Extracellular Vesicles Derived From Human Adipose-Derived Stem Cell Prevent the Formation of Hypertrophic Scar in a Rabbit Model

Abstract

Background Preventing scar formation during wound healing has important clinical implications. Numerous studies have indicated that adipose-derived stem cell culture mediums, which are rich in cytokines and extracellular vesicles (EVs), regulate matrix remodeling and prevent scar formation after wound healing. Therefore, using a rabbit scar model, we tried to demonstrate which factor in adipose-derived stem cell culture mediums plays a major role in preventing scar formation (EVs or cytokines), as well as revealing the underlying mechanism. Methods Human adipose-derived stem cells (hASCs) were isolated from the subcutaneous adipose tissue of a healthy female donor. The surface CD markers of third-passage hASCs were analyzed by flow cytometry. The adipogenic differentiation capacity of the hASCs was detected using Oil O staining. A cultured medium of third- to five-passage hASCs was collected for EV and EV-free medium isolations. Extracellular vesicles were characterized using transmission electron microscopy, NanoSight, and the Western blotting for surface markers CD63, TSG101, and Alix. The EV-free medium was characterized by Western blotting for vascular endothelial growth factor A (VEGFA), platelet derived growth factor B (PDGFB), and transforming growth factor β 1 (TGFβ1). Eight-millimeter-diameter wounds were created on the ventral side of both ears of 16 New Zealand rabbits. A total of 0.1 mL of the human adipose-derived stem cell–extracellular vesicle (hASC-EV) or EV-free medium was locally injected into wounds made on the right ears during wound healing. Meanwhile, equal amounts of phosphate buffer saline were injected into the left ears as a control. Biopsies of the wounded skin and surrounding tissue were excised on postoperative day 28 and subjected to hematoxylin and eosin (H&E), Masson, and α-SMA immunofluorescence staining. The protein expression of α-SMA and collagen I in both scar tissues and the normal skin were evaluated via Western blotting. Results The hASCs expressed high levels of 49d, CD90, CD105, and CD73 but did not express CD34 or CD45. The hASCs differentiated into adipocytes under an adipogenic induction medium. Under transmission electron microscopy, the hASC-EVs were circular, bilayer membrane vesicles and approximately 95% of the particles were between 50 and 200 nm in size. The hASC-EVs expressed the same surface markers as EVs, including CD63, TSG101, and Alix and displayed little expression of VEGFA, PDGFB, and TGFβ1. The EV-free medium had a high expression of VEGFA, PDGFB, and TGFβ1 but displayed no expression of CD63, TSG101, and Alix. In vivo, the hASC-EV treatment prevented the formation of hypertrophic scars on postoperative day 28 and suppressed collagen deposition and myofibroblast aggregation. However, the EV-free medium did not prevent the formation of hypertrophic scars on the same time point and had little effect on collagen deposition and myofibroblast aggregation when compared with the control gr...

Assessment of human adipose‐derived stem cell on surface‐modified silicone implant to reduce capsular contracture formation

“…We then characterized the phenotype of hASCs via fluorescenceactivated cell sorting analysis in accordance with the low expression of surface makers including CD14, CD34, and CD45 and high expression of major surface marker including CD73 and CD105. [53][54][55][56] As predicted, high expression of CD73 and CD105 and low expression of CD14, CD34, and CD45 were observed (Figure S3), consistent with that in prior studies. 53,57,58 To observe cell viability and the adhesion pattern of hASCs grown on a culture plate, PDMS, and IA-PDMS, the Live/Dead assay was used.…”

Exosomes and Other Extracellular Vesicles with High Therapeutic Potential: Their Applications in Oncology, Neurology, and Dermatology

“…Wang et al demonstrated that intravenous injection of ASC-Exos enhanced extracellular matrix (ECM) reconstruction through regulating the ratios of collagen type III: type I, TGF-β3:TGF-β1, and MMP3:TIMP1 in a mouse skin incision model [180]. Zhu et al presented the use of human adipose-derived stem cells (hASCs) as a new approach to preclude hypertrophic scar development [181]. Local hASCc injection successfully prevented hypertrophic scar formation by suppressing myofibroblast aggregation and collagen deposition in a rabbit scar model.…”

An Emerging Frontier in Intercellular Communication: Extracellular Vesicles in Regeneration

“…For example, EVs derived from human adipocyte stem cells (hASC-EVs) promote ECM remodeling and scarless healing of dorsal skin incisions in mice by inhibiting myofibroblast differentiation and increasing the ratios of collagen III to collagen I and TGFβ-3 to TGFβ-1, similar to levels in fetal scarless wound healing ( Wang et al, 2017 ). hASC-EVs also reduced hypertrophic scarring during wound healing in rabbit ears, by suppressing myofibroblast differentiation and collagen deposition ( Zhu et al, 2020 ). In an example of EVs’ therapeutic potential, Dinh and colleagues showed that inhalation of lung spheroid cell-derived EVs inhibits collagen deposition and improves alveolar repair in mouse and rat models of pulmonary fibrosis, possibly by transporting miR-30a, an anti-fibrotic miRNA, to matrix-secreting cells ( Berschneider et al, 2014 ; Dinh et al, 2020 ).…”

“…In vitro , EVs derived from fetal mouse neural stem cells (NSCs) promote NSC differentiation through miR-9 targeting of Hes1, suggesting EVs could be used in conjunction with stem cell transplantation to treat neurodegenerative disease ( Yuan et al, 2021 ). EVs also inhibit differentiation, for example, by preventing differentiation of pro-fibrotic myofibroblasts during inflammation, as discussed in Section 4.3 ( Wang et al, 2017 ; Zhu et al, 2020 ).…”