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.

Keywords: collagen; dental pulp; dentin; hydrogel; mesenchymal stem cells; regeneration.

Figure 1.

Characterization of polymerized methacrylated type I collagen and canine dental pulp stem cells (DPSCs). (A) Stiffness analyses of 3 mg/mL and 10 mg/mL collagen hydrogels before and after crosslinking by rheology analysis. (B) Representative scanning electron microscopy images of 2 concentrations of collagen hydrogel. The white line indicates the interface adaptation between the 2 different concentrations of collagen. (C) Canine DPSC stemness characterization by flow cytometry. Values are presented as mean ± SD.

Figure 2.

Low-stiffness collagen hydrogel enhanced endothelial differentiation of dental pulp stem cells (DPSCs). (A) Representative images and quantitative data of cell viability stained by cell viability imaging kit (red: apoptotic cells, blue: total cells) in complete α-MEM, angiogenic induction medium, and odonto/osteogenic induction medium. (B) Representative immunofluorescence images and quantification of vWF after 14 d of angiogenic induction (red: vWF, blue: DAPI). (C) Relative mRNA expression of CD31, vWF, and VEGFR2 after 7 and 14 d of angiogenic induction. Values are presented as mean ± SD. ANG, angiogenic induction medium; CON, control (complete α-MEM medium); OGN, odonto/osteogenic induction medium.

Figure 3.

High-stiffness collagen hydrogel augmented odonto/osteogenic differentiation of dental pulp stem cells. (A) Representative images of Alizarin red staining and quantification after 21 d odonto/osteogenic induction. (B) Relative messenger RNA expression of DSPP, ALP, Col1, and Runx2 after 7, 14, and 21 d of odonto/osteogenic induction. Values are presented as mean ± SD. CON, control (complete α-MEM medium); OGN, odonto/osteogenic induction medium.

Figure 4.

In vivo animal model and histology hematoxylin and eosin (H&E) staining of pulp–dentin regeneration. (A) The 1-mm-thick tooth slices (a) were cut from the cervical region of a noncarious human third molar after injecting 2 concentration collagen scaffolds with cells into the pulp chamber concentrically within the pulp chamber (b, c), implanted subcutaneously in SCID mouse (d). (B) Representative H&E staining images of regenerated pulp/dentin-like tissue after 6 wk. Blue arrows indicate the perfused vessels.

Figure 5.

Representative immunofluorescence (IF) staining and quantification for pulp–dentin regeneration in vivo. (A) Representative IF staining images for CD31. (B) Representative IF staining images for vWF. (C) Quantification of vessel-like structure. (D) IF staining of microvessels positive for GFP (green) and CD31 (red). Cell nuclei were stained in blue. White arrows indicate CD31-positive lumen-like structures originating from GFP-transduced dental pulp stem cells (DPSCs). (E) Representative IF staining for DSPP. (F) DSPP-positive cell percentage showed that the Col3+Col10+DPSCs+GF group had the most vascular-like structure formation and DPSC-differentiated odontoblasts. (G) Tetracycline autofluorescence of newly formed dentin (in vivo) in implanted tooth slices observed in Col3+Col10+DPSCs and Col3+Col10+DPSCs+GF groups. The white arrows indicate the newly formed dentin. Values are presented as mean ± SD.