence. The samples were incubated at room temperature in the dark for an additional 5 minutes. After staining, the samples were washed with 1× PBS to remove excess dye. Imaging was conducted immediately under a fluorescence microscope using 490 nm excitation to visualize both live (green fluorescence) and dead (red fluorescence) cells. Immunofluorescence staining of ECM Components in GelMA Hydrogels To analyze ECM component deposition (FN, LAMA4, and COL1 A2) by DPSCs in GelMA hydrogels, immunofluorescence staining was performed. First, GelMA hydrogels containing DPSCs were washed with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde for 15 minutes at room temperature. After fixation, the samples were washed three times with PBS to remove residual fixative. The hydrogels were then permeabilized using 0.1% Triton X-100 in PBS for 10 minutes to facilitate antibody penetration. Following permeabilization, the samples were blocked with 5% bovine serum albumin (BSA) in PBS for 1 hour at room temperature to prevent nonspecific binding. For primary antibody incubation, the samples were incubated overnight at 4 °C with specific antibodies against fibronectin (FN), laminin subunit alpha-4 (LAMA4), and collagen type I alpha-2 (COL1 A2), each diluted in 1% BSA in PBS according to the manufacturer’s instructions. After incubation, the hydrogels were washed three times with PBS to remove unbound primary antibodies. The samples were then incubated with fluorescently labeled secondary antibodies diluted in 1% BSA in PBS for 1 hour at room temperature in the dark. After secondary antibody incubation, the hydrogels were washed three times with PBS to remove excess antibody. Finally, the samples were counterstained with DAPI to visualize cell nuclei and mounted on glass slides for imaging. Fluorescence images were captured using a confocal microscope to observe the localization and intensity of ECM components within the GelMA hydrogels. Dental pulp regeneration animal experiment The experimental protocol was approved by the Institutional Review Board (IRB) of the Fourth Military Medical University (IACUC-20241397). To evaluate the potential of PDGFRβ+ DPSCs in promoting dental pulp regeneration, GelMA hydrogels encapsulating PDGFRβ+ DPSCs were prepared and transplanted into immunocompromised mice. A random number table was used to randomly group animals. Investigators were not blinded to the animal groups, but were blind to the implants. Hydrogel samples, including GelMA alone, GelMA loaded with P1-DPSCs, GelMA loaded with PDGFRβ+ DPSCs, and GelMA loaded with PDGFRβ+ DPSCs combined with an integrin receptor inhibitor, were seeded into dentin matrix blocks that had been prepared as scaffolds. These dentin matrix blocks were obtained from teeth and treated by demineralizing with gradually reduced EDTA concentrations (from 17 to 5%) for 10 minutes, followed by thorough washing with deionized water. The treated dentin matrix blocks were stored in PBS with penicillin (50 U/mL) and streptomycin (50 mg/mL) at 4 °C until use. For each group, the GelMA hydrogel was loaded into the treated dentin matrix canals, with one end sealed using iRoot BP Plus (Innovative BioCeramix Inc., Canada). These constructs were then subcutaneously implanted into the left and right dorsal regions of 6-week-old Balb/c nude mice. Mice were housed in specific pathogen-free facilities under standardized conditions throughout the experiment. After 6 weeks of transplantation, mice were euthanized by cervical dislocation following isoflurane anesthesia (R510-22-10; RWD), the samples were harvested, fixed in 4% paraformaldehyde, and embedded in paraffin for histological analysis. Hematoxylin and eosin (H&E) staining and immunofluorescence staining for ECM proteins and vascular markers were performed to evaluate tissue regeneration and angiogenesis within the regenerated pulp-dentin complex. The work has been reported in line with the ARRIVE guidelines 2.0. Statistical analysis Single-cell sequencing data were analyzed using R (V.3.62). Continuous variables (mean ± SD) were compared using the Wilcoxon test, while categorical data (%) were compared using χ^2-test or Fisher’s exact test. A two-sided P < 0.05 was deemed statistically significant. SPSS 22.0 software was used for further data analysis. Differences between groups were assessed using one-way ANOVA, with the Tukey test for pairwise comparisons. A P < 0.05 indicated statistical significance. Results Cellular definition and ECM remodeling by DPSCs in dental pulp development Analysis of single-cell sequencing data allowed us to categorize various cell types within dental pulp samples, with a focus on defining the roles of dental pulp stem cells (DPSCs) during different stages of pulp development (Fig. [67]1A). This data-driven approach highlighted the significant involvement of DPSCs in both early developmental and mature phases of pulp tissue, consistent with their recognized role in dental pulp homeostasis and vascular formation. Our investigation into cell-cell communication showed that interactions between DPSCs and other cell types in developing pulp tissues were primarily mediated by extracellular matrix (ECM) components and integrin receptors (Fig. [68]1B). This ECM-integrin interaction suggests that DPSCs contribute to the structural organization necessary for supporting vascular growth and cellular communication, aligning with findings that emphasize the importance of fibronectin (FN1) and integrin interactions in promoting angiogenesis. Finally, Fig. [69]1C displays GO analysis results indicating a high expression of ECM-related genes in DPSCs during pulp development (including extracellular matrix organization, extracellular structure organization and external encapsulating structure organization etal). These genes are critical for ECM remodeling, a process that facilitates cell adhesion and migration, supporting DPSC functions in establishing a vascular network essential for regenerative applications. These results collectively underscore the critical role of PDGFRβ+ DPSCs in ECM remodeling and their potential to initiate angiogenesis within the pulp tissue, a foundation for advancing pulp regeneration strategies. Fig. 1. [70]Fig. 1 [71]Open in a new tab Cellular Definition and ECM Remodeling by DPSCs in Dental Pulp Development. A: Cell type definition in dental pulp by single-cell sequencing. B: ECM-integrin interactions in DPSC communication. C: High ECM gene expression in DPSCs during pulp development Comparative immunofluorescence analysis of ECM components in developing and mature permanent dental Pulp In Fig. [72]2, we analyzed the expression of key extracellular matrix (ECM) components, including fibronectin (FN1), laminin subunit alpha-4 (LAMA4), and collagen type I alpha 2 (COL1 A2), through immunofluorescence staining in clinically collected samples of developing and mature permanent dental pulp. The results showed a significantly higher expression of FN1 (Fig. [73]2A, D), LAMA4 (Fig. [74]2B, E), and COL1 A2 (Fig. [75]2C, F) in the developing dental pulp samples compared to mature pulp tissues. These findings suggest that during the developmental phase, there is an active ECM remodeling process, characterized by increased deposition of structural proteins such as fibronectin, laminin, and collagen, which are essential for supporting cellular adhesion, migration, and potential angiogenesis. This high ECM component expression aligns with the need for structural integrity and signaling during early tissue organization, potentially contributing to the developmental environment that supports DPSC activity and dental pulp angiogenesis. Fig. 2. [76]Fig. 2 [77]Open in a new tab Comparative Immunofluorescence Analysis of ECM Components in Developing and Mature Permanent Dental Pulp. A–C: Immunofluorescence staining of CD31 (green) and FN/LAMA4/COL1 A2 (red) in mature and young permanent dental pulp tissues. B–F: Average fluorescence intensity(AFI) analysis of FN1, LAMA4, and COL1 A2 immunofluorescence results (*P < 0.05, **P < 0.01, ***P < 0.001) Role of PDGFRβ+ DPSCs in establishing an ECM-based angiogenesis microenvironment Extracellular matrix (ECM) components are essential for forming an angiogenesis microenvironment, as indicated by our findings that ECM-related elements in developing permanent dental pulp may support angiogenesis. DPSCs, known to regulate this angiogenic environment, are central to this process. In previous studies, we successfully identified and isolated PDGFRβ+ DPSCs via flow cytometry and confirmed their significant role in dental pulp angiogenesis in both in vitro and in vivo models. To further investigate the involvement of PDGFRβ+ DPSCs in establishing a angiogenesis microenvironment in developing pulp, we compared the gene expression profiles of PDGFRβ+ DPSCs with P1-DPSCs (primary DPSCs at first passage) using transcriptomic sequencing. GO analysis indicated that genes highly expressed in PDGFRβ+ DPSCs were notably enriched in ECM-related functions, such as collagen fibril organization, ECM structural integrity, collagen and ECM binding, and structural resilience (e.g., collagen-containing ECM, fibrillar collagen trimer) (Fig. [78]3A, D). Additionally, GSEA analysis showed that PDGFRβ+ DPSCs had elevated expression in pathways associated with angiogenesis and ECM regulation (Fig. [79]3B, C). These findings underscore that PDGFRβ+ DPSCs are key cells in constructing an ECM-based angiogenesis microenvironment, potentially facilitating dental pulp angiogenesis during development. Fig 3. [80]Fig 3 [81]Open in a new tab Role of PDGFRβ+ DPSCs in Establishing an ECM-Based Angiogenesis Microenvironment. A: GO enrichment analysis of highly expressed genesin PDGFRβ+ DPSCs (bubble Chart). B, C: GSEA Enrichment Plot for angiogenesis and ECM regulation. D: GO enrichment analysis of highly expressed genesin PDGFRβ+ DPSCs (chordal graph) Validation of PDGFRβ+ DPSCs in ECM formation As noted previously, stem cells, particularly DPSCs, play a crucial role in ECM formation, which supports tissue structure and facilitates angiogenesis by binding and storing growth factors. This ECM serves as both a structural and signaling scaffold critical for vascular development​. We further validated the role of PDGFRβ+ DPSCs in ECM formation through in vitro analysis. Western blot results demonstrated significantly elevated expression of fibronectin (FN), laminin subunit alpha-4 (LAMA4), and collagen type I alpha-2 (COL1 A2) in PDGFRβ+ DPSCs compared to primary passage DPSCs (P1-DPSCs) (Fig. [82]4A–D). Additionally, scaffold-free 3D culture enabled PDGFRβ+ DPSCs to self-assemble into spheroids, closely mimicking an in vivo environment and promoting cell interaction and ECM deposition. Results showed that FN, LAMA4, and COL1 A2 expression was significantly higher in these 3D spheroids, underscoring the critical role of PDGFRβ+ DPSCs in establishing an ECM-based angiogenesis microenvironment (Fig. [83]4E-F). Fig. 4. [84]Fig. 4 [85]Open in a new tab Validation of PDGFRβ+ DPSCs in ECM Formation. A–D: Western blot was used to detect FN, LAMA4, and COL1 A2 expression in P1-DPSCs and PDGFRβ+ DPSCs(Uncropped western blottings for Supplementary File2). E, F: scaffold-free 3D culture was used to detect FN, LAMA4, and COL1 A2 expression in P1-DPSCs and PDGFRβ+ DPSCs (*P < 0.05, **P < 0.01, ***P < 0.001) Animal experiments demonstrated the effects of PDGFRβ+DPSCs on ECM formation To further elucidate the role of PDGFRβ+ cells in dental pulp vascular development, PDGFRβ-CreER^T2/ROSA-DTA double-transgenic mice were successfully constructed for the conditional ablation of PDGFRβ+ cells. By injecting tamoxifen, Cre recombinase was induced to enter the nucleus, promoting the transcription and translation of diphtheria toxin A subunit (DTA) in cells expressing PDGFRβ, thereby achieving specific conditional ablation of PDGFRβ+ cells (Fig. [86]5A). The mice were divided into three groups: young permanent teeth control group (4 weeks old), mature permanent teeth control group (8 weeks old), and tamoxifen-treated group (8 weeks old). Immunofluorescence analysis revealed that in the tamoxifen-treated group, the dental pulp tissue exhibited significantly reduced expression of ECM proteins FN, LAMA4, and COL1 A2, as well as a notable decrease in vascular density (Fig. [87]5B–F). These in vivo findings reinforce the importance of PDGFRβ+ cells in ECM composition and angiogenesis, supporting their critical role in establishing a functional angiogenesis microenvironment within developing dental pulp. Fig. 5. [88]Fig. 5 [89]Open in a new tab Animal experiments demonstrated the effects of PDGFRβ+DPSCs on ECM Formation. A: PDGFRβ-CreER^T2/ROSA-DTA double-transgenic mice were successfully constructed for the conditional ablation of PDGFRβ+ cells. B–D: AFI of immunofluorescence staining of FN, LAMA4, and COL1 A2. E: Immunofluorescence staining of CD31 (red) and PDGFR (green) in dental pulp tissues. F: Immunofluorescence staining of FN, LAMA4, and COL1 A2 (red) and CD31 (green) in dental pulp tissues (*P < 0.05, **P < 0.01, ***P < 0.001) Evaluation of GelMA Hydrogels for encapsulating PDGFRβ+ DPSCs GelMA (methacrylated gelatin) is a widely studied hydrogel with excellent biocompatibility and biodegradability, promoting cell adhesion and proliferation, making it suitable for stem cell encapsulation in clinical applications. We explored the use of GelMA to encapsulate PDGFRβ+ DPSCs, assessing cell viability and performance to support the potential clinical application of PDGFRβ+ DPSCs in dental pulp regeneration (Fig. [90]6A). Fig. 6. [91]Fig. 6 [92]Open in a new tab Evaluation of GelMA Hydrogels for Encapsulating PDGFRβ+ DPSCs. A: Schematic diagram of GelMA encapsulated PDGFR β+ DPSCs experimental protocol. B, C: SEM images of GelMA. D, E: Live (green)/dead (red) staining and cell viability analysis of DPSCs in GelMA (*P < 0.05, **P < 0.01, ***P < 0.001) To determine the optimal degree of methacrylation, we compared GelMA hydrogels with 30%, 60%, and 90% methacrylation levels (Fig. [93]6B, [94]C). SEM images demonstrated distinct structural differences across the GelMA formulations, with lower methacrylation levels providing a more porous network conducive to cell interactions (Fig. [95]6B, [96]C). To identify the best crosslinking density for supporting DPSC viability, we encapsulated DPSCs in each hydrogel formulation and performed live/dead cell assays to assess cell viability. Results showed that DPSCs remained viable across all formulations, with the 30% methacrylated GelMA exhibiting the highest percentage of live cells (Fig. [97]6D, [98]E). In conclusion, GelMA with 30% methacrylation is the optimal choice for DPSC encapsulation, providing a supportive environment for cell survival and offering a promising foundation for future clinical applications. ECM protein secretion and angiogenic support via integrin signaling by PDGFRβ+ DPSCs in GelMA Hydrogels To evaluate the ECM deposition capability of PDGFRβ+ DPSCs within GelMA hydrogels, we performed immunofluorescence staining to compare ECM protein expression between PDGFRβ+ DPSCs and P1-DPSCs. The results showed that PDGFRβ+ DPSCs secreted and deposited significantly higher levels of ECM proteins, including fibronectin (FN), laminin (LAMA4), and collagen (COL1 A2), within the GelMA matrix compared to P1-DPSCs (Fig. [99]7A–D). This increased ECM deposition indicates that PDGFRβ+ DPSCs in GelMA hydrogels create a supportive environment that could potentially enhance angiogenesis. Fig. 7. [100]Fig. 7 [101]Open in a new tab ECM Protein Secretion and Angiogenic Support via Integrin Signaling by PDGFRβ+ DPSCs in GelMA Hydrogels. A: FN, LAMA4 and COL1 A2 in GelMA Hydrogels was stained and images were taken using 3D confocal microscopy. B–D: AFI of immunofluorescence staining of FN, LAMA4, and COL1 A2. E–G: The tube formation assay of HUVECs was performed by counting branch points and capillary length.. H: RT-PCR were used to detect key genes expression involved in the integrin receptor related signaling pathway in HUVECs (*P < 0.05, **P < 0.01, ***P < 0.001) To assess the pro-angiogenic effects of this ECM-enriched environment, we conducted an in vitro tube formation assay by seeding HUVECs on the surface of the PDGFRβ+ DPSC-laden GelMA hydrogels. The results demonstrated that HUVECs on PDGFRβ+ DPSC-loaded GelMA hydrogels formed significantly more tube-like structures compared to controls, indicating enhanced angiogenic potential (Fig. [102]7E–G). Furthermore, we investigated the activation of integrin-mediated signaling pathways, as previous cell communication analyses suggested that ECM-integrin interactions play a role in promoting angiogenesis. After pre-treatment of HUVECs with integrin receptor inhibitors, the angiogenic effect of PDGFRβ+ DPSCs laden GelMA hydrogels was significantly inhibited. And PCR analysis of HUVECs cultured on PDGFRβ+ DPSC-laden GelMA hydrogels revealed significant upregulation of downstream integrin signaling pathways, including FAK/SRC, PI3 K, and RHO (Fig. [103]7H). These pathways are known to facilitate cytoskeletal rearrangement and endothelial cell adhesion, further supporting the role of PDGFRβ+ DPSCs in establishing an ECM-based microenvironment that promotes angiogenesis. In vivo evaluation of PDGFRβ+ DPSC-loaded GelMA for dental pulp–dentin complex regeneration To investigate the regenerative potential of GelMA hydrogels loaded with PDGFRβ+ DPSCs in supporting complex tissue regeneration, such as the dental pulp–dentin complex, we conducted an in vivo study. Four experimental groups were established: (1) GelMA alone, (2) GelMA loaded with P1-DPSCs, (3) GelMA loaded with PDGFRβ+ DPSCs, and (4) GelMA loaded with PDGFRβ+ DPSCs combined with an integrin receptor-specific inhibitor. Each hydrogel formulation was placed within treated dentin matrix blocks and subcutaneously implanted into immunocompromised mice for 6 weeks (Fig. [104]8A). Fig. 8. [105]Fig. 8 [106]Open in a new tab In Vivo Evaluation of PDGFRβ+ DPSC-Loaded GelMA for Dental Pulp–Dentin Complex Regeneration. A: Treatment of dentin matrix blocks and vivo experiments. B–F: HE, MASSON and Immunofluorescence staining to visualize the ECM protein deposition and blood vessel formation at 6 weeks of subcutaneous implantation. (*P < 0.05, **P < 0.01, ***P < 0.001) The results showed disorganized tissue formation in the GelMA-only group, without pulp-like structure. In the GelMA loaded with P1-DPSCs group, limited ECM deposition was observed, and angiogenesis was sparse. However, in the GelMA loaded with PDGFRβ+ DPSCs group, there was a significant increase in ECM protein deposition, and early vascular structures were more pronounced within the regenerated pulp-like tissue, suggesting enhanced angiogenesis. In contrast, the group with PDGFRβ+ DPSC-loaded GelMA combined with the integrin inhibitor showed a marked reduction in angiogenesis, indicating that the angiogenic effect was inhibited (Fig. [107]8B–F). These findings confirm that GelMA loaded with PDGFRβ+ DPSCs promotes angiogenesis and supports the formation of an organized pulp-like tissue, indicating its clinical potential for pulp regeneration. The results suggest that the pro-angiogenic effects may be mediated through ECM-integrin receptor signaling pathways. Discussion Dental pulp regeneration holds significant therapeutic promise, as restoring pulp vitality can preserve tooth structure, maintain sensory function, and support long-term dental health [[108]1]. Traditional endodontic treatments remove the infected pulp but lack the ability to regenerate vital tissue within the tooth, underscoring the need for effective pulp regeneration strategies. Advances in stem cell biology have highlighted the potential of DPSCs to regenerate pulp tissue by providing cellular components necessary for a functional pulp-dentin complex [[109]2]. Yet, achieving sustainable angiogenesis within regenerated tissue remains a primary challenge for successful pulp regeneration. Previous studies have shown that PDGFRβ+ DPSCs promote dental pulp vascular development through the FN-integrin signaling pathway. Building on these findings, we aimed to investigate dental pulp developmental mechanisms to inform clinical strategies for pulp regeneration [[110]9, [111]16]. Our review of single-cell sequencing data revealed that communication between DPSCs and various other cell types in young permanent dental pulp tissue is predominantly mediated by ECM-integrin signaling pathways. Further transcriptomic sequencing data suggest that PDGFRβ+ DPSCs, a relatively specific stem cell subpopulation in young permanent dental pulp, exhibit high expression of genes associated with extracellular matrix composition and angiogenesis. PDGFRβ+ DPSCs offer distinct advantages in dental pulp regeneration due to their enhanced ECM production and vascular-supportive properties. Compared to general DPSCs, PDGFRβ+ DPSCs not only exhibit robust proliferative capacity but also play a crucial role in ECM remodeling, which is essential for establishing a supportive scaffold within pulp tissue [[112]9]. The influence of the local microenvironment is crucial in the angiogenesis of in vivo grafts, with numerous studies indicating that extracellular matrix components play a pivotal role [[113]30]. In our study, PDGFRβ+ DPSCs demonstrated higher secretion levels of ECM proteins such as fibronectin (FN), laminin (LAMA4), and collagen (COL1 A2) when encapsulated in scaffold-free 3D culture. These ECM components provide structural support and biochemical signals that are critical for guiding angiogenesis, an essential factor in pulp regeneration [[114]12, [115]30, [116]31]. The advantage of scaffold-free 3D culture in validating PDGFRβ+ DPSCs’ ability to construct extracellular matrix (ECM) lies in its closer approximation to the in vivo three-dimensional microenvironment, providing a more accurate reflection of natural cell behavior [[117]32–[118]34]. Compared to traditional 2D cultures, scaffold-free 3D culture enables PDGFRβ+ DPSCs to self-assemble into spheroidal or tissue-like structures without additional support, promoting greater production and secretion of ECM components. This setting allows for more natural cell-cell interactions, with ECM deposition and organization more closely resembling in vivo tissue characteristics. Consequently, it offers deeper insights into the role of PDGFRβ+ DPSCs in ECM regulation and their contribution to the angiogenesis microenvironment. This ECM production by PDGFRβ+ DPSCs creates a microenvironment conducive to angiogenesis, which is necessary for pulp tissue viability and functionality. The use of hydrogels to deliver stem cells for dental pulp regeneration has shown promise, but challenges remain in optimizing both the material properties and cellular components for sustained regenerative outcomes [[119]35]. GelMA hydrogels are biocompatible and offer a tunable matrix that can support cell adhesion and differentiation, making them ideal for encapsulating stem cells [[120]6, [121]36, [122]37]. However, achieving consistent angiogenesis and functional tissue formation has been difficult with standard DPSC-loaded hydrogels. Our study demonstrates that PDGFRβ+ DPSCs, when loaded into GelMA hydrogels, can overcome some of these limitations by providing ECM-mediated support for angiogenesis. Compared to traditional DPSCs, PDGFRβ+ DPSCs showed superior angiogenic support, suggesting that their specific ECM-producing abilities could enhance the effectiveness of hydrogel-based pulp regeneration approaches. Previous studies on biomaterials loaded with stem cells have primarily focused on material synthesis to enhance cellular bioactivity. However, this study emphasizes the role of stem cell loading in directly improving the microenvironment of the material itself. By leveraging cell interactions both in vitro and in vivo, our approach aims to create an angiogenesis microenvironment within the scaffold, facilitating angiogenesis. Our findings underscore the importance of the ECM-integrin signaling pathway in angiogenesis. The ECM-integrin signaling pathway plays a crucial role in cellular processes such as adhesion, migration, and differentiation. Integrins, as transmembrane receptors, mediate cell-ECM interactions by binding to ECM proteins like fibronectin, collagen, and laminin, which activate intracellular signaling cascades [[123]38–[124]40]. Key pathways involved include FAK/SRC, PI3 K/AKT, and Rho GTPase signaling, which regulate cytoskeletal dynamics, cell survival, and gene expression [[125]12, [126]41–[127]43]. This signaling network not only provides structural support but also transduces mechanical and biochemical cues from the ECM, ultimately influencing tissue remodeling and angiogenesis. In regenerative applications, ECM-integrin signaling is essential for creating a microenvironment that supports cell function and tissue repair. The integrin pathway, activated through ECM proteins secreted by PDGFRβ+ DPSCs, plays a key role in endothelial cell adhesion, migration, and survival. In our study, HUVECs cultured on PDGFRβ+ DPSC-loaded GelMA hydrogels showed significant upregulation of FAK/SRC, PI3 K, and RHO signaling pathways, which are known to facilitate cytoskeletal reorganization and cellular interactions necessary for vessel formation. This highlights how ECM-integrin interactions can modulate cell behavior in the angiogenic process, aligning with recent literature that supports integrin’s role in mediating endothelial responses through ECM-derived signals. These results suggest that PDGFRβ+ DPSCs contribute to the formation of a angiogenesis microenvironment in dental pulp regeneration. Conclusion Our study illustrates that PDGFRβ+ DPSCs are a promising cell source for dental pulp regeneration, particularly due to their capacity for ECM production and angiogenesis support. The findings emphasize the importance of ECM-integrin signaling in forming a functional angiogenesis microenvironment within pulp tissue. By encapsulating PDGFRβ+ DPSCs in GelMA hydrogels, we provide a viable strategy for creating a vascularized, ECM-rich pulp tissue suitable for clinical application. These insights contribute to the development of more effective regenerative therapies for dental pulp and other vascularized tissues. Supplementary Information [128]Additional file 1.^ (17.6KB, docx) [129]Additional file 2.^ (8MB, tif) [130]Additional file 3.^ (58MB, xls) Acknowledgements