Table 1.
Summary table of parameters for the light irradiation.
Table 2.
List of antibodies for immunofluorescence staining.
Table 3.
Histological scoring system.
Fig 1.
Enhanced expression of hypoxia-induced survival factors and angiogenic growth factors in hASC L-spheroids.
(A) The light source used was LED (660 nm) designed to fit over a microplate (12.5 × 8.5 cm) for cell culture. (B) Formation of hASC L-spheroids. hASCs morphology on non–tissue culture–treated 24-well plates at day 3. Scale bar = 500 μm. (C) Western blot analysis and quantification of HIF1-α in hASCs cultured as spheroids, L-spheroids and monolayers (*p < 0.01, compared to the L-spheroid group). (D) Angiogenesis-related protein analysis of L-spheroids (*, p < 0.05, compared to the spheroid group, t-test, n = 3 in each group). (E) ELISA measurement of spheroids cultured for 3 days. Concentrations of VEGF are presented as pg-corrected for 104 cells. (*, p < 0.05, compared with spheroid 6J/cm2 group, t-test, n = 3 in each group).
Fig 2.
Endothelial phenotyping of L-spheroids.
(A) Monitoring cell surface markers via immunofluorescence staining. L-spheroids cultured for 3 days were cryosectioned and stained with anti human CD34, CD31, and KDR antibodies. Scale bar = 500 μm. (B) Flow cytometry analysis.
Fig 3.
Survival of transplanted hASCs in the wound bed.
(A) For the ASCs, L-spheroid and L-spheroid + LLLT groups, DAPI (blue) and caspase 3 (apoptotic marker; red)-positive cells were detected after immunostaining at 14 days. The hASCs were stained with HNA (green). Apoptosis of transplanted hASCs (arrows) was reduced in the L-spheroid + LLLT group. (B) The ratio of HNA-positive cells (transplanted hASCs) to DAPI-positive cells (total cells) in the wound bed (*p < 0.01). (C) Ratio of caspase-3-positive cells plus HNA-positive cells (apoptotic transplanted hASCs) to HNA-positive cells (transplanted hASCs) in the wound bed (*p < 0.01).
Fig 4.
Enhanced secretion of angiogenic growth factors from hASCs 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 indicated the expression of bFGF, VEGF, and HGF at 14 days. The results of the Western blot were analyzed as relative density (*p < 0.05, compared to the spheroid + LLLT group).
Fig 5.
Endothelial cell and smooth muscle cell differentiation of transplanted cells.
(A) The implants were removed on day 14 after transplantation and were stained with anti human CD31 and human αSMA antibody. The scale bars indicate 200 μm. (B) Vessel density in the wound bed (*p < 0.05 compared to the spheroid + LLLT group). (C) Western blot indicates the expression of HIF-1α and CD31 at 14 days. (D) Western blot analysis quantification (*p < 0.01, compared to the spheroid + LLLT group).
Fig 6.
Differentiation of ASCs into epithelial cells.
Immunofluorescence images show cytokeratin-positive epithelial cells (red) at 14 days. The scale bar indicates 20 μm.
Fig 7.
Evaluation of the wound closure.
(A) The prepared excisional wound splinting model. (B) Photographs of the wounds. (C) The percentage of the wound area was calculated using photographs of the wounds at 1, 7, and 14 days. *p < 0.05 versus the L-spheroid group. (D, E) Histological analysis of the wound bed. Wounds were stained with (D) H&E and (E) Masson’s trichrome at 14 days. The wound edges are indicated with arrowheads. The closed arrows indicate skin appendages (hair follicles). The scale bar indicates 500 μm. (F) The regeneration of skin appendages was investigated by counting the number of skin appendages per wound section. (G) Histological scoring was performed using the criteria presented in Table 3. *p < 0.05, versus the L-spheroid group.