The Effects of Aqueous Ozone on Normal Platelet Function and Growth Factor Release in Diabetics and non-Diabetics

Abstract

Background: Gaseous ozone therapy is a new modality that has shown increasingly promising results in the healing of diabetic foot ulcers and an increased release of growth factors from platelets but presents health risks. Since aqueous ozone is safer and may also increase the release of growth factors, it was used in this multifaceted five-part study to determine its safety and effect on the release of platelets in diabetic participants. The data is expected to provide a proof of concept for a future study to quantify the effect of the external application of aqueous ozone on diabetic foot ulcers. Methods: Experiment 1: real time platelet aggregation and adenosine triphosphate (ATP) release from the platelets of four non-diabetic participants was analysed via the Chrono-log lumi-aggregometer after immediate exposure and a 2hr incubation with phosphate buffered saline (PBS), ozonized PBS (O3PBS) or adenosine diphosphate (ADP). Experiment 2: Six non-diabetic and six diabetic’s blood plasma collected in citrate tubes was treated with either PBS, O3PBS, or ADP and incubated for either 30 min or 2 hrs. Analysis of the growth factors was performed via platelet derived growth factor (PDGF-BB) ELISA assay kit. Experiment 3: Protocol is identical to experiment 2 however the level of transforming growth factor β (TGF-β) was analysed via an ELISA assay kit. Experiment 4: Four non-diabetic and four diabetic’s blood plasma was treated with either PBS, O3PBS or ADP and incubated for either 2hrs or 4hrs. Analysis of oxidative stress was determined via a protein carbonyl assay. Experiment 5: two diabetic volunteers with a diabetic foot ulcer consented to an aqueous ozone wash three times per week for 5 weeks or until the ulcer had healed. Results: The level of ATP release was higher in the O3PBS condition compared to the PBS condition in both immediate and 2 hr incubation groups (p < 0.001). No significant difference was seen in level of aggregation between O3PBS and ADP and PBS groups (p = 0.1, p = 0.5). In experiment 2 the level of PDGF was higher in the ADP group compared to O3PBS and PBS group after 2hrs incubation (p < 0.001). No other significant differences were seen in any other conditions. No significant differences in level of TGF-β were seen in experiment 3. The level of protein carbonyl in experiment 4 was significantly higher for both O3PBS and ADP in the diabetic after 2hrs of incubation (p < 0.05) compared to PBS. Aqueous ozone significantly decreased ulcer size of both diabetic participants after 5 weeks of treatment (p < 0.008) in experiment 5. Discussion: These five experiments give evidence towards a healing effect of aqueous ozone without causing notable oxidative stress or aggregation in platelets of diabetics. Although O3PBS could cause release of dense-alpha granules, demonstrated by ATP release, this healing effect may not be due to growth factor interaction as previously thought. For the purpose of this study however, we have demonstrated some evidence that aqueous ozone may be a beneficial treatment for diabetic foot ulcers but via a different pathway then previously predicted.