Regenerative Potential Nanomedicine of Adipocyte Stem Cell-Derived Exosomes in Senescent Skin Tissue

Abstract

Introduction: Skin is the first-line barrier defense against infection, irradiation, and toxins, but is prone to natural aging (intrinsic aging) and environmental factors (extrinsic aging). Hence, there is an increasing urgency to explore an effective treatment for aging skin. This study was focused on testing the potential of utilizing adipocyte stem cell derived exosomal as nanomedicine to regenerate the dermal layer and counteract signs of skin aging.

Methods: The harvested stem cells from adipose tissues were isolated, cultured, and then starved. The centrifugation of cell cultures medium yielded the human adipose-derived stem cells conditional medium (HADSCs-CM). Collagen secretion and fibroblast viability of human fibroblasts (Hs68) were measured in the presence of HADSCs-CM. The dermal layer, vascular endothelial growth factor (VEGF), and collagen levels were evaluated on the mice animal models between the treatments with and without HADSCs-CM.

Results: Western blotting, transmission electron microscopy (TEM), and dynamic light scattering (DLS) confirmed that the functional particles in HADSCs-CM were exosomes. When Hs68 fibroblasts were treated with HADSCs-CM, both cell viability and collagen secretion increased in a dose-dependent manner. Following the post-ultraviolet A (post-UVA) exposure, the mice exposed to the HADSCs-CM have decreased dermal thickness and VEGF expression and increased collagen volume compared to the non-HADSCs-CM exposed mice (control group).

Conclusion: HADSCs-CM significantly alleviated signs of skin senescence, including reduced dermal thickness, decreased VEGF expression, and enhanced collagen production. Exosomes, identified in the HADSCs-CM, are the functional component of these regenerative effects. This study highlights that the exosomal nanomedicine found in HADSCs-CM could regenerate skin, boost collagen production, improve fibroblast cell viability, and contain functional exosomes.

Keywords: adipocyte stem cells; collagen; dermal regeneration; exosomal nanomedicine; skin aging.

Graphical abstract

Figure 1

(A) Hs68 cell viability in different NaB concentration after 48 hrs from 0 to 16 mM. (B) Intracellular ROS of Hs68 in different NaB dosage treatments. (C) SA-β-gal stained Hs68 after 0 and 4 mM NaB treatments. n=3, *p<0.05, and **p<0.01.

Figure 2

Examination of different concentrations of HADSCs-CM on Hs68 cell viability. n=3, *p<0.05, **p<0.01, and ***p<0.005.

Figure 3

Effects of HADSCs-CM in preventing Hs68 fibroblast senescence. (A) Pre-treatment effect of HADSCs-CM in decreasing Hs68 fibroblasts cell viability. (B) Consequences of HADSCs-CM pre-treatment in increasing collagen release from Hs68 fibroblasts. (C) Post-treatment effect of HADSCs-CM in lowering Hs68 fibroblasts cell viability. (D) Consequences of HADSCs-CM post-treatment in increasing collagen release from Hs68 fibroblasts. n=3, *p<0.05 and **p<0.01 comparison to control group; and #p<0.05, ##p<0.01 comparison to negative group.

Figure 4

Aged skin model from nude mice. (A) Dermal thickness after 0 (control group), 20, and 50 J/cm² UVA irradiation (×200). Thickness-increasing percentage was measured by SmileView. (B) Dermal collagen expression after UVA irradiation. An increasing percentage of collagen emission was measured by Image J. (C). Different concentrations of HADMSC-CM affected dermal thickness after 50 J/cm² UVA irradiation. Dermal thickness measured by SmileView. (D) ×10, ×20, and ×40 concentrated HADSCs-CM affected VEGF secretion, increasing on senescent skin nude mice. Expression of VEGF was measured by Image J. n=3, *p<0.05 or **p<0.01 compared with the control group; #p<0.05, ##p<0.01 compared with the negative group.

Figure 5

Identification of exosomes. (A) TEM took the exosome microphotograph at x400k magnification. Dynamic Light Scattering measures the exosome’s size distribution. Cell marker CD81 was detected on exosomes by Western blotting. (B) Cell viability was affected by a variety of concentrations of HADSCs-Exos. (C) BODIPY^TM TR Ceramide and DAPI distinguished the phospholipid layers of exosomes and the cell nucleus of Hs68. Merged photo shows exosomes and Hs68 cell interactions. n=3, *p<0.05, **p<0.01, and ***p<0.005.