Effect of negative-pressure wound therapy on the circulating number of peripheral endothelial progenitor cells in diabetic patients with mild to moderate degrees of ischaemic foot ulcer

Abstract

Objective

To investigate the effect of negative-pressure wound therapy (NPWT) on the circulating number of endothelial progenitor cells (EPCs) in diabetic patients with mild to moderate degrees of ischemic foot ulcer.

Methods

We selected 84 diabetic patients who had a foot ulcer with a duration of at least four weeks and who had an ankle-brachial index of 0.5–0.9. Patients were assigned to one two groups according to 2:1 randomization: NPWT group (n = 56) and non-NPWT (patients who did not receive NPWT) group (n = 28). The control group (NC group) was composed of 18 patients who had normal glucose tolerance and lower extremity ulcer without arteriovenous disease. NPWT was performed on the ulcer after debridement for one week for patients in both the NPWT group and the NC group, and the patients in the non-NPWT group received conventional treatment process. The circulating number of EPCs was measured before and after various treatments, and the factors influencing their changes were analysed.

Results

After NPWT, the circulating number of EPCs significantly increased in both the NPWT group and the NC group ((85.3 ± 18.1) vs. (34.1 ± 12.5)/10⁶ cells; (119.9 ± 14.4) vs. (66.1 ± 10.6)/10⁶ cells, both P < 0.05). In contrast, the circulating number of EPCs had no significant change in the non-NPWT group ((45.2 ± 19.4) vs. (34.7 ± 16.8)/10⁶ cells, P > 0.05). In addition, the circulating levels of vascular endothelial growth factor (VEGF) and the protein expressions of VEGF and stromal cell-derived factor-1α (SDF-1α) in the granulation tissue significantly increased after NPWT in both the NPWT and the NC group, but there was no significant change in the non-NPWT group. Compared with the non-NPWT group, the changes in VEGF and SDF-1α levels in the sera and granulation tissue were all significantly higher in both the NPWT and NC groups (P < 0.05, P < 0.01, respectively). There was no significant difference in changes in the circulating number of EPCs in the peripheral blood and levels of VEGF and SDF-1α in the sera and granulation tissue between the NPWT and NC groups. Correlation analysis showed that the change in the circulating number of EPCs was correlated with the changes of VEGF and SDF-1α levels in the sera and granulation of the NPWT and NC groups (P < 0.05).

Conclusion

NPWT may increase the circulating number of EPCs in diabetic patients with mild to moderate ischaemic foot ulcer as in non-diabetic controls, which may be attributed to the upregulation of systemic and local VEGF and SDF-1α levels.

Keywords

Diabetic foot ulcer endothelial progenitor cell negative-pressure wound therapy cytokines

Introduction

A prospective analysis of a large cohort of patients with diabetes and patients with diabetic foot ulcer (DFU) shows that the annual incidence of DFU is 8.1% in Chinese patients older than 50 years who have diabetes; DFU is one of the main causes of non-traumatic amputation.¹ Adequate surgical debridement, effective antibiotic therapy, correction of metabolic abnormalities, and proper wound management are essential for healing of DFUs.^2,3 Despite painstaking management, DFUs frequently fail to heal completely. Therefore, a series of new treatment methods were recently applied to DFUs.^4,5 Negative-pressure wound therapy (NPWT) is an adjuvant therapy that uses negative pressure to evacuate fluid from open wounds through a sealed dressing and a tube that is connected to a suction device.^6,7 NPWT has been widely adopted to treat many acute and chronic wounds, including diabetic foot infections and pressure ulcers.^8,9 Blood supply of the wound is one of the key factors affecting the DFU healing outcome. Some randomized controlled trials have confirmed that NPWT can effectively promote DFU wound healing,^5,10 and one of its mechanisms may be related to its role in promoting wound vessel formation and improving wound circulation.¹¹ However, the specific effect path is not clear. Endothelial progenitor cells (EPCs) are precursor cells of vascular endothelial cells, which are stimulated by physiological or pathological factors, and can be mobilized from the bone marrow to the peripheral blood and transfer finally to the ischaemic site to repair the damaged blood vessels.^12,13 In recent years, there have been several studies focusing on the role of EPCs in DFU healing.^14,15 The application of EPCs to the wounds of diabetic rats can increase the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor as well as the formation of neovascularization in the wound, thus promoting healing of the wound.¹⁶ Seo et al.¹⁷ found that the circulating number of EPCs in patients with a DFU during one or two courses of NPWT adjuvant therapy increased significantly, suggesting that wound healing may be promoted by increasing the number of peripheral blood EPCs and related physiological effects. However, previous studies could not determine whether the changed number of peripheral blood EPCs after NPWT in the observed subjects was attributed to a direct effect of NPWT.

The purposes of the present study therefore were to determine whether local NPWT has an direct effect on the number of peripheral blood EPCs in DFU patients with mild to moderate ischaemia and to assess whether the effect of NPWT on the number of circulating EPCs differed between patients with DFU and patients with a lower extremity ulcer who had normal glucose tolerance.

Subjects and methods

Study subjects

From January 2013 to December 2015, a total of 84 patients with DFU were admitted to the Department of Endocrinology, the First Affiliated Hospital of Anhui Medical University. There were 49 male and 35 female patients, with an average age of 55.7 ± 11.9 years and a history of diabetes for 3–18 years. Sixteen patients had type 1 diabetes mellitus and 68 patients had type 2 diabetes mellitus. All the patients met the following inclusion criteria: (1) the ulcer course was four weeks or longer; (2) the ulcer area (ULA) was 2–20 cm² and Wagner grade 2–4; (3) the ankle-brachial index (ABI) was 0.5–0.9; and (4) the fasting plasma glucose (FPG) concentration was <10 mmol/L. The DFU patients were assigned to one of two groups according to 2:1 randomization: an NPWT treatment group (NPWT group) composed of 56 patients and a group that did not receive NPWT group (non-NPWT group) composed of 28 patients. In the NPWT group, 13 patients received statin treatment. In the non-NPWT group, seven patients received statin treatment. In addition, 18 patients with lower extremity ulcer in the Department of Burns, the First Affiliated Hospital of Anhui Medical University were selected as the control group (NC group). This group consisted of 10 male and 8 female patients, with normal glucose tolerance and without lower extremity arteriovenous disease; their ulcer course was four weeks or longer, their average age was 56.8 ± 9.3 years and five patients received statins. The ratio of sex, age, and statin use among the three groups matched. None of the subjects had serious heart, liver, or kidney dysfunction or cancerous ulcer wounds, and none had a previous history of ulceration ulcers. None of the subjects had previously used NPWT, and none received treatment with glucocorticoids, immunosuppressive agents, oestrogens, or exogenous cytokines, such as epidermal growth factor, erythropoietin, or recombinant human granulocyte colony-stimulating factor, within the past six months. No statins were added during the study (retained before use). This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Anhui Medical University, and informed consent of the subjects was obtained.

Research methods

All subjects were given routine systemic treatment, including anti-infective, antihypertensive, hypoglycaemic (for diabetic patients), correcting hypoalbuminaemia and anaemia treatment. Debridement was performed to remove the dark necrotic soft tissue and bone tissue. Patients receiving NPWT underwent a persistent negative pressure aspiration with use of VAC® negative pressure-assisted healing treatment system (Kinetic Concepts, Inc., USA) to adjust the negative pressure to 125 mm Hg (1 mm Hg = 0.133 kPa) for one week. Patients in the non-NPWT group received conventional dressing treatment according to the specific circumstances of the wound and did not use drugs or dressings containing exogenous cytokines. Cubital venous blood and wound granulation tissue samples of subjects receiving NPWT were collected before NPWT and one week after NPWT (the last day of NPWT). Cubital venous blood and wound granulation tissue samples from the non-NPWT patients were taken on the day of debridement and one week after surgery. The number of cells that were CD34⁺, CD133⁺, and KDR⁺ were counted with flow cytometry (BD FACSCalibur) and represented the number of EPCs. Inflammatory markers of peripheral blood were measured one week before and after treatment, including serum C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), peripheral white blood cell (WBC) count, and serum VEGF, SDF-1α, and albumin (ALB) levels. The ULA was measured with the use of a digital camera combined with software analysis. The ABI was measured before and after treatment by using a Doppler flow detector. The percutaneous oxygen partial pressure (TcPo₂) around the ulcer before and after treatment was measured by using the percutaneous oxygen partial pressure detector. Serum VEGF and SDF-1α were measured by the ELISA method. All patients had all the described measurements made; Δ represents change in the observed variable after one week of therapy and before treatment.

Western blot analysis was used to detect the expression of VEGF and SDF-1α in the wound tissue before and after treatment. Wound granulation tissue (100 mg) was homogenized and centrifuged at 12,000 r/min for 10 min at 4°C. The protein concentration was determined by use of the bicinchoninic acid (BCA) method. Protein was extracted and denatured at 100°C for 5 min, and then sampling, electrophoresis, film transfer, and closure were conducted. VEGF and SDF-1α primary antibodies (Sigma), diluted 1:300 and 1:400, respectively, were added and incubated overnight. The secondary antibody (Sigma), diluted 1:1000 and 1:1500, respectively, was added for 2-h incubation for ECL autography the next day. The bands obtained from Western blotting were scanned into images, and the grey value of the target bands were analysed with an image processing system. Then, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping protein and was determined following the same procedure as mentioned earlier. The ratios of VEGF and SDF-1α protein to its own GAPDH in granulation tissue were used as the relative expression level of each target gene.

Statistical analysis

The SPSS statistical software package for Window version 17.0 (SPSS, Chicago, IL, USA) was used for all statistical analyses. Each variable was examined for normal distribution, and significantly skewed variables were log transformed. Results are expressed as mean ± SD. Paired-samples t-test was used before and after treatment in each group. One-way ANOVA was used for comparison among multiple groups, and SLD analysis was used for comparison between the two groups. Pearson correlation analysis and multiple linear stepwise regression analysis were used to explore the factors affecting the changes of EPCs before and after treatment. All P-values are two-sided, and a value of <0.05 was considered statistically significant.

Results

Characteristics of the study population

A total of 102 patients participated in the study, and all patients completed the experiment including the described measurements. No significant side effects or other adverse events were observed during treatments. The baseline characteristics of the three groups of patients are summarized in Table 1. In addition, diabetic foot wounds of all patients in the NPWT group and NC group were healed during follow-up, and none of the subjects received amputation after NPWT. However, in the NC group, one case received minor amputation and the other received major amputation during follow-up.

Table 1.

Comparisons of clinical indices between pre-treatment and post-treatment in three groups ((mean±SD), n (%)).

Variables	NPWT	Non-NPWT	NC
Number
Pre-treatment	56	28	18
Post-treatment	56	28	18
Gender male, %
Pre-treatment	33 (58.9)	16 (57.1)	10 (55.6)
Post-treatment	33 (58.9)	16 (57.1)	10 (55.6)
Age, year
Pre-treatment	57.8±10.6	56.4±11.8	56.8±9.3
Post-treatment	57.8±10.6	56.4±11.8	56.8±9.3
Duration, year
Pre-treatment	10.8±7.2	11.2±8.1	–
Post-treatment	10.8±7.2	11.2±8.1	–
FPG, mmol/L
Pre-treatment	10.7±3.6^b	10.6±4.2^b	5.5±0.5
Post-treatment	8.1±2.8^d	8.3±2.9d	5.4±0.6
Hb_A1c, %
Pre-treatment	9.4±1.8^b	9.3±1.7^b	5.6±0.6
Post-treatment	–	–	–
ALB, g/L
Pre-treatment	32.8±3.1	33.1±1.9	34.9±0.8
Post-treatment	33.1±2.9	33.2±2.4	35.1±1.2
TcPo₂, mmHg
Pre-treatment	38.9±11.7^b	39.1±13.8^b	70.4±7.8
Post-treatment	42.4±12.3	39.2±13.2	70.6±7.2
ABI
Pre-treatment	0.75±0.12^b	0.76±0.11^b	1.11±0.06
Post-treatment	0.76±0.14	0.76±0.13	1.12±0.05
CRP, mg/dl
Pre-treatment	35.2±0.6	34.2±0.5	35.5±1.1
Post-treatment	22.5±0.9^c	27.6±0.7^c	21.9±0.7^c
WBC, ×10⁹/L
Pre-treatment	11.2±0.3	10.5±0.5	11.7±0.8
Post-treatment	8.5±0.5^d	9.6±0.9^c	7.7±1.1^d
ESR, mm/h
Pre-treatment	58±17	55±15	57±12
Post-treatment	43±15^c	49±12	33±13^c
SDF-1α, pmol/ml
Pre-treatment	2851.6±997.3	2945.2±1021.2	2810.3±756.2
Post-treatment	3037.4±1003.2	2957.1±989.4	3026.9±639.5
VEGF, pmol/ml
Pre-treatment	266.7±78.7	269.5±90.4	258.5±69.4
Post-treatment	321.5±82.5^c	275.6±94.3	365.3±78.7^c
EPCs,×10^–6 cell
Pre-treatment	34.1±12.5^a	34.7±16.8^a	66.1±10.6
Post-treatment	85.3±18.1^d	45.2±19.4	119.9±14.4^d
ULA, cm²
Pre-treatment	10.2±4.1	10.7±4.4	12.1±6.6
Post-treatment	9.3±5.4	10.1±5.2	11.2±7.2

Note: Age expressed as means±SD and the P-value was evaluated by ANOVA. The P-values were evaluated by χ² test for gender composition.

NC: control group; NPWT: negative-pressure wound therapy group; non-NPWT: without receiving NPWT group; Duration: course of diabetes mellitus; FPG: fasting plasma glucose; Hb_A1c: glycated haemoglobin A1c; ALB: serum albumin; TcPo₂: transcutaneous oxygen pressure; ABI: ankle brachial index; CRP: C-reactive protein; WBC: white blood cell; ESR: erythrocyte sedimentation rate; SDF-1α: stromal cell derived factor alpha 1; VEGF: vascular endothelial growth factor; EPCs: endothelial progenitor cells; ULA: ulcer area; vs. pre-treatment in NC group.

^aP < 0.05, ^bP < 0.01; vs. Pre-treatment in each group, ^cP < 0.05, ^dP < 0.01.

Comparisons of clinical indices between pre-treatment and post-treatment in the three groups

There were no significant differences in age, gender composition, serum ALB level, CRP level, WBC count, ESR level, UL, and serum SDF-1α and VEGF levels among the three groups before treatment. No significant differences between the NPWT group and the non-NPWT group were seen with respect to the duration of diabetes, the levels of FPG, glycated haemoglobin (Hb_A1c), TcPo₂, and ABI and the number of EPCs. However, the values of TcPO2, ABI, and the number of EPCs in the NPWT group and the non-NPWT group were significantly lower than those in the NC group (P < 0.05 or P < 0.01), and the levels of FPG and Hb_A1c were significantly higher than those in NC group (P < 0.01) (Table 1).

Compared with pre-treatment, the values for CRP, ESR, and WBC count significantly decreased (P < 0.05 or P < 0.01), while the circulating number of EPCs and the level of VEGF in peripheral blood significantly increased (P < 0.01) after treatment in both the NPWT and NC groups. In addition, the level of FPG significantly decreased after treatment in the NPWT group (P < 0.05). In the non-NPWT group, compared with pre-treatment, the levels of FPG and CRP and the WBC count after treatment were significantly lower (P < 0.05), but the circulating number of EPCs, the levels of VEGF in peripheral blood, and ESR remained significantly unchanged (P > 0.05). Similarly, no significant difference was seen between pre-treatment and post-treatment with respect to the values of TcPo₂, ABI, ULA, serum ALB concentrations, and SDF-1α levels in peripheral blood in three groups (P > 0.05) (Table 1).

Comparisons of Δ value of clinical indices before and after treatment among three groups

Compared with the non-NPWT group, the levels of ΔTcPo₂, ΔSDF-1α, ΔVEGF, and ΔEPCs in peripheral blood all significantly increased in the NPWT group (P < 0.05 or P < 0.01), while the levels of ΔCRP, ΔWBC, and ΔESR significantly decreased (P < 0.05 or P < 0.01) in the NPWT group. No significant difference in the levels of ΔFPG was observed between the non-NPWT group and the NPWT group (P > 0.05). Compared with the NC group, the levels of ΔTcPo₂ significantly increased in the NPWT group (P < 0.05), but no significant differences were found with respect to the levels of ΔCRP, ΔWBC, ΔESR, ΔSDF-1α, ΔVEGF, and ΔEPCs in peripheral blood (P > 0.05). In addition, there were no significant differences in the levels of ΔALB, ΔABI, and ΔULA among three groups (P > 0.05) (Table 2).

Table 2.

Comparisons of Δ value of clinical indices before and after treatment among three groups (mean±SD).

Variables	NPWT	Non-NPWT	NC
Number	56	28	18
ΔFPG, mmol/L	–2.63 ± 0.62^b	–2.34 ± 0.64^b	–0.09 ± 0.03
ΔALB, g/L	0.21 ± 0.08	0.11 ± 0.08	0.17 ± 0.09
ΔTcPo₂, mmHg	4.47 ± 0.81^ac	0.11 ± 0.02	0.18 ± 0.08
ΔABI	0.01 ± 0.01	0.01 ± 0.02	0.01 ± 0.01
ΔCRP, mg/dl	–10.68 ± 0.14^d	–3.58 ± 0.24^b	–11.57 ± 0.31
ΔWBC, ×10⁹	–2.59 ± 0.14^d	–0.87 ± 0.23^b	–3.78 ± 0.12
ΔESR, mm/h	–15.14 ± 2.74^d	–6.04 ± 2.89^b	–13.92 ± 1.83
ΔSDF-1α,pmol/ml	135.71 ± 52.43^d	13.39 ± 15.24^b	104.31 ± 49.22
ΔVEGF, pmol/ml	56.17 ± 10.14^d	6.15 ± 10.28^b	97.21 ± 22.17
ΔEPCs, ×10^–6 cell	51.18 ± 5.96^d	10.51 ± 6.38^b	53.76 ± 8.21
ΔULA, cm²	0.86 ± 0.22	0.58 ± 0.13	0.91 ± 0.27

NC: control group; NPWT: negative-pressure wound therapy group; non-NPWT: without receiving NPWT group; FPG: fasting plasma glucose; ALB: serum albumin; TcPo₂: transcutaneous oxygen pressure; ABI: ankle brachial index; CRP: C-reactive protein; WBC: white blood cell; ESR: erythrocyte sedimentation rate; SDF-1α: stromal cell-derived factor alpha 1; VEGF: vascular endothelial growth factor; EPCs: endothelial progenitor cells; ULA: ulcer area; Δ: The change value of various index before and after treatment; vs. NC group, ^aP < 0.05, ^bP < 0.01; vs. non-NPWT group, ^cP < 0.05, ^dP < 0.01.

Comparisons of the protein expressions and the Δ value of VEGF and SDF-1α in granulation before and after treatment in three groups

Compared with pre-treatment, the protein expressions of VEGF and SDF-1α in granulation tissue significantly increased after NPWT in both the NPWT group and the NC group (P < 0.05), but no significant difference was observed in the non-NPWT group (P > 0.05) (Figure 1). In addition, compared with the non-NPWT group, the levels of ΔVEGF and ΔSDF-1α protein in granulation tissue were all significantly higher in the NPWT group and the NC group (P < 0.05). There were no significant differences in the levels of ΔVEGF and ΔSDF-1α in the granulation tissue between the VAC and NC groups (P > 0.05).

Figure 1.

The protein expressions of VEGF and SDF-1α in the granulation tissue before and after treatment in three groups. Bands 1, 2, and 3 represent non-NPWT, NPWT, and NC groups before treatment, respectively; bands 4, 5, and 6 represent non-NPWT, NPWT, and NC groups after treatment, respectively; the images in (a) and (b) were representatives of VEGF and SDF-1α expression detected by Western blotting analysis in the granulation tissue before and after treatment, respectively. Each bar represents mean±SD (n = 28 for non-NPWT group, n = 56 for NPWT group, n = 18 for NC group, respectively) vs. before treatment, ^aP < 0.05.

Pearson correlation analysis of the Δ value of the circulating number of EPCs with the Δ value of other clinical indices in three groups

Pearson correlation analysis showed that the levels of ΔEPCs in the NPWT and NC groups were negatively correlated with those of ΔWBC, ΔCRP, and ΔESR and positively correlated with the levels of ΔSDF-1α and ΔVEGF in the sera and ΔSDF-1α and ΔVEGF in the granulation tissue (P < 0.05). In the NPWT group, the level of ΔEPCs was positively correlated with the level of ΔTcPo₂ (P < 0.05). In the non-NPWT group, the levels of ΔEPCs were negatively correlated with those of ΔWBC and ΔCRP (P < 0.05). In both the VAC and NC groups, we found that the level of ΔSDF-1α in the sera was positively correlated with the level of ΔSDF-1α in the granulation tissue (r = 0.683, P < 0.01), and the level of ΔVEGF in the sera was positively correlated with that of ΔVEGF in the granulation tissue (r = 0.521, P < 0.01). However, in the non-NPWT group, we did not observe any significant correlations of the levels of ΔSDF-1α and ΔVEGF in the sera with those of ΔSDF-1α and ΔVEGF in the corresponding granulation tissue (P > 0.05) (Table 3).

Table 3.

Pearson correlation analysis of ΔEPCs with Δ value of other clinical indices in three groups (r).

Variable	NPWT (n=56)		Non-NPWT (n=28)		NC（n=18）
Variable	r^a	P	r^a	P	r^a	P
△FPG	–0.112	0.324	–0.145	0.263	0.052	0.597
△TcPo₂	0.198	0.042	0.101	0.344	0.098	0.363
△ABI	0.072	0.514	0.175	0.131	0.102	0.251
△WBC	–0.193	0.047	–0.201	0.031	–0.205	0.032
△CRP	–0.206	0.025	–0.193	0.042	–0.189	0.048
△ESR	–0.195	0.039	–0.181	0.093	–0.275	0.014
△ULA	–0.161	0.235	–0.173	0.144	–0.186	0.056
△S-SDF-1α	0.211	0.027	0.192	0.062	0.197	0.037
△S-VEGF	0.414	0.002	0.113	0.207	0.286	0.012
△T-SDF-1α	0.204	0.033	0.176	0.142	0.198	0.041
△T-VEGF	0.312	0.008	0.104	0.432	0.296	0.011

^aCorrelation coefficients and P values were determined using Spearman’s correlation analysis.

NC: control group; NPWT: negative-pressure wound therapy group; non-NPWT: without receiving NPWT group; FPG: fasting plasma glucose; TcPo₂: transcutaneous oxygen pressure; ABI: ankle brachial index; CRP: C-reactive protein; WBC: white blood cell; ESR: erythrocyte sedimentation rate; SDF-1α: stromal cell-derived factor alpha 1; VEGF: vascular endothelial growth factor; EPCs: endothelial progenitor cells; ULA: ulcer area; Δ: The change value of various index before and after treatment.

Multivariate linear stepwise regression analysis

In three groups, the level of ΔEPCs was referred to as a dependent variable, and the Δ values of other clinical indices were referred to as independent variables. The multivariate linear stepwise regression analysis revealed that the levels of ΔWBC and ΔCRP were independent factors associated with the level of ΔEPCs in the non-NPWT group (P < 0.05); meanwhile, the levels of ΔVEGF in the sera and of ΔVEGF and ΔSDF-1α in the granulation tissue were independent factors associated with the level of ΔEPCs in the NPWT group (P < 0.05). In addition, the levels of ΔVEGF in the sera and ΔVEGF in the granulation tissue were independent factors associated with the level of ΔEPCs in the NC group (P < 0.05) (Table 4).

Table 4.

Multiple linear regression analysis of ΔEPCs with Δ value of other clinical indices in three groups.

Group	Variable	β	SE	t	P	95% CI
Non-VAC	△WBC	‒0.269	0.022	‒3.027	0.011	0.236–0.951
Non-VAC	△CRP	‒0.294	0.051	‒4.103	0.003	0.121–0.835
VAC	△S-VEGF	0.337	0.061	5.026	0.000	0.027–0.475
	△T-VEGF	0.283	0.049	3.182	0.009	0.013–0.789
	△T-SDF-1α	0.195	0.076	2.191	0.028	0.058–0.942
NC	△S-VEGF	0.292	0.057	2.975	0.012	0.035–0.702
NC	△T-VEGF	0.202	0.068	2.017	0.032	0.168–0.933

NC: control group; NPWT: negative-pressure wound therapy group; non-NPWT: without receiving NPWT group; WBC: white blood cell; C-reactive protein; VEGF: vascular endothelial growth factor; SDF-1α: stromal cell derived factor alpha 1; EPCs: endothelial progenitor cells; Δ: The change value of various index before and after treatment; S: serum; T: tissue.

Discussion

In the present study, we found that NPWT for one week could significantly increase the number of EPCs in peripheral blood in diabetic patients with mild to moderate ischaemic foot ulcer and patients with non-diabetic and non-ischaemic lower extremity ulcers, whereas there was no significant change in the circulating number of EPCs in DFU patients with mild to moderate ischaemia treated with conventional wound therapy. Therefore, the results of this study suggest that NPWT could directly increase the mobilization ability of EPCs migrating from the bone marrow to the peripheral blood in DFU patients with mild to moderate ischaemia. In addition, we demonstrated that there was no significant difference in the increased amplitude of EPC number in peripheral blood between DFU patients with mild to moderate ischaemia and patients with non-diabetic and non-ischaemic lower extremity ulcer, suggesting that the mobilization ability of bone marrow EPCs during NPWT was similar between DFU patients without serious ischaemia and patients with non-ischaemic lower extremity ulcer with normal glucose tolerance, but still need more research to prove.

Thus far, few studies have examined the effect of NPWT on the circulating number of EPCs in DFU patients, especially with varying severity of ischaemic foot ulcer. In the previous study, Seo et al. revealed that NPWT could induce EPC mobilization in diabetic patients with foot infection or skin defects.¹⁷ Our results enhanced the strength of their data. However, the study reported by Seo et al. did not have control group that did not receive NPWT; therefore, it was impossible to determine whether the changed number of peripheral blood EPCs in the observed subjects was the direct effect of NPWT. To our knowledge, our study is the first clinical study in the literature to examine the direct effect of NPWT on the circulating number of EPCs in diabetic patients with mild to moderate ischaemic foot ulcer.

However, the factors influencing mobilization of bone marrow EPCs during NPWT are not clear. Previously reported stimuli for EPC mobilization include various pathophysiological conditions, growth factors and cytokines, and drugs.^18–20 Furthermore, surgical intervention and musculoskeletal trauma are also known to promote transient EPC mobilization that cannot be differentiated from an inflammatory response induced by injury.²¹ Of the various EPC-mobilizing stimuli, ischaemia is considered to be one of the strongest and to be mediated by EPC-mobilizing cytokines such as VEGF and SDF-1α. It has been found that VEGF and SDF-1α are major cytokines that promote mobilization of bone marrow EPCs and homing to ischaemic wounds.²² It is confirmed that VEGF and SDF-1α have a synergistic effect in promoting EPC angiogenesis in vitro and in animal experiments.^23,24 Previous studies suggest that the effect of NPWT on the systemic and local levels of VEGF/VEGF receptor and SDF-1α. Seo et al. reported that the systemic level of VEGF decreased during NPWT, whereas the level of SDF-1α was maintained.¹⁷ Labler et al. found that VAC therapy increases local vascular endothelial growth factor levels in traumatic wounds.²⁵ Wang et al. demonstrated that VAC could significantly increase the expression of adhesion molecule-1, migration inhibitory factor, VEGF, and collagen I in severe traumatic wounds in pigs.²⁶ Tanaka et al. revealed that NPWT induced early wound healing via increased and accelerated expression of VEGF receptors in rabbit wound closure model.²⁷ These studies suggested the EPC mobilization during NPWT might be induced by changes in the systemic and local levels of some cytokines.

In our study, the level of peripheral blood VEGF and the protein expressions of VEGF and SDF-1α in local wound tissue were found to be significantly increased after NPWT. The multivariate regression analysis showed that the levels of ΔVEGF in the sera and ΔVEGF and ΔSDF-1α in the granulation tissue were independent factors associated with the level of ΔEPCs in the NPWT group. Similarly, the levels of ΔVEGF in the sera and ΔVEGF in the granulation tissue were independent factors associated with the level of ΔEPCs in the NC group. These results suggested that NPWT may increase the levels of VEGF and SDF-1α both in peripheral blood and in local wound tissue to improve the mobilization ability of EPCs in bone marrow and to wound homing effect in diabetic and non-diabetic patients, thus promoting the angiogenesis and granulation tissue formation of ulcer wounds.

It has also been observed in this study that NPWT may be more effective in improving systemic inflammatory status in subjects than is conventional wound treatment, and correlation analysis showed that the level of ΔEPCs was negatively correlated with the change value of various inflammation indices, including ΔWBC, ΔCRP, and ΔESR, indicating that with the improvement of the wound inflammation, the mobilization capacity of bone marrow EPCs may be enhanced. However, further analysis found that this change may not be a key factor affecting the mobilization capacity of bone marrow EPCs after NPWT. In addition, this group of data also showed that the number of circulating EPCs in diabetic patients with mild to moderate ischaemic foot ulcer was significantly lower compared with that in patients with non-diabetic and non-ischaemic lower extremity ulcer. Menegazzo et al.²⁸ have found that a variety of pathological factors including hyperglycaemia can affect the number and activity of EPCs in diabetic patients, which supported the results of this study.

It is noteworthy that, in this study, TcPo₂ from the surroundings of the wound significantly increased in DFU patients with mild to moderate ischaemia after NPWT, but no similar change was found in non-diabetic and non-ischaemic lower extremity ulcer patients. TcPo₂ is a well-recognized indicator reflecting the microcirculation state of tissue.²⁹ It has been found that homing EPCs in DFU wounds can effectively promote neovascularization in ischaemic wounds and improve wound circulation through paracrine pathways and activation of Wnt signalling pathways.^30,31 As the mobilization capacity of EPCs in bone marrow in DFU patients with mild to moderate ischaemia after NPWT is similar to that of non-diabetic and non-ischaemic lower extremity ulcers, it can be speculated that there may be differences in wound homing effect and differentiation ability in bone marrow EPCs between subjects in the above two groups after NPWT. However, further research is required to clarify this issue.

In conclusion, we found that NPWT can significantly directly increase the number of circulating EPCs in patients with diabetes with mild to moderate ischemic foot ulcer, and its ability to mobilize bone marrow EPCs is similar to that of non-diabetic and non-ischaemic lower extremity ulcers. Changes in local and systemic cytokines, including VEGF and SDF-1α, are the main factors influencing bone marrow EPC mobilization after NPWT. It should also be noted that a limitation of this study was that only early phases of NPWT were analysed and unable to clarify how mobilized EPCs contribute to neovascularization. Therefore, the assessment of the effect of prolonged NPWT on EPC mobilization and the exact mechanism involved in the vasculogenic potential of NPWT need to be clarified in further study.

Footnotes

Acknowledgements

We thank the participants of this study including the physicians, nurses, administrative staff, and researchers from the Department of Endocrinology in the First Affiliated Hospital of Anhui Medical University.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Funding for this project was provided by Clinical Medical Special Foundation of Chinese Society (13061050490).

Authors’ contributions

Shichang Mu and Qiaoqiao Hua contributed equally to this work.

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