Extracellular Vesicles-Induced Cell Homing and Odontogenesis via microRNA Signaling for Dentin Regeneration

Abstract

Reparative tertiary dentinogenesis requires the recruitment and odontogenic differentiation of dental pulp stem cells (DPSCs). Extracellular vesicles (EVs) as bioactive molecules have gained attention in regenerative medicine for their ability to mediate tissue repair through intercellular communication, influencing cell recruitment, proliferation, and differentiation. This study aimed to evaluate the effects of EVs on DPSC homing and odontogenic differentiation for dentin regeneration. DPSC-derived EVs were cultured in either growth (EV-G) or odontogenic differentiation (EV-O) conditions and isolated using a modified precipitation method. EVs were characterized by nanoparticle tracking analysis, scanning electron microscopy, antibody array, and cellular uptake assay. Treatment with 5 × 10⁸ EVs/mL significantly enhanced DPSC chemotaxis and proliferation compared with a no-treatment control and a lower dosage of EV (5 × 10⁷ EVs/mL). Gene expression and biochemical analyses revealed that EV-O up-regulated odontogenic markers including collagen type 1A1 (COL1A1), runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALP). EV-O enhanced dentin regeneration by approximately 55% over vehicle controls in a rabbit partial dentinotomy/pulpotomy model. We identified key microRNAs (miR-21-5p, miR-221-3p, and miR-708-3p) in EV-O involved in cell homing and odontogenesis. In conclusion, our EV-based cell homing and odontogenic differentiation strategy has significant therapeutic potential for dentin regeneration.

Keywords: cell homing; dental pulp stem cells; dentin regeneration; dentinogenesis; extracellular vesicles; microRNA; odontogenesis.

Figure 1

Characterization of rabbit dental pulp stem cell-derived extracellular vesicles (DPSC-EVs). DPSC-EVs were prepared from regular growth medium (EV-G) or odontogenic differentiation medium (EV-O). (A) Nanoparticle tracking Analysis (NTA) (n = 3). Red error bars indicate ±1 standard error of the mean. Latex beads (100 nm diameter) were used as a reference particle. (B) Mean size, distribution, and concentration of EVs. N.A.: not applicable. (C) Scanning electron microscopy (SEM) of EV-O. (D) Antibody array having eight positive markers (CD63, EpCAM: epithelial cell adhesion molecule, ANXA5: annexin A5, TSG101: tumor susceptibility gene 101, FLOT1: flotillin-1, ICAM: intercellular adhesion molecule 1, ALIX: programmed cell death 6 interacting protein, and CD81) and four controls (cis-Golgi matrix protein (GM130) as a negative marker, 2 positive controls (+Ctrl), and a blank control). The images of SEM and antibody array for EV-G are available in our previous manuscript [11].

Figure 2

The effect of dental pulp stem cell-derived extracellular vesicles (DPSC-EVs) on cell viability, proliferation, chemotaxis, and odontogenesis. DPSC-EVs were prepared from regular growth medium (EV-G) or odontogenic differentiation medium (EV-O). EV-G-L and EV-O-L: 5 × 10⁷ particles/mL, EV-G-M and EV-O-M: 5 × 10⁸ particles/mL, EV-G-H and EV-O-H: 5 × 10⁹ particles/mL. (A) Cellular uptake of DPSCs with or without PKH67 green fluorescence at 2 days. (B) Cell viability at 1 day (n = 3–16). (C) Cell proliferation at 4 days (n = 6). (D,E) Chemotaxis at 2 days (n = 3): (D) representative confocal images of EV-O-M (green: Calcein AM) and (E) quantified fluorescence. The confocal images for control and EV-G are available in our previous manuscript [11]. (F–H) Odontogenic markers at 10 days: (F) collagen type 1A1 (COL1A1; n = 4), (G) runt-related transcription factor 2 (RUNX2; n = 4), and (H) alkaline phosphatase (ALP; n = 4). bOM: basal odontogenic induction medium. cOM: complete odontogenic induction medium. 4-MU: 4-methylumbelliferone. Data are represented as mean ± standard deviation. Colors in bar graphs: gray for No EV, green for EV-G, and orange for EV-O. Lines above bars: p-values between two groups.

Figure 3

Dentin regeneration in a rabbit incisor partial dentinotomy/pulpotomy model. (A) A standard rabbit dental table. (B) Cavity creation using a dental bur in the maxillary incisors. (C) Validation of pulpal approach using computed tomography (CT). (D) Coverage of resin-modified glass ionomer cement after injecting hydrogel only (vehicle control) or extracellular vesicles (EVs)-loaded hydrogel (EV-G or EV-O). (E–L) Representative histologic images with hematoxylin and eosin stain at 4 weeks. (E) Intact mandibular incisor: (F) pulp and (G) incisal dentin. (H) Injured maxillary incisor (vehicle control): (I) pulp and (J) incisal dentin. (K) EV-G. (L) EV-O. (M) Inter- and intra-observer correlation coefficients. (N) Modified histological scoring system for dentin regeneration (n = 5). Cem: cement. Den: dentin, OL: odontoblast layer, PCT: pulp cavity trace, Pul: pulp, Control: vehicle control (injury + hydrogel), EV-G: EVs harvested from growth medium, EV-O: EVs harvested from odontogenic differentiation medium. Data are represented as mean ± standard deviation. A line above bars: p-value between control and EV-O.