Injectable Tissue-Specific Hydrogel System for Pulp–Dentin Regeneration

Abstract

The quest for finding a suitable scaffold system that supports cell survival and function and, ultimately, the regeneration of the pulp–dentin complex remains challenging. Herein, we hypothesized that dental pulp stem cells (DPSCs) encapsulated in a collagen-based hydrogel with varying stiffness would regenerate functional dental pulp and dentin when concentrically injected into the tooth slices. Collagen hydrogels with concentrations of 3 mg/mL (Col3) and 10 mg/mL (Col10) were prepared, and their stiffness and microstructure were assessed using a rheometer and scanning electron microscopy, respectively. DPSCs were then encapsulated in the hydrogels, and their viability and differentiation capacity toward endothelial and odontogenic lineages were evaluated using live/dead assay and quantitative real-time polymerase chain reaction. For in vivo experiments, DPSC-encapsulated collagen hydrogels with different stiffness, with or without growth factors, were injected into pulp chambers of dentin tooth slices and implanted subcutaneously in severe combined immunodeficient (SCID) mice. Specifically, vascular endothelial growth factor (VEGF [50 ng/mL]) was loaded into Col3 and bone morphogenetic protein (BMP2 [50 ng/mL]) into Col10. Pulp–dentin regeneration was evaluated by histological and immunofluorescence staining. Data were analyzed using 1-way or 2-way analysis of variance accordingly (α = 0.05). Rheology and microscopy data revealed that Col10 had a stiffness of 8,142 Pa with a more condensed and less porous structure, whereas Col3 had a stiffness of 735 Pa with a loose microstructure. Furthermore, both Col3 and Col10 supported DPSCs’ survival. Quantitative polymerase chain reaction showed Col3 promoted significantly higher von Willebrand factor (VWF) and CD31 expression after 7 and 14 d under endothelial differentiation conditions ( P < 0.05), whereas Col10 enhanced the expression of dentin sialophosphoprotein (DSPP), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and collagen 1 (Col1) after 7, 14, and 21 d of odontogenic differentiation ( P < 0.05). Hematoxylin and eosin and immunofluorescence (CD31 and vWF) staining revealed Col10+Col3+DPSCs+GFs enhanced pulp–dentin tissue regeneration. In conclusion, the collagen-based concentric construct modified by growth factors guided the specific lineage differentiation of DPSCs and promoted pulp–dentin tissue regeneration in vivo.