Increased growth factors play a role in wound healing promoted by noninvasive oxygen-ozone therapy in diabetic patients with foot ulcers

Abstract

Management of diabetic foot ulcers (DFUs) is a great challenge for clinicians. Although the oxygen-ozone treatment improves the diabetic outcome, there are few clinical trials to verify the efficacy and illuminate the underlying mechanisms of oxygen-ozone treatment on DFUs. In the present study, a total of 50 type 2 diabetic patients complicated with DFUs, Wagner stage 2~4, were randomized into control group treated by standard therapy only and ozone group treated by standard therapy plus oxygen-ozone treatment. The therapeutic effects were graded into 4 levels from grade 0 (no change) to grade 3 (wound healing). The wound sizes were measured at baseline and day 20, respectively. Tissue biopsies were performed at baseline and day 11. The expressions of vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and platelet-derived growth factor (PDGF) proteins in the pathologic specimens were determined by immunohistochemical examinations. The effective rate of ozone group was significantly higher than that of control group (92% versus 64%, P < 0.05). The wound size reduction was significantly more in ozone group than in control group (P < 0.001). After treatment, the expressions of VEGF, TGF-β, and PDGF proteins at day 11 were significantly higher in ozone group than in control group. Ozone therapy promotes the wound healing of DFUs via potential induction of VEGF, TGF-β, and PDGF at early stage of the treatment. (Clinical trial registry number is ChiCTR-TRC-14004415).

Figures

Figure 1

Criteria of therapeutic effects and wound size determination. Panel (a) (A) Grade 0: no change or worse than before. (B) Grade 1: wound size reducing less than 1/2. (C) Grade 2: wound size reducing more than 1/2, the secretion obviously less than before, there is little necrosis, fresh granulation generated. (D) Grade 3: wound healing, completely epithelialized. Panel (b) Ulcer areas were calculated from film transparency tracings using grid paper.

Figure 2

Wound size reduction (cm², x-±s). Before treatment there was no significant difference in ulcer area between the ozone group and the control group (11.74 ± 0.72 versus 10.82 ± 0.93, P = 0.439). After treatment the ulcer area in both groups was significantly smaller than before. The wound area reduction (Δarea) was significantly more in the ozone group than in the control group (6.84 ± 0.62 versus 3.19 ± 0.65 cm2, P < 0.001). *P < 0.05, ***P < 0.001.

Figure 3

Collagen fibers in tissue specimens by Masson's staining (×40). Before treatment there was no difference in collagen fibers between the ozone group and the control group (0.92 ± 0.04 versus 0.88 ± 0.05, P = 0.433). After treatment there were more collagen fibers than before in both groups (P < 0.001). The collagen fibers were significantly more in the ozone group than in the control group (4.48 ± 0.43 versus 3.07 ± 0.23, P = 0.012). *P < 0.05, ***P < 0.001.

Figure 4

Changes of growth factors in wound exudates at 0, 3, 7, and 11 d after treatment. (a) VEGF levels (pg/mL) in wound exudates significantly increased in both groups after treatment. (b) TGF-β levels (pg/mL) in wound exudates significantly increased in both groups at 7 d and 11 d after treatment. And at 11 d ozone group has higher TGF-β level than control. (c) PDGF levels (pg/mL) in wound exudates significantly increased at 7 and 11 d after treatment in both groups with higher levels in ozone group than in control. *P < 0.05 versus the same group at day 0. #P < 0.05 versus control group at the same day.

Figure 5

Contents of VEGF, TGF-β, and PDGF in tissue specimens (pg/mg). Before treatment there were no significant differences in the contents of VEGF, TGF-β, and PDGF in the wound between the ozone group and the control group (19.95 ± 0.53 versus 17.93 ± 0.84, P = 0.056; 4.48 ± 0.43 versus 5.17 ± 0.49, P = 0.304; 14.23 ± 0.68 versus 15.50 + 0.78, P = 0.235). But after treatment the contents of VEGF, TGF-β, and PDGF were all significantly higher in the ozone group than in the control group (34.86 ± 3.00 versus 26.44 ± 2.02, P = 0.032; 14.95 ± 1.39 versus 10.45 ± 1.07, P = 0.019; 31.44 ± 3.33 versus 20.78 ± 2.69, P = 0.023). *P < 0.05, ***P < 0.001.

Figure 6

Expressions of VEGF, TGF-β, and PDGF by immunohistochemical examinations (×40). (a) Expressions of VEGF pre- and posttreatment in two groups. (b) Expressions of TGF-β pre- and posttreatment in two groups. (c) Expressions of PDGF pre- and posttreatment in two groups. Before treatment there were no significant differences in the expressions of VEGF, TGF-β, and PDGF protein between the ozone group and the control group (0.83 ± 0.06 versus 0.82 + 0.04, P = 0.892; 0.88 ± 0.05 versus 0.94 ± 0.08, P = 0.495; 0.91 + 0.04 versus 0.92 ± 0.04, P = 0.802). But after treatment the expressions of VEGF, TGF-β, and PDGF were all significantly higher in the ozone group than in the control group (3.34 ± 0.27 versus 2.03 ± 0.16, P < 0.001; 7.83 ± 0.49 versus 6.10 ± 0.45, P = 0.018; 4.09 ± 0.14 versus 3.06 ± 0.13, P < 0.001). *P < 0.05, ***P < 0.001.