Enhancement of Wound Healing by Conditioned Medium of Adipose-Derived Stromal Cell with Photobiomodulation in Skin Wound

Abstract

Background and objectives: The objective of this study was to investigate whether conditioned medium from photobiomodulation (PBM) irradiated adipose-derived stromal cell (ASC) spheroids prior to implanting could stimulate angiogenesis and tissue regeneration to improve functional recovery of skin tissue in an animal skin wound model.

Methods and results: ASC were split and seeded on chitosan-coated 24 well plate at a density of 7.5×104 cells/cm2, and allowed to adhere at 37°C. Within 3 days of culture, ASC formed spheroids by PBM irradiation. Conditioned medium (CM) fractions were collected from the PBM-ASC to yield nor adipose-derived stromal cell spheroid (spheroid) and PBM-spheroid, respectively, centrifuged at 13,000 g at 4℃ for 10 min, and stored prior to use for ELISA, protein assay, or in vivo wound-healing assays. Phosphate-buffered saline, cultured CM from ASCs, PBM irradiation prior to implanting conditioned medium from ASC, cultured CM from ASC spheroid, and PBM-spheroid-CM (PSC) were transplanted into a wound bed in athymic mice to evaluate therapeutic effects of PSC in vivo. PSC enhanced wound closure in a skin injury model compared to PBS, CM, PBM-CM, and spheroid-CM. The density of vascular formations increased as a result of angiogenic factors released by the wound bed and enhanced tissue regeneration at the lesion site.

Conclusions: These results indicate that implant of PSC can significantly improve functional recovery compared to PBS, CM, PBM-CM, or spheroid-CM treatment. Implant of PSC may be an effective form of paracrine mediated therapy for treating a wound bed.

Keywords: Adipose-derived stromal cell; Angiogenesis; Conditioned medium; Photobiomodulation; Spheroid; Wound healing.

Conflict of interest statement

Potential Conflict of Interest

The authors have no conflicting financial interest.

Figures

Fig. 1

ASC formed spheroids by Low-Level light irradiation. (A) Brief overview of our experiment proce-dure. The light source used was LED (660 nm) designed to fit over a microplate (12.5×8.5 cm) for spheroid formation. ASC morphology on 24 well polystyrene plate at 72 h. Scale bar=500 μm. (B) Western blot analysis and quantification of hypoxia-induced survival factor as hypoxia-inducible factor (HIF)-1α in ASC cultured as spheroids, PBM.AS, and monolayers. (C) Enhanced secretion of angiogenic growth factors from PBM.AS in the wound bed. Angio-genesis-related protein analysis of PBM.AS (*, p4 cells (*, p< 0.05, compared with spheroid 6 J/cm2 group, t-test, n=3 in each group).

Fig. 2

Enhanced secretion of angiogenic growth factors in the wound bed. (A) Immunostaining was performed with anti-bFGF and anti-VEGF or anti-HGF antibody (red) at 14 days. The scale bar indicates 100 μm. (B) Western blot indicating the expression of bFGF, VEGF, and HGF at 14 days.

Fig. 3

Angiogenic efficacy in the wound bed. (A) Implants were removed on day 14 after implanted and stained with anti CD31 and αSMA antibodies. The scale bar indicates 200 μm. (B) Western blot showing the expression of CD31 and αSMA at 14 days. Beta-actin, also known as a “housekeeping” protein, is used as a loading control. (C) Immunofluorescence images showing cytokeratin-positive epithelial cells (red) at 14 days. The scale bar indicates 20 μm.

Fig. 4

Evaluation of wound closure. (A) The prepared excisional wound splinting model. Photographs of wounds. (B) Percentage of wound area was calculated using photographs of wounds at 1, 7, and 14 days. *p