Adipose-derived stem cells combined with platelet-rich plasma enhance wound healing in a rat model of full-thickness skin defects

Abstract

Background: Wound healing is impaired in patients with diabetes due to the multifactorial etiology of the disease, which limits the therapeutic efficacy of various approaches. This study hypothesizes that the combination of adipose-derived stem cells (ADSCs) and platelet-rich plasma (PRP) might achieve optimally efficient diabetic wound healing.

Methods: ADSCs were isolated from the adipose tissues of Sprague-Dawley (SD) rats. PRP was prepared by using a two-step centrifugation technique. A diabetic wound model was established on the backs of SD rats to evaluate the effect of ADSCs incorporated into PRP. Hematoxylin and eosin staining, immunofluorescence, and immunohistochemistry were performed to observe the changes in neovascularization. ELISA and Western blot were utilized to detect the angiogenesis-related protein expression levels. The proliferation of endothelial cells was assessed by the MTS assay.

Results: ADSCs incorporated into PRP induced a higher wound closure rate than ADSCs, PRP, and negative control. The expression levels of VEGF, p-STAT3, and SDF-1 in the ADSC+PRP group were higher than those in the other groups. Moreover, the proliferation of endothelial cells was strongly stimulated by treatment with the combination of ADSC-conditioned medium (ADSC-CM) and PRP.

Conclusions: PRP enhanced diabetic wound healing induced by ADSCs, and its promoting effect involved neovascularization.

Keywords: Adipose-derived stem cell; Diabetic wound healing; Neovascularization; Platelet-rich plasma.

Fig. 1

Timeline of the experimental design. PBS, PRP, ADSCs, or ADSC+PRP was injected into the wound area, and the wound size was measured and digitally photographed each day until day 14

Fig. 2

Characterization of ADSCs and the optimal concentration of PRP for combination with ADSCs. a Immunophenotypical analysis of ADSCs by flow cytometry. Passage 3 ADSCs, which were positive for CD29 and CD99 and negative for CD34, expressed typical MSC surface antigens. b Multipotent differentiation properties of ADSCs. Passage 3 ADSCs were cultured in adipogenic and osteogenic differentiation media for 21 days. Then, differentiation was evaluated by staining lipid droplets with oil red O (adipogenic, right), and calcium nodules were detected by Alizarin red staining (osteogenic, left). Representative images of 3 independent experiments are shown. Scale bars = 100 μm. c Migration efficiency was evaluated by crystal violet staining and microscopy and quantified with ImageJ. The analysis generally suggested that the optimal concentration of PRP in combination with ADSCs was 20%, with efficiencies ranging from 5 to 100%. All experiments were performed in triplicate and were repeated three times to confirm the findings (*p < 0.05). One-way ANOVA and Tukey’s post hoc test showed statistically significant differences overall between the eight groups. Values were expressed as mean ± SEM (n = 5 high-powered fields per well, three wells per group). Significance was set to *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 3

Wound healing in a diabetic rat excisional wound model and histology of wound beds posttreatment. a Representative images of the wounds show that compared to PBS, PRP, and ADSCs, treatment with ADSC+PRP accelerated wound healing, and full epithelialization was observed by day 10. Scale bar = 1 cm. b The wound closure percentage was evaluated postwounding. The ADSC+PRP group exhibited significantly accelerated wound healing compared to that exhibited by the other groups. Values were expressed as mean ± SEM (n = 12 animals per group, 24 wounds total per group). Statistics were performed using one-way ANOVA and showed statistically significant differences between groups, all six combinations of groups (PBS and PRP, PBS and ADSCs, PBS and ADSC+PRP, PRP and ADSCs, PRP and ADSCs+PRP, and ADSCs and ADSCs+PRP) were statistically significant with *p < 0.05 or better, as evaluated by Tukey post hoc test. c Hematoxylin and eosin staining on day 14 postwounding showed a thicker epidermis and increased appendages in the dermis of the ADSC+PRP group compared with the other groups. Scale bars = 50 μm. d Collagen deposition in the dermis was visualized with Masson’s trichrome staining and microscopy. The analysis generally demonstrated that compared to PBS, PRP, and ADSCs, treatment with ADSC+PRP promoted epithelialization. Scale bars = 50 μm (significance was set to *p < 0.05, **p < 0.01, and ***p < 0.001)

Fig. 4

Evaluation of blood vessel formation. a Early angiogenesis in the wound beds was visualized on day 3 postwounding by hematoxylin and eosin staining. Staining showed an increased number of blood vessels forming in the wound in the ADSC+PRP group compared with the other groups. The black arrows indicate blood vessels. Scale bars = 50 μm. b Representative images of tissue sections from diabetic rats immunostained for CD31 (green), CD34 (pink), and α-SMA (red) and quantification of c the capillary density and the proportion of CD31+, CD34+, and α-SMA+ cells 7 and 14 days postwounding (× 200, scale bars = 50 μm; × 400, scale bars = 20 μm; *p < 0.05; n = 3 wounds per group)

Fig. 5

Effects of ADSC+PRP in promoting angiogenesis and vasculogenesis. a ELISA of VEGF in skin tissue lysates postwounding showed higher levels of VEGF protein production in the wounds of the ADSC+PRP group than those of the other groups. All experiments were performed in triplicate and were repeated three times to confirm the findings. Values were expressed as mean ± SEM. Statistical analysis was evaluated using one-way ANOVA and Tukey post hoc test (significance was set to *p < 0.05, **p < 0.01, and ***p < 0.001). b ADSC+PRP significantly enhanced the proliferation of endothelial cells compared with the ADSC-CM, PRP, or control group; the similar phenomenon was also presented in high-glucose medium. All experiments were performed in triplicate and were repeated three times to confirm the findings. Values were expressed as mean ± SEM. One-way ANOVA and Tukey’s post hoc test showed statistically significant differences overall between the eight groups (significance was set to *p < 0.05, **p < 0.01, and ***p < 0.001). c Protein expression levels of p-STAT3 on wound tissues were detected by Western blot analysis. d Representative images of tissue sections from diabetic rats immunostained for SDF-1 14 days postwounding (× 400, scale bars = 20 μm; *p < 0.05; n = 3 wounds per group)

Fig. 6

Graphical abstract. Graph showing the combined application of ADSCs and PRP could enhance diabetic wound healing