Canine dental pulp stem cell-derived exosomes: Proteomic characterization and first xenogeneic application for post-castration wound healing in cats.

Abstract

Background and aim: Mesenchymal stem cell-derived exosomes (MSC-Exos) have emerged as promising cell-free therapeutics because they retain the regenerative and immunomodulatory properties of their parent cells while minimizing the risks associated with cell transplantation. Canine dental pulp stem cell-derived exosomes (cDPSC-Exos) represent an attractive veterinary regenerative platform because dental pulp is readily accessible through minimally invasive procedures. However, their molecular composition and xenogeneic therapeutic potential remain largely unexplored. This study aimed to characterize canine dental pulp stem cells (cDPSCs) and their derived exosomes, define the proteomic profile of cDPSC-Exos, and evaluate their safety and therapeutic potential for post-castration wound-healing in cats.

Materials and methods: cDPSCs were isolated from canine dental pulp and characterized according to mesenchymal stem cell (MSC) criteria using morphology, colony-forming ability, proliferation, trilineage differentiation, and reverse transcription-quantitative polymerase chain reaction analysis of MSC surface and stemness markers. Exosomes were isolated from conditioned medium and characterized by nanoparticle tracking analysis and CD9 expression. High-resolution mass spectrometry was used for proteomic profiling, followed by functional pathway and protein interaction analyses. A randomized controlled pilot study involving six healthy male cats (n = 3/group) compared topical cDPSC-Exos gel (3.3 × 10⁸ particles/g) with Bioplacenton® following castration. Wound-healing was evaluated using macroscopic wound scores, hematological parameters, and laser speckle contrast imaging.

Results: The isolated cDPSCs fulfilled the defining characteristics of MSCs, including multilineage differentiation and expression of MSC-associated markers. cDPSC-Exos exhibited typical exosome characteristics, with a mean particle diameter of 111 ± 24 nm. Proteomic analysis identified 312 proteins, including 22 proteins associated with inflammation regulation, angiogenesis, extracellular matrix organization, cell migration, and tissue remodeling. Western blot analysis confirmed the presence of vimentin and β-actin, supporting the proteomic findings. Topical administration of cDPSC-Exos was well tolerated, without adverse reactions, and significantly improved wound redness, swelling, and pain scores during early healing (p < 0.05), while hematological variables and tissue perfusion remained comparable to the positive control.

Conclusion: This study provides the first comprehensive proteomic characterization of cDPSC-Exos and the first evidence supporting their xenogeneic application in feline post-castration wound-healing. The findings demonstrate that cDPSC-Exos constitute a safe and promising cell-free regenerative therapy capable of modulating early wound repair through multiple biological pathways. Larger controlled studies are warranted to validate their clinical efficacy and optimize therapeutic protocols.