Oxygen-Ozone (O₂-O₃) Therapy in Peripheral Arterial Disease (PAD): A Review Study

Figures & data

Table 1 In vitro Studies Concerning Ozone Therapy

Author, Year	Aim of the Study	Ozone Therapy	Results
Costanzo et al, 2015^Citation18	Investigation of the effects of low ozone concentrations on structural and functional cell features	Time: 10 min Concentration of O2/O3 mixture: Gr1: 1 µg O3/mL O2 Gr2: 10 µg O3/mL O2 Gr3: 20 µg O3/mL O2 Control group – yes: GrC: air Gr0: 100% O2	Cell viability and proliferation: no significant differences Cell adhesion: ↑ Gr1 vs GrC & Gr0 after 30 min and after 1 hour; after 2 h, 4 h, 6 h: no significant difference ↑ Gr2 vs GrC & Gr0 ↓ Gr3 vs GrC & Gr0 Cell flattening: - no significant difference between GrC, Gr0, Gr1, Gr3 - percentage of flat cells significantly higher in Gr2	Mitochondrial membrane potential: no significant difference Distribution of protein in the cell: no significant difference Pyruvate dehydrogenase complex: no significant difference	Morphological features: Gr1: smaller mitochondria with lower amount of cristae Gr2: well developed & abundant cristae Gr2: higher density of fibrillary component in both nucleoplasm and nucleolus Gr2: higher content of Hsp70 protein in nucleolus (vs GrC), lower content of Hsp70 protein in cytoplasm (vs GrC) Gr2: higher content of mtHsp70 protein inside mitochondria (vs GrC)
Valacchi et al, 1999^Citation17	Investigation whether the use of heparin or citrate added to blood, before ozonation, affects the release of platelet factors differently	Before ozonation, blood samples were enriched with either heparin or citric acid. Time: 30 sec Concentration of 3% O2/O3 mixture: Gr1: 20 µg O3/mL O2 Gr2: 40 µg O3/mL O2 Gr3: 80 µg O3/mL O2 Control group – yes: GrC: air Gr0: 100% O2	Total antioxidant status: no significant difference Protein thiol group: ↓ Gr1 + heparin, Gr2 + heparin, Gr3 + heparin ↓ Gr2 + heparin vs Gr2 + citric acid ↓ Gr3 + heparin vs Gr3 + citric acid The higher dose of 03, the larger reduction. Thiobarbituric acid reactive substances: ↑ Gr1, Gr2, Gr3 (no significant difference between heparin and citric acid) The higher dose of 03, the larger increase.	PDGF: - larger increase for heparin + GrC, Gr1, Gr2, Gr3 heparin vs citric acid at all times - for citric acid increase only in Gr3 at time point of 1h, 2h, 4h; in Gr2 at time point of 4 h - for heparin increase in all groups at all time points -the higher dose of 03, the larger increase of PDGF TGF-β1: - heparin: significant increase for Gr1, Gr2, Gr3 at 1h; Gr2 & Gr3 at 2h; Gr1, Gr2 & Gr3 at 4h -heparin: the higher dose of 03, the larger increase of TGFβ1 - citric acid: no significant difference	IL-8: - ↑ for Gr1 + heparin, Gr2 + heparin, Gr3 + heparin for 4h - ↑ for Gr2 + citric acid, Gr3 + citric acid for 4h - the higher dose of 03, the large increase of IL-8 at time point of 4h TXB2: - ↑ for Gr2 & Gr3 at 1h & 2h (both heparin and citric acid) - at 4h ↑ for Gr1, Gr2, Gr3 (both heparin and citric acid)

Abbreviations: Gr, group of cells; IL-8, interleukin 8; PDGF, platelet-derived growth factor; TGF-β1, transforming growth factor beta 1; TXB2, thromboxane B2.

Table 2 Animal Studies Concerning Ozone Therapy

Author, Year	Aim of the Study	Animals	Experimental Groups	Ozone Therapy	Results
Akcilar et al, 2015^Citation19	To determine the role of ozone administration in hypertension	n = 23 male Sprague Dawle rats with DOCA-salt-induced hypertension	GrC: n = 7, control group GrH: n = 8, hypertension GrHO: n = 8, hypertension + ozone therapy	Time: 10 days Method: intraperitoneal Dose: 3% O3/O2 mixture 1.1 mg/kg body weight	ET-1 (pg/mL): p = 0.0001 GrC: 20.9 ± 1.24 GrH: 29.8 ± 3.95 GrHO: 18.2 ± 0.51 p = 0.0001 GrH vs GrHO Renin (mU/mL): p = 0.001 GrC: 38.6 ± 1.44 GrH: 26.0 ± 0.58 GrHO: 38.7 ± 0.96 p = 0.0001 GrH vs GrHO NO (nmol/mL): p = 0.04 GrC: 1.97 ± 0.14 GrH: 1.12 ± 0.25 GrHO: 2.29 ± 0.32 p = 0.038 GrH vs GrHO	Systolic blood pressure (mmHg): p = 0.0001 GrC: 119.9 ± 1.43 GrH: 150.9 ± 1.96 GrHO: 136.4 ± 0.75 p = 0.0001 GrHO vs GrC Diastolic blood pressure (mmHg): p = 0.001 GrC: 83.3 ± 1.92 GrH:94.7 ± 0.74 GrHO: 88.5 ± 1.38 Mean arterial pressure (mmHg): p < 0.05 GrC: 101.0 ± 1.24 GrH: 121.3 ± 1.11 GrHO: 111.1 ± 1.09 p = 0.001 GrHO vs GrC	ET-1 mRNA expression: - heart: p = 0.004 GrC: 0.089 ± 0.29 GrH: 4.39 ± 0.88 GrHO: 0.89 ± 0.13 - vascular tissue: p = 0.003 GrC: 0.03 ± 0.01 GrH: 0.29 ± 0.009 GrHO: 0.03 ± 0.001 ET-1 mRNA expression was significantly higher in GrH than in GrHO: - in the heart (p = 0.004) - in the vascular tissue (p = 0.002)
Di Filippo et al, 2010^Citation20	Investigation of the ozone therapy effect on damage linked to acute myocardial	n = 20 Sprague Dawle rats after 25 minutes occlusion and 2-hour reperfusion of the left descending coronary artery	- GrC: n = 5, control group - Gr1: n = 5 - Gr2: n = 5 - Gr3: n = 5	Time: 30 min prior to ischemia/reperfusion (IR) procedure Method: intraperitoneal Dose of O2/O3 mixture: Gr1: 100 µg/kg Gr2: 150 µg/kg Gr3: 300 µg/kg	Area at risk/left ventricle: GrC: 53 ± 2% Gr1, Gr2, Gr3: 52 ± 3.2%, p > 0.05	Infract size/left ventricle: GrC: 33 ± 1.5% Gr2: no change, p > 0.05 Gr2: 22 ± 2%, p < 0.05 Gr3: p < 0.01	GrC: low level of immunoprotection from CD-34 and CD117/c-kit, low level of eNOS Gr2: increase amount of CD-34 and CD117/c-kit, high level of eNOS (p < 0.01)
Koca et al, 20102^Citation2	To investigate protective role of hyperbaric oxygen precondition and ozone precondition on IR injury	n = 32 male Wistar Albino rats after 2-hours occlusion and 22-hours reperfusion of unilateral femoral artery	- GrS: n = 8, sham - GrC: n = 8, IR - GrHBOT: n = 8, IR + hyperbaric oxygen precondition - GrO3: n = 8, IR + ozone precondition	Time: 72, 48, 24, 0 hours prior to IR Method: intraperitoneal Dose of 3% O2/O3 mixture: 4 doses of 0.7 mg/kg	Lipid peroxidation: - higher level in GrC than GrS, p < 0.01 - lower level in GrHBOT & GrO3 (GrHBOT < GrO3) than GrC, p < 0.01	Oxidative damage: - higher level in GrC than GrS, p<0.01 - lower level in GrHBOT & GrO3 (GrO3 < GrHBOT) than GrC, p<0.01	Antioxidant enzymes: - higher level of SOD in GrC (vs GrS, p < 0.01) and lower level of GSH-Px (p < 0.01) - GrHBOT i GrO3 (vs GrC) had higher level of SOD and GSH-Px (p < 0.01) (GrHBOT > GrO3)
Ozkan et al, 2015^Citation23	To evaluate and compare the effect of hypothermia and ozone therapy on ischaemia reperfusion injury of skeletal muscle	n=18 male Wistar Albino rats after 2-hours occlusion and 22-hours reperfusion of common iliac artery	- GrS: n=6, sham - GrC: n = 6, IR - GrH: n = 6, IR + hypothermia - GrO3: n = 6, IR + ozone therapy - GrH + O3: n = 6, IR + hypothermia + ozone therapy	Time: 72, 60, 48, 36, 24, 12 hours prior to IR Method: intraperitoneal Dose of 3% O2/O3 mixture: 6 doses of 0.7 mg/kg	Lipid peroxidation - MDA (mmol/g protein): - GrS: 0.63 ± 0.16 - GrC: 4.23 ± 0.68 - GrO3: 2.10 ± 0.49* - GrH + O3: 1.73 ± 0.45* *p < 0.05 vs GrC Oxidative stress - SOD (U/g protein): - GrS: 918.7 ± 257.7 - GrC: 312.6 ± 66.1 - GrO3: 991.9 ± 251.6*	- GrH + O3: 727.3 ± 115.1*^# *p < 0.05 vs GrC ^#p < 0.05 vs GrO3 Oxidative stress - GSH-Px (U/g protein): - GrS: 90.9 ± 11.3 - GrC: 29.5 ± 6.9 - GrO3: 87.6 ± 17.1* - GrH + O3: 86.4 ± 20.1* *p < 0.05 vs GrC Nitrosative stress – NOx (g/g protein):	- GrS: 0.25 ± 0.02 - GrC: 0.59 ± 0.11 - GrO3: 0.34 ± 0.2* - GrH + O3: 0.31 ± 0.7* *p < 0.05 vs GrC Inflammation – IL-1β (pg/p protein): - GrS: 13.0 ± 2.8 - GrC: 60.6 ± 12.4 - GrO3: 24.6 ± 3.4* - GrH + O3: 23.3 ± 0.7*^# *p < 0.05 vs GrC ^#p < 0.05 vs GrO3

Abbreviations: DOCA, deoxycorticosterone acetate; eNOS, endothelial nitric oxide synthase; ET-1, endothelin-1; Gr, group of subjects; GSH-Px, glutathione peroxidase; IL-1β, interleukin 1 beta; IR, ischemia/reperfusion procedure; MDA, malondialdehyde; n, number of subjects; NO, nitric oxide; NOx, nitrite-nitrate; SOD, superoxide dismutase.

Table 3 Clinical Trials of Oxygen-Ozone Therapy

Author, Year	Aim of the Study	Participants	Ozone Therapy	Results
Clavo et al, 2004^Citation24	To assess the effect of ozone therapy on the blood flow in the middle cerebral and common carotid arteries	n = 7 subjects: 2 healthy volunteers + 5 patients with cerebral vascular disease Age: 58 years (34–78 years)	Autohemotherapy: 200 mL blood + 200 mL O2/O3 mixture (concentration 60 µg/mL) 3 autohemotherapy sessions on alternate days over one week	Middle cerebral artery: - systolic velocity: t0 = 90.9 ± 6.1 cm/s t1 = 111 ± 7.3 cm/s, p = 0.001 t2 = 104.3 ± 8 cm/s, p = 0.035 - diastolic velocity: t0 = 41.1 ±4.4 cm/s t1 = 54.6 ± 4.6 cm/s, p < 0.001 t2 = 48.6 ± 5 cm/s, p = 0.023 t0 – baseline t1 – after third session t2 – one week later	Common carotid artery: - blood flow: t0 = 233 ± 19 mL/min t1 = 407 ± 38 mL/min, p < 0.001 t2 = 301 ± 22 mL/min, p = 0.039 The percentage increase in common carotid artery blood flow was directly correlated with age (r = 0.35, p = 0.004) and inversely correlated with the initial values (r = 0.691, p = 0.009).
Wu et al, 2013^Citation25	To determine the association between major ozonated autohemotherapy and functional recovery of patients with acute cerebral infarction	n = 86 acute cerebral infarction patients GrO3: autohemotherapy GrC: conventional therapy	10 ± 3 daily autohemotherapy sessions: 100 mL blood + 100 mL O2/O3 mixture (concentration 47 µg/mL)	Modified Rankin Scale: GrO3: t0 = 4.1 ± 0.8; t1 = 2.5 ± 1.1 GrC: t0= 4.1 ±0.9; t1 = 3.1 ± 1.3 GrO3 vs GrC p < 0.05 National Institutes of Health Stroke Scale (NIHSS): GrO3: t0 = 11.7 ± 3.5; t1 = 5.4 ± 3.5 GrC: t0 = 11.5 ± 3.8; t1 = 7.3 ± 4.1 GrO3 vs GrC p < 0.05 Central motor conduction time: - upper limb: GrO3 significantly shorter time in comparison with GrC (p < 0.05) - lower limb: no significant difference between groups t0 – baseline t1 – after 10 ± 3 sessions	Cortical potential rise rate: no significant difference between groups Motor-evoked potential amplitude: - upper limb: GrO3: significantly higher in comparison with GrC (p < 0.05) - lower limb: no significant difference between groups Correlation analysis: - positive correlation between NIHSS improvement rate and central motor conduction time improvement rate of the upper limb (r = 0.78, p < 0.005) - positive correlation between NIHSS improvement rate and the improvement rate of motor-evoked potential amplitude of the upper limb (r = 0.85, p < 0.05)
Martinez-Sanchez et al, 2012^Citation26	Investigation of the therapeutic efficacy of ozone in patients with coronary artery disease, treated with antithrombotic therapy, Aspirin and policosanol.	n = 53 patients with coronary artery disease GrO3: 26 patients, regular anti-thrombotic therapy + ozone therapy GrC: 27 patients, regular anti-thrombotic therapy GrN: reference group of healthy subjects	20 daily sessions: - rectal insufflation - dose: 200 mL O2/O3 mixture, concentration 40 µg/mL	Prothrombin time (s): GrO3: t0 = 11.2 ± 0.6 s; t1 = 24.6 ± 6.6 GrC: t0 = 11.0 ± 0.4 s; t1 = 18.4 ± 4.6 t1: GrO3 vs GrC p < 0.01 Bleeding time: no difference (GrO3 vs GrC) Advanced oxidation protein products (µM): GrO3: t0 = 23.90 ± 0.84; t1 = 18.26 ± 3.61 GrC: t0 = 23.12 ± 5.31; t1 = 24.25 ± 7.18 t1: GrO3 vs GrC p < 0.05 Malondialdehyde (µM): GrO3: t0 = 12.04 ± 0.44; t1 = 8.38 ± 3.63 GrC: t0 = 13.71 ± 3.01; t1 = 10.82 ± 2.52 t1: GrO3 vs GrC p < 0.05 Peroxidation potential (µM): GrO3: t0 = 16.25 ± 3.62; t1 = 6.16 ± 2.51 GrC: t0 = 20.41 ± 8.16; t1 = 19.00 ± 7.03 t1: GrO3 vs GrC p < 0.05 t1: GrO3 vs GrN p > 0.05 t0 – baseline t1 – after 20 sessions	Ferric reducing ability of plasma (µM): GrO3: t0 = 191.3 ± 57.0; t1 = 471.9 ± 115.8 GrC: t0 = 146.2 ± 62.3; t1 = 386.0 ± 104.3 t1: GrO3 vs GrC p < 0.05 Reduced glutathione (mM): GrO3: t0 = 1.18 ± 0.23; t1 = 1.55 ± 0.63 GrC: t0 = 1.5 ± 0.23; t1 = 1.08 ± 0.45 t1: GrO3 vs GrC p < 0.05 Superoxide dysmutase (U/mL): GrO3: t0 = 37.75 ± 7.13; t1 = 16.27 ± 8.39 GrC: t0 = 40.48 ± 10.12; t1 = 35.27 ± 14.21 t1: GrO3 vs GrC p < 0.05 Catalase (U/l): GrO3: t0 = 839.5 ± 165.3; t1 = 543.4 ± 172.3 GrC: t0 = 880.4 ± 250.1; t1 = 288.0 ± 99.6 t1: GrO3 vs GrC p < 0.05 t1: GrC vs GrN p > 0.05 No difference between GrC and GrO3 for total hydroperoxides.
Martinez-Sanchez et al, 2005^Citation27	Evaluation of the effectiveness of ozone in the treatment of patients with type 2 diabetes suffering from diabetic foot complications and its effects on oxidative stress, hyperglycaemia and some markers of endothelial damage.	n = 100 patients with diabetic foot GrO3: 51 patients, ozone therapy GrA: 49 patients, antibiotic therapy GrC: 50 healthy subjects, control group	20 daily sessions: - rectal insufflation (ozone dose 10 mg, concentration 50 mg/l); - local ozone treatment (concentration 60 mg/l, time 1 hour) - Oleozon: ozonised sunflower oil	Blood glucose concentration: GrO3 > GrA (p < 0.05) Blood glucose within reference range (3.33–8.33 mM): GrO3: t0 = 27%, t1 = 56% patients GrA: t0 = 30%, t1 = 26% patients Lesions area reduction: GrO3: 74.58 ± 0.35% GrA: 50.3 ± 0.17%, GrO3 vs GrA p = 0.017 GrO3: 2.66 ± 0.03 cm^2/day GrA: 1.21 ± 0.01 cm^2/day, GrO3 vs GrA p = 0.005 t0 - baseline t1 –24 h after the last session	Total recovery time: GrO3: 21 ± 10 days GrA: 45 ± 11 days, GrO3 vs GrA p = 0.002 Length of hospitalisation: GrO3: 26 days (6–58 days) GrA: 34 days (7–83 days), GrO3 vs GrA p = 0.010 Biomarkers of oxidative damage: GrO3: improvement (t1 vs t0) of the following biomarkers: fructolysine, advanced oxidation protein products, peroxidation potential, superoxide dismutase, catalase. GrO3: improvement to the GrC levels (t1 vs t0) of the following biomarkers: nitrites/nitrates, total hydroperoxides. GrO3: no change for the following biomarkers: reduced glutathione, malondialdehyde, glutathione peroxidase
Tafil-Klawe et al, 2002^Citation28	Comparison of the effect of ozone and classical balneological methods on health condition of patients with obliterative artheromatosis and on serum activity of three lysosomal enzymes.	n = 94 subjects GrO3: 32 patients with lower limb ischemia Age: 65.9 ± 6.9 years GrB: 32 patients with lower limb ischemia Age: 62 ± 8 years GrC: 30 healthy subjects, control group Age: 60 years	GrO3: everyday ozone therapy for 10 days: - intravenous infusions: 500 mL of normal saline satiated with ozone (60 µg O3/mL) - 30-minute aerosol oxygen-ozone baths: ozone concentration in a chamber was 19 µg/l GrB: whirlpool massage therapy and carbonic acid baths, everyday therapy for 10 days	GrO3: - arylsulphatase: t1 = 1.71 ± 0.40; t2 = 1.88 ± 0.42 (p < 0.05 vs t1), t3 = 2.71 ± 0.68 - acid phosphatase: t1 = 14.11 ± 2.56; t2 = 12.21 ± 1.9 (p < 0.05 vs t1), t3 = 9.97 ± 1.48 - cathepsin D: t1 = 1.75 ± 0.38; t2 = 3.41 ± 0.69 (p < 0.05 vs t1), t3 = 5.39 ± 1.19 - alfa-1-antitrypsin: t1 = 1.62 ± 0.22; t2 = 1.39 ± 0.22 (p < 0.05 vs t1), t3 = 1.19 ± 0.15 (p > 0.05 vs GrC: 1.07 ± 0.12) t1 – 24 h hours before first therapy t2 – one hour following therapy t3 – on 10th day of therapy	GrO3: - BMI reduction by 2.4% - ankle arm index (right extremity): improved by 13.8% - ankle arm index (left extremity) improved by 14.9% - intermittent claudication distance in the corridor: improve by 34.2% - intermittent claudication distance on a track: improvement by 50.6% - total cholesterol: reduction by 5.0% - HDL: no change - LDL: reduction by 7.2% - triglycerides: reduction by 11.1%
Marfella et al, 2009^Citation31	Analysis of ozone therapy effect on TNF-α level, endothelial progenitor cells (EPCs) level and on limb perfusion.	n = 151 patients with critical limb ischemia, candidates not appropriate for standard revascularization. Age > 40 years GrO3: 77 patients, ozone therapy GrC: 74 patients receiving injections with 10 mL sterile saline	Intragluteal injection of autologous blood exposed to oxygen/ozone gas mixture (ozone concentration: 15.35 g/m;^Citation3 flow rate: 240 mL/min) Two treatments were given on consecutive days, followed by a third one on day 7. Subsequent treatments given at 1-week intervals for a least 22 weeks.	Pain perception: GrO3: reduced pain perception in 36 patients GrC: reduced pain perception in 12 patients Wound healing: GrO3: deterioration in wound size t1 vs t0 (p<0.05), at t1 GrO3 vs GrC (p<0.05) GrO3: complete ulcer healing in 32 patients at 6 months Ankle-Branchial Index: No difference among groups at any of the time points t0 – baseline t1 – 22 weeks	TcPO2: GrO3: significant increase at t1 vs t0 (p<0.01), GrO3 vs GrC (p<0.001) GrO3: higher number of patients with TcPO2> 30 mmHg (t0 = 31.1%, t1 = 84.4%) GrC: lower number of patients with TcPO2> 30 mmHg (t0 = 35.1%, t1 = 15.1%) TNF-α level: GrO3: t0 = 225.5±28.2 ng/l, t1 = 114.8±23.1 ng/l (p<0.001) GrC: no difference EPCs: GrO3: increase of CD34/KDR(+); No difference in CD34(+), CD133(+) or total number of mononuclear cells
Zhang et al, 2014^Citation30	Assessment of effectiveness of ozone therapy on the healing and the expression of growth factors of the wounds at the early stage after treatment of diabetic foot ulcer.	n = 50 patients with type II diabetes mellitus, as well as with diabetic foot ulcer of Wagner classification stages 2, 3, 4 GrO3: 25 patients, ozone therapy GrC: 25 patients, standard treatment including debridement, wound dressing, moisture maintenance of the wound	Ozone gas bath: - concentration: 52 mg/mL - time: 30 min/day, 20 days	Effective rate of wound healing: GrO3: 92% GrC: 64%, GrO3 vs GrC p = 0.037 Wound size reduction: GrO3: 6.84 ± 0.62 cm^2 GrC: 3.19 ± 0.65 cm^2, GrO3 vs GrC p < 0.001 Collagen contents of the wound (a.u.): GrO3: 4.48 ± 0.43 GrC: 3.07 ± 0.23, GrO3 vs GrC p = 0.012 VEGF levels in wound exudates at 7 day following up (pg/mL): GrO3: 27.89 ± 5.53 GrC: 22.25 ± 4.05, GrO3 vs GrC p < 0.05 PDGF levels in wound exudates at 7 day following up (pg/mL): GrO3: 21.31 ± 3.08 GrC: 13.39 ± 2.33, GrO3 vs GrC p < 0.05 TGF-β in wound exudates at 11 day following-up (pg/mL): GrO3: 9.81 ± 2.61 GrC: 8.45 ± 1.74, GrO3 vs GrC p < 0.05	VEGF levels in tissues at 11 day following up (pg/mL): GrO3: 34.86 ± 3.00 GrC: 26.44 ± 2.02, GrO3 vs GrC p = 0.032 PDGF levels in tissues at 11 day following up (pg/mL): GrO3: 31.44 ± 3.33 GrC: 20.78 ± 2.69, GrO3 vs GrC p = 0.023 TGF-β in wound tissues at 11 day following-up (pg/mL): GrO3: 14.95 ± 1.39 GrC: 10.45 ± 1.07, GrO3 vs GrC p = 0.019 Expression of VEGF proteins in tissues at 11 day following up (a.u.): GrO3: 3.34 ± 0.27 GrC: 2.03 ± 0.16, GrO3 vs GrC p < 0.001 Expression of PDGF proteins in tissues at 11 day following up (a.u.): GrO3: 4.09 ± 0.14 GrC: 3.06 ± 0.13, GrO3 vs GrC p < 0.001 Expression of TGF-β proteins in tissues at 11 day following-up (a.u.): GrO3: 7.83 ± 0.49 GrC: 6.10 ± 0.45, GrO3 vs GrC p = 0.018
Zhou et al, 2016^Citation32	Comparison of the effectiveness of standard endovenous laser therapy with endovenous laser therapy combined with ozone gas bathing in patients with lower limb venous ulcers.	n = 92 patients with lower limb venous ulcers GrO3: 50 patients Age: 61.1 years ± 11.2 GrC: 42 patients, control group Age: 60.2 years ± 9.7	Preconditioning with ozone gas bath: - concentration: 60 mg/l - time: 1 hour, once a day until the necrosis and infection were improved an suitable for skin puncture (mean: 8.6 days, range: 8–13 days)	Ratio of complete occlusion of the treated vein: no significant difference at any of time point (1, 3, 6, 12 months of follow-up) Ratio of recovery at the 12 months following up: GrO3: 92.00% GrC: 76.19%, GrO3 vs GrC p = 0.03	Ratio of ulcer recur at the 24 months following up: GrO3: 6.52% GrC: 25.00%, GrO3 vs GrC p = 0.03 Patient’s satisfaction assessment: GrO3: 90.00% GrC: 71.43%, GrO3 vs GrC p = 0.03

Note: p values above 0.05 are shown in bold.

Abbreviations: BMI, body mass index; EPCs, endothelial progenitor cells; Gr, study group; HDL, high-density lipoprotein; KDR, kinase-insert domain-containing receptor; LDL, low-density lipoprotein; n, number of subjects; p, statistical difference; PDGF, platelet-derived growth factor; t, time point; TcPO₂, transcutaneous oxygen pressure; TGF-β, transforming growth factor-beta; TNF-α, tumor necrosis factor-alpha; VEGF, vascular endothelial growth factor.

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