Table 1.

In vitro Studies Concerning Ozone Therapy

Author, Year	Aim of the Study	Ozone Therapy	Results
Costanzo et al, 201518	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, 199917	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.