Enhanced wound healing effect of canine adipose-derived mesenchymal stem cells with low-level laser therapy in athymic mice

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Abstract

Background

Adipose-derived mesenchymal stem cells (ASCs) are attractive cell source for skin tissue engineering. However, one obstacle to this approach is that the transplanted ASC population can decline rapidly in the recipient tissue.

Objective

The aim of this study was to investigate the effects of low-level laser therapy (LLLT) on transplanted canine ASCs in a skin wound animal model.

Methods

LLLT, ASC transplantation (ASCs) and ASC transplantation with LLLT (ASCs + LLLT) were applied to the wound bed in athymic mice. Wound healing was assessed by gross evaluation and by hematoxylin and eosin staining. The survival, differentiation and secretion of vascular endothelial growth factor and basic fibroblast growth factor of the ASCs were evaluated by immunohistochemistry and Western blotting.

Results

The ASCs and ASCs + LLLT groups stimulated wound closure and histological skin regeneration. The ASCs + LLLT group enhanced the wound healing, including neovascularization and regeneration of skin appendages, compared with the ASCs group. The ASCs contributed skin regeneration via differentiation and secretion of growth factors. In the ASCs + LLLT group, the survival of ASCs was increased by the decreased apoptosis of ASCs in the wound bed. The secretion of growth factors was stimulated in the ASCs + LLLT group compared with the ASCs group.

Conclusion

These data suggest that LLLT is an effective biostimulator of ASCs in wound healing that enhances the survival of ASCs and stimulates the secretion of growth factors in the wound bed.

Introduction

Wound healing can be delayed because of the wound location, personal history of disease or large area of skin defect resulting from trauma, burn or surgery. To accelerate skin regeneration, many skin tissue engineering techniques, such as the use of various scaffolds, cells and growth factors, have been researched. The ultimate goal for skin tissue engineers is to regenerate skin such that the complete structural and functional properties of the wound area are restored to the levels before injury. However, only a subset of the functions can be restored with existing tissue engineering techniques [1].

Mesenchymal stem cells (MSCs), which are found in many adult tissues, are an attractive cell therapy source for the regeneration of damaged tissues because they are able to self-renew and are capable of differentiating into various cells and tissues [2]. In cutaneous repair, MSCs significantly accelerate wound closure, exhibiting increased re-epithelialization, neovascularization and regeneration of skin appendages [3]. However, several studies claim that the effects of stem cell therapy are not dramatic in the absence of scaffolds or stimulators [3], [4]. Recently, various scaffolds or growth factors were studied to increase the skin regeneration effect of stem cells [5], [6], [7].

Low-level laser therapy (LLLT) has been used for various purposes, such as relief of pain and inflammation and improvement in the local circulation, for a long time. Moreover, many studies have demonstrated positive biostimulatory effects of LLLT on cells. LLLT can stimulate cellular responses and promote the migration and proliferation of various cells [8], [9], [10]. The proliferation, growth factor secretion and differentiation of MSCs were also enhanced by LLLT [11], [12], [13]. However, little is known about the effects of LLLT on transplanted stem cells in animal models.

This study was performed to determine the effect of LLLT on transplanted adipose-derived mesenchymal stem cells (ASCs) in a skin wound animal model. The ASC transplantation and LLLT were applied in the wound beds of athymic mice. We compared the skin regeneration effects between the ASC transplantation (ASCs) group and the ASC transplantation with LLLT (ASCs + LLLT) group.

Section snippets

Culture of ASCs

We use canine ASCs that were supplied from Ryu (Department of Veterinary Surgery, College of Veterinary Medicine, Seoul National University); these ASCs were previously characterized [14]. The ASCs were cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM, Gibco®, Life Technologies, NY, USA) with 10% fetal bovine serum (FBS, Gibco®, Life Technologies, NY, USA), 100 units/ml penicillin and 100 μg/ml streptomycin at 37.0 °C, in a 5% CO2 incubator. At passage 4 or 5, green fluorescent

Wound closure

The excisional wound splinting model was prepared, and the silicon splints remained tightly adherent to the skin and restricted wound contraction during the experimental period (Fig. 1A). Our data showed that the ASCs and ASCs + LLLT groups accelerated wound closure (Fig. 1B). At 3, 7, 10 and 14 days after surgery, the ASCs and ASCs + LLLT groups exhibited significantly smaller wound areas than did the other groups. At 10 days, the ASCs + LLLT group showed a significantly smaller wound area than the

Discussion

In this study, ASCs accelerated wound closure with increased re-epithelialization, granulation, neovascularization and regeneration of skin appendages. In addition to the hair follicles and sebaceous glands, some cytokeratin positive-ASCs were observed in regenerated epidermis. Additionally, some VEGF or bFGF positive-ASCs were detected in the wound bed. VEGF is most effective and specific growth factor that regulate angiogenesis [18]. bFGF is important growth factor in wound healing because of

Acknowledgement

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (2011-0009338).

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