Intradermal injection of human adipose-derived stem cells accelerates skin wound healing in nude mice

Jonathan Rodriguez, Fabien Boucher, Charlotte Lequeux, Audrey Josset-Lamaugarny, Ondine Rouyer, Orianne Ardisson, Héléna Rutschi, Dominique Sigaudo-Roussel, Odile Damour, Ali Mojallal, Jonathan Rodriguez, Fabien Boucher, Charlotte Lequeux, Audrey Josset-Lamaugarny, Ondine Rouyer, Orianne Ardisson, Héléna Rutschi, Dominique Sigaudo-Roussel, Odile Damour, Ali Mojallal

Abstract

Background: The use of stem cells from adipose tissue or adipose-derived stem cells (ASCs) in regenerative medicine could be an interesting alternative to bone marrow stem cells because they are easily accessible and available in large quantities. The aim of this study was to evaluate the potential effect of ASCs on the healing of 12 mm diameter-excisional wounds (around 110 mm(2)) in nude mice.

Methods: Thirty nude mice underwent surgery to create one 12-mm excisional wound per mouse (spontaneous healing, n = 6; Cytocare® 532, n = 12; ASCs, n = 12). The Galiano wound model was chosen to avoid shrinkage and thus slow the spontaneous healing (SH) of mouse skin, making it closer to the physiology of human skin healing. Transparent dressings were used to enable daily healing time measurements to be taken. Immunohistochemistry, histological and blood perfusion analysis were carried out on the healed skin.

Results: The in vivo results showed the effectiveness of using ASCs on reducing the time needed for complete healing to 21.2 days for SH, 17.4 days for vehicle alone (Cytocare® 532) and 14.6 days with the addition of ASCs (p < 0.001). Moreover, cutaneous perfusion of the healed wound was significantly improved in ASC-treated mice compared to SH group, as shown by laser Doppler flowmetry and the quantitation of blood vessels using immunohistochemistry of αsmooth muscle actin.

Conclusions: The tolerance and efficacy of cryopreserved ASCs to accelerate the complete closure of the wound by increasing the maturation of the skin and its blood perfusion, shows their therapeutic benefit in the wound healing context.

Figures

Fig. 1
Fig. 1
Immunophenotyping and trilineage differentiation of ASC. a ASCs were stained with antibodies and analyzed by flow cytometry. ASCs expressed markers CD73, CD90, and HLA ABC and were negative for CD45, CD14, and HLA DR. Representative histograms are shown. b ASCs were cultured in adipogenic, osteogenic or chondrogenic differentiation medium. Cells were fixed and stained with Oil Red O for adipogenesis, Alizarin red for osteogenesis and Alcian blue for chondrogenesis. Representative images are shown. Scale bar = 100 μm. ASCs adipose-derived stem cells, CD cluster of differentiation, HLA human leukocyte antigen
Fig. 2
Fig. 2
Influence of ASCs on wound healing kinetics. a After surgery, nude mice were intradermally injected or not with Cytocare® 532 or ASCs. Representative images are shown for 0, 7, 14, and 21 days post-surgery. Scale bar 5 mm. b Percentage of wound healing was monitored every day until complete wound closure. Results are expressed as means, with error bars indicating SEM. Statistical analysis was performed using the unpaired t-test. * p < 0.05, **p < 0.01 and † p < 0.001 compared to spontaneous healing. c Days to complete wound closure were noted. Results are expressed as means of days needed to reach complete closure and error bars indicate SEM. The Kruskal-Wallis test was followed by Dunn’s multiple comparison tests to estimate the significance of differences for between-group comparisons. *** p < 0.001 compared to spontaneous healing. ASCs adipose-derived stem cells
Fig. 3
Fig. 3
Wound histology after Masson’s trichrome and Picro Sirius red staining. Tissue sections obtained from the wound area at day 27 after cell injection were stained with Masson’s trichrome (left panel) and Picro Sirius red (right panel). Representative micrographs of wound histological images are shown. ASCs adipose-derived stem cells
Fig. 4
Fig. 4
Quantitative and functional aspects of blood perfusion in the healed tissue. a Quantification of perfusion at day 27 after surgery was performed on the healed tissue compared with normal skin using laser Doppler imaging. b Percentage of vasodilation after ACh iontophoresis and c after SNP iontophoresis in SH-, Cytocare® 532- and ASC-treated mice. d One-way ANOVA was followed by a multiple comparison test to estimate the significance of differences for between-group comparisons. Significance was defined at P < 0.05 and ** p < 0.01 vs. control. Tissue sections obtained from the wound area 27 days after cell injection were stained with antibody against alpha smooth muscle actin and blood vessels in the total wound area were quantified. Representative images of the staining are displayed for each group. The Kruskal-Wallis test was followed by Dunn’s multiple comparison test to estimate the significance of differences for between-group comparisons. * p < 0.05 compared to spontaneous healing. All data are expressed as mean ± SEM. Ach acetylcholine, ASCs adipose-derived stem cells, SH spontaneous healing, SNP sodium nitroprusside

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