PDGF-BB overexpressing dental pulp stem cells improve angiogenesis in dental pulp regeneration

Abstract

Introduction: Angiogenesis represents a critical challenge in dental pulp regeneration due to the tissue's restricted nutrient supply through a 0.5-mm apical foramen. While dental pulp stem cells (DPSCs) hold regenerative potential, their limited vascularization capacity impedes clinical applications. Through Single-cell RNA sequencing (scRNA-seq) analysis of human dental pulp, we discovered a PDGF (+) mesenchymal subset exhibiting enhanced angiogenic signatures, suggesting targeted cell selection could overcome this bottleneck.

Methods: ScRNA-seq identified PDGF (+) subpopulation in human pulp samples, validated through multiplex immunohistochemical of the localization of PDGF/CD73/CD31. PDGF-BB-overexpressing DPSCs were engineered via lentiviral vectors. Functional assessments included: 1) CCK-8/Edu/cell cycle/transwell assays for proliferation and migration ability 2) HUVECs co-culture models analyzing chemotaxis and tube formation 3) Vascularized tissue formation in rat kidney capsule transplants.

Results and discussion: The CD73 (+) PDGF (+) subpopulation demonstrated spatial correlation with CD31 (+) vasculature. PDGF-BB overexpression enhanced DPSCs' proliferative capacity and migration capacity. Co-cultured HUVECs exhibited increased tube formation with PDGF-BB group. In vivo transplants generated more vascular structures containing CD31 (+) endothelia. These findings establish PDGF-BB engineering as an effective strategy to amplify DPSCs' angiogenic potential, while emphasizing the therapeutic value of functionally-defined stem cell subpopulations in pulp regeneration.

Keywords: dental pulp regeneration; dental pulp stem cells; endothelial; single-cell RNA sequencing; vascularization.

FIGURE 1

Identification and characterization of PDGF(+) MSCs in dental pulp angiogenesis. (A) UMAP visualization of seven distinct cell populations identified through single-cell RNA sequencing analysis. (B) Dotplot representation of characteristic marker genes used for cell type annotation and cluster identification. (C) Proportional distribution of PDGF (+) and PDGF (−) subpopulations within the MSC population, presented as a percentage pie chart. (D, E) Cell interaction analysis of each cell in dental pulp tissue. MSC (+) PDGF (+) cells exhibited significantly stronger intercellular interaction with endothelial cells compared to MSC (+) PDGF (−) cells.(F) Comparative analysis of angiogenesis-associated gene expression patterns between PDGF (+) and PDGF (−) subpopulations, presented as module scores (****p < 0.0001). (G) The results of GO enrichment analysis of DEGs upregulated in PDGF (+) cells compared to PDGF (−) cells, highlighting significant biological processes. (H) mIHC staining of human dental pulp tissue showing spatial distribution of CD31 (white; endothelial cells), CD73 (red; mesenchymal stem cells), PDGF-BB (green), and DAPI (blue; nuclei). Scale bar = 100 μm.

FIGURE 2

Lentiviral transfection significantly enhanced both the gene and protein expression levels of PDGF-BB in DPSCs. (A, B) Schematic diagram of the lentiviral plasmid for overexpression of PDGF-BB and lentiviral transfection process. (C) Observation under Fluorescence microscope after transfection of 72 h. Scale bar = 200 μm. (D) Western blot analysis were used to measure PDGF-BB protein level. (n = 3) (E) qRT-PCR analysis were used to measure PDGF-BB mRNA level. (n = 3) (F) ELISA analysis were used to measure PDGF-BB secretion level. (n = 3) (G) Immunofluorescence detection of PDGF-BB expression. Scale bar = 50 μm. (ns, no significant difference; ****p < 0.0001).

FIGURE 3

PDGF-BB overexpressing enhanced the proliferation and migration ability of DPSCs. (A, B) Representative images and quantitative analysis of EdU incorporation assay. Cells in the process of DNA replication are stained red, and the percentage of EdU-positive cells was calculated as the ratio of EdU-positive cells to the total number of nuclei stained with DAPI (blue). Scale bar = 100 μm (n = 5). (C) Cell proliferation curves were analyzed using the CCK-8 assay. (n = 3) (D) Cell cycle distribution analyzed by flow cytometry. (E) Quantitative analysis of the cell cycle distribution. The percentages of cells in G0/G1, S, and G2/M phases for each group are presented as bar graphs. (n = 3) (F, G) Representative images and quantitative analysis of the transwell migration assay. Scale bar = 200 μm. (n = 5) (ns, no significant difference; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

FIGURE 4

PDGF-BB overexpressing enhances DPSC-mediated angiogenesis by upregulating VEGFA expression. (A) Schematic diagram of Control, GFP and PDGF-BB cells coculture with HUVECs for in vitro experiments. (B, C) Transwell co-culture system was used to evaluate the recruitment ability of DPSC, DPSC-GFP, and DPSC-PDGF on HUVECs migration. Scale bar = 200 μm. (n = 5) (D, E) Tube formation assay of HUVECs stimulated with conditioned media from the three experimental groups. Representative images and quantitative analysis of tube-like structures are shown. Scale bar = 200 μm. (n = 5) (F, G) mRNA and protein expression levels of VEGFA in different groups. (n = 3) (H) ELISA analysis were used to measure VEGFA secretion level. (n = 3) (I) Confocal microscope images demonstrate enhanced VEGFA signals in PDGF-BB compared to Control and GFP groups. Scale bar = 50 µm. (ns, no significant difference; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

FIGURE 5

In vivo transplantation of PDGF-BB overexpressing DPSCs demonstrated enhanced angiogenic potential. (A) Fluorescence microscopy images of cells seeding on CPC scaffold after culture 24 h in vitro. Scale bar = 100 μm.(B) Schematic diagram of the surgery for sub kidney capsule transplantation of tissue-engineered composites. (C, D) HE staining of tissue sections and statistical analyses of blood vessel numbers. Scale bar = 200 μm, 50 μm (partial enlargement). (BV, blood vessel; CT, collagenous tissue; n = 10) (E, F) Masson staining of tissue sections and statistical analyses of the percentage of collagen-positive area. Scale bar = 200 μm, 100 μm (partial enlargement). (G, H) Immunofluorescence staining for the angiogenesis marker CD31 expression in groups and statistical analyses of CD31 positive area. Scale bar = 50 μm. (ns, no significant difference; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).