Wound healing effect of adipose-derived stem cells: A critical role of secretory factors on human dermal fibroblasts

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Summary

Background

Adipose-derived stem cells (ADSCs) are a population of pluripotent cells, which have characteristics similar to bone marrow-derived mesenchymal stem cells. Whereas ADSCs have potential applications for the repair and regeneration of various damaged tissues, few studies have dealt with the effect of ADSCs on fibroblasts, which play a key role in skin biology.

Objective

In this study, we investigated the possible roles of ADSCs in skin wound healing process, especially in the aspect of fibroblast activation—proliferation, collagen synthesis and migratory properties.

Methods and results

ADSCs promoted human dermal fibroblast (HDF) proliferation, not only by cell-to-cell direct contact, which was confirmed by co-culture experiment, but also by paracrine activation through secretory factors, resolved by transwell co-culture and culturing with conditioned medium of ADSCs (ADSC-CM). ADSC-CM enhanced the secretion of type I collagen in HDFs by regulating the mRNA levels of extracellular matrix (ECM) proteins: up-regulation of collagen type I, III and fibronectin and down-regulation of MMP-1. Moreover, ADSC-CM showed stimulatory effect on migration of HDFs in in vitro wound healing models. Additional to those in vitro evidences, wound healing effect of ADSCs was also verified with in vivo animal study, resulted that ADSCs significantly reduced the wound size and accelerated the re-epithelialization from the edge.

Conclusion

Collectively, these data suggest that ADSC is constitutionally well suited for dermal wound healing and secretory factors derived from ADSCs promote wound healing via HDFs and ADSCs can be used for the treatment of photoaging and wound healing.

Introduction

Adipose-derived stem cells (ADSCs) are a population of pluripotent mesenchymal cells, which can differentiate into various lineages [1], [2], [3]. Some clinical applications of ADSCs for compensation of diverse tissue defects have been suggested [4], [5], [6]. However, few studies have dealt with the effects of ADSCs on fibroblasts, which play a key role in skin biology such as wound healing, scar and photoaging. On the other hand, several lines of evidences reported that bone marrow-derived mesenchymal stem cells (BM-MSCs) and some growth factors accelerate wound healing [7], [8], [9]. ADSCs have surface markers and gene profiling similar to BM-MSCs. Given their convenient isolation compared with BM-MSCs and extensive proliferative capacities ex vivo, ADSCs hold great promise for use in wound repair and regeneration.

In a highly coordinated biological process of dermal wound healing, skin fibroblasts interact with surrounding cells such as keratinocytes, fat cells and mast cells. Fibroblasts produce extracellular matrix (ECM), glycoproteins, adhesive molecules and various cytokines [10]. By these syntheses and cell-to-cell, cell-to-cytokine interdependencies, skin fibroblasts contribute to the fibroblast–keratinocyte–endothelium complex that not only repairs wounds but also maintains the integrity and youth of skin. In the early phase of wound healing, fibroblasts migrate into the affected area and move across fibrin-based provisional matrix. Since the provisional fibrin-based matrix is relatively devoid of fibroblasts, the processes of migration, proliferation and ECM production are the key steps in the regeneration of a functional dermis [11]. Fibroblasts produce collagen-based ECM that ultimately replaces the provisional fibrin-based matrix and help re-approximate wound edges through their contractile properties.

In an attempt to explore the contribution of ADSCs to the cutaneous wound healing, we investigated whether conditioned medium of ADSCs (ADSC-CM), together with cell-to-cell contact between ADSCs and human dermal fibroblasts (HDFs), promote proliferation. In addition, secretion of type I collagen was examined followed by measuring the mRNA levels of ECM proteins and effect on migration activity of HDFs was tested in in vitro wound healing model. As an alternative therapeutic strategy for damaged tissue, we studied the effect of ADSCs on wound size and skin re-epithelialization in animal model.

Section snippets

Isolation and culture of ADSCs and HDFs

Human subcutaneous adipose tissue samples were acquired from elective liposuction of 23 healthy females with informed consents as approved by the institutional review boards. The obtained samples were digested with 0.075% collagenase type II (Sigma–Aldrich, St. Louis, MO) under gentle agitation for 45 min at 37 °C, and centrifuged at 300 × g for 10 min to obtain the stromal cell fraction. The pellet was filtered with 70 μm nylon mesh filter, and resuspended in phosphate-buffered saline (PBS). The

Characterization of ADSCs

The average number of processed lipoaspirated (PLA) cells isolated was 2.4 ± 1.4 × 106 cells (n = 23)/ml of lipoaspirated fat. ADSCs expanded easily in vitro by culturing PLA cells and exhibited a fibroblast-like morphology (Fig. 1A). In flow cytometry, characteristic expressions of stem cell-related surface markers were confirmed. ADSCs expressed CD73, CD90, and CD105, and were lacking in CD34 and CD49d (Fig. 1B). Adipogenic, osteogenic, and chondrogenic differentiation was also confirmed by

Discussion

In this study, ADSCs were superior to HDFs in promoting HDF proliferation and ADSC-CM was also superior to HDF-CM in up-regulating type I collagen secretion by HDFs (Figs. 2A and 4A). In our preliminary proteomic analysis and other reports [16], [17], ADSCs secrete several collagens, fibronectin and growth factors. The concentrations of type I collagen and fibronectin were found to be at least 1000-fold higher than those of several growth factors. In addition, ADSCs promote the mRNA expression

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