Self-Organizing 3D Dermal Papilla Cell Spheroids Yield Therapeutic Extracellular Vesicles that Target Hypertrophic Scar Regression via the miR-26a-5p/CCNE2 Axis

Abstract

Background Hypertrophic scarring remains a critical challenge in regenerative medicine because of the limited efficacy of current antifibrotic therapies. Although dermal papilla cells (DPCs) exhibit intrinsic scar-inhibitory potential, their therapeutic utility is constrained by rapid replicative senescence and poor scalability in traditional monolayer cultures, necessitating innovative strategies to enhanceincrease cellular functionality and manufacturing feasibility. Methods A self-feeder layer 3D (SFL-3D) platform was established to reprogram primary human DPCs into rejuvenated tdDPC spheroids via autocrine–paracrine signalling activation. tdDPC-derived extracellular vesicles (tdDPC-EVs) were isolated from culture supernatants by differential centrifugation. The antifibrotic effects of tdDPC-EVs were systematically evaluated using human scar fibroblasts (HSFs) through scratch wound healing assays, CCK-8 proliferation assays, and fibrotic marker analysis (Western blotting and immunofluorescence staining for α-SMA and collagen I). Bioinformatics was used to predict key pathways involved in hypertrophic scar (HS) pathogenesis, whereas gain/loss-of-function studies investigated the miR-26a-5p/CCNE2 regulatory axis. Therapeutic validation was performed in a rabbit ear hypertrophic scar model with histopathological and molecular profiling. Results Compared with conventional 3D cultures, the SFL-3D system demonstrated superior proliferative support, enabling stable tdDPC expansion beyond 10 passages while maintaining high viability and enhanced EV biogenesis. miR-26a-5p-enriched tdDPC-EVs attenuated fibrosis through two mechanisms: (1) silencing CCNE2 to block PI3K/AKT-driven collagen overproduction and (2) suppressing α-SMA+ myofibroblast differentiation. In the rabbit ear HS model, tdDPC-EV administration reduced the scar elevation index and restored the collagen I/III ratio to near-physiological levels. Conclusions This study positions tdDPC-EVs as a scalable acellular therapy that overcomes the replicative senescence and manufacturing limitations of cellular approaches. The antiscarring efficacy of these EVs, which is mediated by the miR-26a-5p/CCNE2/PI3K/AKT axis, highlights their clinical potential as precision-targeted strategies for hypertrophic scar management. The SFL-3D platform further provides a translatable framework for EV-based regenerative therapeutics.