Extracellular Vesicles Enriched with Mitochondrial Components from Intermittently Cold-Exposed Adipose Tissue Drive Metabolically Active Adipose Regeneration via miR-296-3p

Current acellular adipose tissue engineering focuses primarily on structural regeneration, whereas functional metabolic recovery remains a major challenge. Extracellular vesicles (EV) derived from adipose tissue (AT-EV) have recently emerged as potent mediators of tissue regeneration, and cold stimulation has been reported to enhance the metabolic reprogramming of AT-EV. We have previously reported that fat grafts from cold-stimulated mice exhibited enhanced survival and adipose regeneration. Yet whether cold-induced AT-EV contribute to in vivo metabolic adipose regeneration remains unclear. Moreover, the cold stimulation temperatures used in traditional mouse models are often intolerable for humans. To address this, we developed a short-term intermittent mild cold exposure (STIMCE) protocol to metabolically activate murine adipose tissue for EV isolation (STIMCE-EV). Following only 3 or 7 days of STIMCE treatment, adipose tissue exhibited increased EV secretion and enrichment of mitochondrial components. Moreover, STIMCE-EV were efficiently incorporated into human acellular adipose matrix (AAM) hydrogel and exhibited controlled release in vitro. Injectable AAM@STIMCE-EV hydrogel transplantation in nude mice showed improved adipogenesis and volume retention in situ after 2 months. Importantly, the AAM@STIMCE-EV grafts improved cold adaptation and enhanced the thermogenic function of the host mice. Mechanistically, mmu-miR-296-3p was identified as the key regulatory factor within STIMCE-EV. Exogenous mmu-miR-296-3p supplementation significantly improved the adipogenic regeneration of AAM. Overall, this study presents a metabolically activated, cell-free adipose regeneration strategy that combines STIMCE-EV with AAM scaffolds. This minimally invasive and injectable system enables functional adipose tissue regeneration with systemic thermogenic metabolic activity, providing a promising translational solution for soft tissue reconstruction.