C57 mice (n=10; 5–8 weeks old; weighing 20±4 g; sex ratio, 1:1) were purchased from the Experimental Animal Center of the Third Military Medical University (Chongqing, China). The mice were housed in the specific-pathogen free environment with a temperature of 24–28°C, relative humidity of 50–60% and natural light cycle. The mice were given sterilized food, and water with bacitracin (4 g/l) and neomycin (4 g/l) ad libitum. All procedures performed in animals were approved by the Animal Care and Use Committee of the Third Military Medical University. The adult healthy mice mated and the vaginal plug was observed at 8:00 in the morning, the day that vaginal plug was identified was recorded as gestational age 0 day. After 15–20 days of pregnancy, the fetal aorta-derived vascular CD133⁺ cells were obtained from aortas of the mouse embryos following a previously reported protocol (15). Briefly, 1×10⁶ cells were separated from the aorta tissue using EDTA (5 mM), centrifuged at 1,000 × g for 3 min at room temperature and incubated with magnetic microbeads (Miltenyi Biotec, Inc., Cambridge, MA, USA) conjugated to the anti-CD133 antibody (cat. no. ab19898; 1:1,000; Abcam, Cambridge, MA, USA) for 30 min at 4°C. CD133⁺ cells were separated using the Quadro MACS^™ Separation Unit (Miltenyi Biotec, Inc.). CD133⁺ cells were then cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS; both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. In order to simulate the environment of high blood sugar levels in vitro, human umbilical vein endothelial cells (HUVECs), which was purchased from American Type Culture Collection (Manassas, VA, USA), were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 30 mM glucose, 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. All cells were maintained at 37°C in 5% CO₂.

Briefly, 80% polylactic acid, 10% collagen protein and 10% silk (PCS) were dissolved to prepare a mixed solution; the concentration of PCS was adjusted by adding distilled water and the final concentration was 5%. Subsequently, 100 mM SRT1720 (Shanghai BetterBioChem Co., Ltd., Suzhou, China) was added to the mixture to form PCSS. An FM-1205 electrospinning device was purchased from Beijing Future Material Sci-tech Co., Ltd. (Beijing, China). Under an operating voltage of 220 kV/m, the mixed solution was ejected from the nozzle of the electro spinner and collected. The surface of the biological material was photographed using an S-3400N-II scanning electron microscope (Hitachi, Ltd., Tokyo, Japan).

The PCSS composite material was dissolved in the PBS at 37°C for 15 days to determine the release of SRT1720. The release of the sample was detected using a UV-VIS spectrometer (Nicolet Evolution 300; Thermo Fisher Scientific, Inc.) at a wavelength of 428 nm and the release ratio was calculated using Beer's law (16).

The cell was seeded on composite materials, then cell bioactivity was evaluated; the proliferation and adhesion of the cells were the important indexes in the evaluation of cell bioactivity (17). To measure the growth of EACCs, after three passages, 5×10⁵ EACCs were seeded onto the surface of PCS composite material (PCS-EACCs) or PCSS composite material (PCSS-EACCs) following immersion in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in 5% CO₂. Cells were then incubated for 72 h and cell growth was determined using a LSM 780 NLO laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany).

Quantitative analysis of the cytokines secreted by EACCs following different treatments was performed using ELISA. TNF-α (cat. no. PT512; Beyotime Institute of Biotechnology, Beijing, China), VEGFA (cat. no. EK0541; Wuhan Boster Biological Technology, Ltd., Wuhan, China), IL-8 (cat. no. EMC104.48) and bFGF (cat. no. EHC130.48; both Neobioscience Technology Company, Shenzhen, China) ELISA kits were used to determine the concentrations of TNF-α, VEGFA, IL-8 and bFGF according to the manufacturer's protocol.

The cell proliferation ratio was detected using the Cell-Light^™ EdU DNA Cell Proliferation kit (Guangzhou RiboBio Co., Ltd., Guangzhou, China), following the manufacturer's protocol.

Cell migration was measured by performing a wound assay. Briefly, 5×10⁶ HUVECs were seeded in the 6-well plate and cultured in RPMI-1640 medium supplemented with glucose, 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin, and incubated overnight at 37°C in 5% CO₂. A wound was created in each well and the plate was washed 3 times with RPMI 1640. Then 2 ml RPMI 1640 medium with 10% FBS was added into each well; after 24 h, the scar areas of each well were observed and photographed using an Olympus BX50 microscope (Olympus Corporation, Tokyo, Japan; magnification, ×200) and were quantified using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA).

EACCs (5×10⁶) were seeded in 6-well plates, 30 nM glucose was added into DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in 5% CO₂ and incubated at 37°C in 5% CO₂ overnight. Following the adherence of the cells to the wells, the cells were treated with dimethyl sulfoxide (0.1%), PCS (50 mM), SRT170 (10 mM) or PCSS (50 mM). After 36 h of incubation, the cell culture media were collected and stored at −20°C.

Cell invasion was measured using a Transwell assay. Transwell chambers were purchased from Corning, Inc. (Corning, NY, USA). Chambers inserted in the lower chamber were coated with diluted Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). A total of 5×10⁵ HUVECs in RPMI 1640 medium were incubated in the upper chamber; the lower chambers were filled with 500 µl RPMI-1640 medium with 10% FBS. Subsequently, the collected cell culture media (100 µl) were added to the lower chambers. Following 12 h incubation, insert membranes were collected. Cells were stained with 0.5% crystal violet at room temperature for 30 min, photographed and counted using an Olympus BX50 microscope (magnification, ×200).

A total of 120 C57 mice (5–8 weeks old; weighing 20±4 g; sex ratio, 1:1) were used in the current study to establish the diabetes model. A total of 100 5–8 week old C57 mice weighing 20±4 g received an intraperitoneal injection of 40 mg/kg streptozotocin (STZ) everyday following 12 h fasting over a period of 5 days to establish the diabetes model. The remaining 20 mice were with injected intraperitoneally with 1 ml PBS and used as controls. After 5 days of continuous STZ injections, fasting blood glucose levels were detected using the ACCU-CHEK Active meter (Roche Applied Science, Rotkreuz, Switzerland). The glucose levels >16.7 mM indicated that the diabetes model was successfully established; the results indicated that all 100 mice were successfully induced as diabetes models and the control mice were normal. Mice with diabetes were anesthetized with 30 mg/kg sodium pentobarbital (10 mg/ml) via intraperitoneal injection. Subsequently, the terminal branches of the femoral artery were ligated and the skin tissue was excised at an area of 6×6 mm at the lateral thigh to establish the ischemic diabetic ulcer. Subsequently, ulcers were covered by EACCs, EACCs grown on the PCS materials (PCS-EACCs), EACCs pretreated with SRT1720 (SRT1720-EACCs), PCSS and EACCs grown on the PCSS materials (PCSS-EACCs; all n=20). C57 mice in the control group (n=20) underwent the same ulcer surgery and the ulcers were left untreated. Breathable medical dressings were used to bandage the wounds.

Bandages were removed 2 days following treatment. Wound recovery was recorded and photographed using a D90 camera (Nikon Corporation, Tokyo, Japan) on days 3, 7 and 14. Wound areas were measured and calculated using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.) to measure wound recovery. On day 14, the tissues surrounding the wounds were collected, fixed with 4% paraformaldehyde at room temperature for 24 h and embedded in paraffin. Tissues were cut into 5-µm-thick sections, and hematoxylin and eosin (H&E) staining was performed using the Hematoxylin and Eosin Staining Kit (cat. no. C0105; Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer's protocol, then the results were observed by an Olympus BX50 microscope (magnification, ×200). In order to further detect the vascular density, the vascular endothelial cell marker CD31 was selected for immunofluorescence staining. The sections were permeabilized with 0.1% triton X-100 for 15 min at room temperature, then incubated by 100% goat serum (cat. no. C0265; Beyotime Institute of Biotechnology) for 30 min at 37°C. The sections were then incubated with anti-CD31 antibodies (cat. no. ab28364; 1:300; Abcam) diluted in 100% goat serum at 4°C overnight, washed 3 times with PBS, incubated with Alexa Fluor^® 680-conjugated donkey anti-rabbit IgG antibodies (cat. no. A10043; 1:500; Thermo Fisher Scientific, Inc.) diluted in 100% goat serum at 37°C for 2 h and washed 3 times with PBS. The images of the sections were observed through a laser scanning confocal microscope (TCS-SP5; Leica Microsystems GmbH; magnification, ×400) and then the size of endothelium was assessed.

All data are presented as the mean ± standard deviation. The nonparametric Mann-Whitney rank-sum test was used to estimate differences between two samples. Intergroup comparisons were performed to assess differences among >2 groups using one-way analysis of variance followed by Bonferroni's correction. P<0.05 was determined to indicate a statistically significant difference.

To improve the uniformity of the matrix material, electrospinning technology was used to form the PCS (80% polylactic acid, 10% collagen protein and 10% silk) and PCSS (100 nM SRT172 was added to the PCS solution to form the PCSS). Scanning electron microscopy was used to determine the uniformity of the materials and it was identified that the silk itself and the gaps between the silk were uniform, and that the structure of the material was also uniform (Fig. 1A). The materials were regular and uniform and there was no difference in thickness (Fig. 1B). To detect the growth of EACCs on the PCSS, EACCs were seeded on the PCSS and observed using a scanning electron microscope. The results indicated that EACCs were able to grow well on the PCSS, indicating that PCSS effectively promote the growth and proliferation of EACCs (Fig. 1C). To detect the release ability of PCSS for SRT1720, a release experiment was performed and the results indicated that the PCSS is able to release SRT1720 slowly and steadily over a period of 15 days (Fig. 1D). These results indicate that the materials designed in the current study not only promote the growth of EACCs but may also be used to promote the steady release of SRT1720 over a prolonged period.

As Sirt1 is closely associated with the biological function of cells, the PCSS used in the current study contained the Sirt1 agonist SRT1720. A number of experiments were conducted to determine the effect of SRT1720 on the biological function of EACCs. To detect cell proliferation, an EdU kit was used. The results demonstrated that SRT1720 significantly promotes the proliferation of EACCs in a high glucose environment (Fig. 2A and B). ELISA experiments were also performed to determine the effects of SRT1720 and PCSS on EACCs in a high glucose environment. TNF-α is an important cytokine that causes cell death (18). In a high glucose environment, the secretion of TNF-α by EACCs increased significantly; however, the secretion of TNF-α by EACCs was significantly decreased following treatment with SRT1720 and PCSS (Fig. 2C). VEGFA induces angiogenesis and promotes cell migration (19). In a high glucose environment, the secretion of VEGFA by EACCs was significantly inhibited; however, treatment with SRT1720 and PCSS significantly increased the secretion of VEGFA by EACCs (Fig. 2D). IL-8 is a multifunctional factor; it is able to stimulate the migration of neutrophils into inflammatory tissue and activate inflammatory cells and is also able to promote fibroblast proliferation. Additionally, IL-8 is a chemotactic cytokine that can promote inflammatory cell chemotaxis and induce cell proliferation (20). The main role of IL-8 is to attract and activate neutrophils, promote the lysosomal enzyme activity of neutrophils and phagocytosis, and have chemotactic effect on basophils and T cells; IL-8 can induce endothelial cell migration and proliferation, further promoting vascular proliferation (20). As angiogenesis facilitates the healing of diabetic ulcers, levels of IL-8 secreted by EACCs in a high glucose environment were measured. The secretion of IL-8 by EACCs was significantly inhibited in a high glucose environment; however, following treatment with SRT1720 and PCSS treatment, levels of secreted IL-8 were significantly increased; furthermore, these levels were markedly higher than in normal EACCs (Fig. 2E). bFGF is a fibroblast growth factor and fibroblasts effectively promote the formation of scars, thereby promoting wound recovery (21). It was demonstrated that the secretion of bFGF by EACCs was significantly inhibited in high glucose; however, following treatment with SRT1720 and PCSS, the secretion of bFGF was restored (Fig. 2F). Notably, the effect of PCSS on bFGF secretion was greater than that of SRT1720. Taken together, these results demonstrate that, although EACCs treated with SRT1720 and PCSS normalize secretion of the four cytokines, PCSS was more effective than SRT1720 at normalizing cytokine secretion. This may be due to the stable release of SRT1720 by PCSS; SRT1720 is released from PCSS at a rate of ~7.14%/day for 2 weeks.

To verify whether EACCs are able to promote the proliferation and migration of vascular endothelial cells paracrine, EACCs were cultured in high glucose medium (Baseline), or co-cultured in high-glucose medium along with PCS, SRT1720 or PCSS. Subsequently, the conditioned media were collected, and the medium from EACCs cultured in normal medium was collected as a normal control. Collected conditioned media were used to treat HUVECs and determine the effect on the proliferation and migration of HUVECs (Fig. 3). The results of the scratch wound assay indicated that, although HUVECs in the Baseline group underwent migration to a certain degree, the migration rate was much lower than in the normal group. However, treatment of HUVECs with the conditioned media from SRT1720- or PCSS-treated EACCs, the migration of HUVECs increased significantly. HUVECs treated with conditioned medium from PCSS-treated EACCs exhibited the highest rate of migration (Fig. 3A and C). To further examine the invasion of HUVECs following treatments with different conditioned media, a Transwell invasion assay was conducted. The results of this assay were consistent with the results of scratch wound assay; conditioned media from SRT1720- or PCSS-treated EACCs significantly promoted the invasion of HUVECs and the effect of the medium from PCSS-treated EACCs was better (Fig. 3B and D). These results indicated that PCSS significantly promoted the paracrine action of EACCs, thus significantly increasing the migration and invasion of HUVECs. Treatment with conditioned medium from SRT1720-treated EACCs also significantly increased the migration and invasion of HUVECs, but to a lesser extent than PCSS.

To clarify whether PCSS-treated-EACCs promote the recovery of diabetic ischemic ulcers; a model of diabetic ischemic ulcers was established by performing STZ injections in C57 mice; subsequently, the material containing EACCs was used to cover the wound (Fig. 4A). To investigate the effect of PCSS-treated-EACCs, the C57 mice were split into 6 different groups (all, n=20). At 0, 3, 7 and 14 days following transplant, wound healing was recorded and photographed. The results indicated that the wounds of the normal control mice had completely recovered by day 7. The wound healing process in the mice with diabetes was slower; the wound healing process in mice from the EACC group was markedly inhibited compared with the control group. However, in the PCS-EACCs group, the wound healing process was quicker compared with the EACCs group. Furthermore, SRT1720-EACCs had a more beneficial effect on the wound healing process than PCS-EACCs. The effect of PCSS on wound healing was almost the same as that of PCSS-EACCs. Treatment with PCSS-EACCs had the best effect on the healing time of ischemic ulcers in diabetic mice and the recovery rate of the mice in this group was similar to that of mice in the control group (Fig. 4B and C). These results may be due to the stable release of SRT1720 by PCSS over a prolonged period of time and that SRT1720 is able to promote the biological function of EACCs. These results further verify the beneficial effect of PCSS-EACCs on the healing of diabetic ischemic ulcers.

To further evaluate the effect of PCSS-EACCs on the repair of diabetic ischemic ulcers, ulcer tissues from each group of mice were collected and stained with H&E. The results indicated that the immune response was effectively suppressed following PCSS-EACCs treatment; additionally, the amount of vascularization of capillaries in ulcer tissues was markedly improved following PCSS-EACCs treatment (Fig. 5A). In order to further detect the effect of PCSS-EACCs on angiogenesis in diabetic ischemic ulcers, immunofluorescence staining was performed to detect CD31 expression, which is a marker of vascular endothelial cell (Fig. 5B). The results suggested that SRT1720-EACCs could effectively promote the proliferation of vascular endothelial cells and therefore angiogenesis. The effect of PCSS-EACCs on angiogenesis was better compared with that of SRT1720-EACCs, due to the stable release of SRT1720 by PCSS. Wound capillary density was measured using a microscope (Fig. 5C). The results indicated that angiogenesis was significantly promoted following transplantation with PCSS-EACCs, restoring blood supply and promoting the healing of the diabetic ischemic ulcer.