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. 2016 Oct 22;7(1):155.
doi: 10.1186/s13287-016-0412-2.

Localization of Human Adipose-Derived Stem Cells and Their Effect in Repair of Diabetic Foot Ulcers in Rats

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Free PMC article

Localization of Human Adipose-Derived Stem Cells and Their Effect in Repair of Diabetic Foot Ulcers in Rats

Rongfeng Shi et al. Stem Cell Res Ther. .
Free PMC article

Abstract

Background: Diabetic foot ulcer (DFU) is an intractable diabetic complication. Patients suffering from diabetes mellitus (DM) frequently present with infected DFUs. In this study, a wound healing model on diabetic rat foot was established to mimic the pathophysiology of clinical patients who suffer from DFUs. Our study aimed to explore the localization of human adipose-derived stem cells (hADSCs) and the role of these cells in the repair of foot ulcerated tissue in diabetic rats, and thus to estimate the possibilities of adipose-derived stem cells for diabetic wound therapy.

Method: Sprague-Dawley rats were used to establish diabetic models by streptozotocin injection. A full-thickness foot dorsal skin wound was created by a 5 mm skin biopsy punch and a Westcott scissor. These rats were randomly divided into two groups: the hADSC-treated group and the phosphate-buffered saline (PBS) control group. The hADSC or PBS treatment was delivered through the left femoral vein of rats. We evaluated the localization of hADSCs with fluorescence immunohistochemistry and the ulcer area and ulcerative histology were detected dynamically.

Result: The hADSCs had a positive effect on the full-thickness foot dorsal skin wound in diabetic rats with a significantly reduced ulcer area at day 15. More granulation tissue formation, angiogenesis, cellular proliferation, and higher levels of growth factors expression were also detected in wound beds.

Conclusions: Our data suggest that hADSC transplantation has the potential to promote foot wound healing in diabetic rats, and transplantation of exogenous stem cells may be suitable for clinical application in the treatment of DFU.

Keywords: Diabetic foot ulcer; Human adipose-derived stem cells; Tissue repair.

Figures

Fig. 1
Fig. 1
Characterization of hADSCs. a Immunofluorescent assay indicated that ADSCs were positive for cell surface markers CD44, CD29, CD105, and CD90 and were negative for CD133, CD34, and CD36. Scale bar, 50 μm. b Flow cytometry also revealed that CD29, CD44, CD90, and CD105 were highly expressed and CD133, CD34, and CD36 were negligible for the cells. c Triplet differentiation assays revealed that ADSCs could differentiate into adipocytes (a), osteocytes (b), and chondrocytes (c), which were stained by Oil Red O, Alizarin red, and Alcian blue, respectively. Scale bar, 25 μm
Fig. 2
Fig. 2
Diabetic foot ulceration healing at different time points after treatment in the hADSC-treated and control groups. a Representative images of ulcerations on diabetic rat feet at D3, D7, and D15 after treatment with hADSCs and PBS. b Mean area of diabetic foot ulceration in hADSC-treated model rats during observation compared with control model rats at D3, D7, and D15 after treatment. Averaged data presented as mean ± SD. *P < 0.05, **P < 0.01. d days, hADSC human adipose-derived stem cell, PBS phosphate-buffered saline. Representative images and mean area of the foot ulcers in the euglycemic rats group were shown in additional file 1
Fig. 3
Fig. 3
Effects of hADSCs on the epithelialization and granulation tissue regeneration at D15 after treatment. a H&E staining of sections showed better dermal re-epithelialization on the foot ulcers in model rats treated with hADSCs compared with control model rats. Scale bar, 100 μm. b Granulation tissues in the hADSC-treated group were thicker than those in the PBS-treated group. Scale bar, 100 μm. c Statistical thickness of granulation tissues by computer-assisted morphometric analysis. d Collagen deposition assessed by Masson Trichrome staining. The hADSC-treated group showed more intense blue staining than the PBS-treated group, suggesting that hADSC treatment accelerated collagen deposition in the granulation tissues. Scale bar, 50 μm. e Quantification analysis of the Masson Trichrome staining section by digital image analysis. Averaged data presented as mean ± SD. *P < 0.05. hADSC human adipose-derived stem cell, PBS phosphate-buffered saline
Fig. 4
Fig. 4
Effects of hADSCs on angiogenesis, cell proliferation, and apoptosis of wounds at D15 after treatment. a Immunohistochemical staining of CD31 showed more blood vessel structures on the wound bed in model rats treated with hADSCs were clearly increased compared with control model rats. Scale bar, 50 μm. b Quantification of blood vessel density expressed as the average number of CD31+ vessels per high-power field. c Cellular proliferation on the wound bed observed by Ki-67 immunofluorescence staining. The hADSC-treated group showed more positive cells than the PBS-treated group, indicating that hADSC treatment promoted cell proliferation in DFU healing. Scale bar, 100 μm. d Quantification analysis of the Ki-67 staining by digital image analysis. e Apoptotic cells detected by TUNEL assays. Few apoptotic cells were detected in the hADSC-treated group, suggesting that hADSC treatment reduced the apoptosis in the process of healing. Scale bar, 100 μm. f Quantification of TUNEL immunofluorescence staining by digital image analysis. Averaged data presented as mean ± SD. *P < 0.05, **P < 0.01. hADSC human adipose-derived stem cell, PBS phosphate-buffered saline, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling
Fig. 5
Fig. 5
Fate of hADSCs after transplantation in DFUs. a hADSCs were labeled by the lentivirus expressing ZsGreen and detected under bright-field and green fluorescence. Scale bar, 25 μm. b Images of transplanted ZsGreen-positive cells at different time points in DFUs. The rate of ZsGreen-positive cells was gradually increased during the first week after transplantation, with the highest intensity on D7. Scale bar, 100 μm. c Rate of ZsGreen-positive cells at different time points. d days, h hours, hADSC human adipose-derived stem cell
Fig. 6
Fig. 6
Effect of hADSC treatment on key growth factors and inflammatory factors in the DFU tissues. a Levels of VEGF, bFGF, and TGF-β in the DFU tissues at different time points were determined by enzyme-linked immunosorbent assay (ELISA).The level of VEGF, bFGF, and TGF-β was obviously higher in the hADSC-treated group compared with the PBS-treated group on D7 but there were no significant differences on D3.The higher level of VEGF and bFGF continued to D15 after treatment. b The R&D Systems Rat Cytokine Antibody Proteome Profiler Array system was used to screen for the inflammation-related cytokines and chemokines in the DFU tissues on D3 after treatment. Quantification of cytokines and chemokines mean pixel density was analyzed by Image-Pro Plus 6.0. *P < 0.05, **P < 0.01. d days, hADSC human adipose-derived stem cell, PBS phosphate-buffered saline

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