Ozone (O3) is an oxidizing natural gas widely applied as adjunctive therapeutic treatment for a variety of pathological conditions. Currently, O3-based therapies rely on the low-dose concept i.e., the administration of low O3 concentrations able to induce a mild oxidative stress stimulating antioxidant and anti-inflammatory response without causing cell damage. In addition, low O3 concentrations are thought to activate cellular and molecular mechanisms responsible for analgesic and regenerative effects. Due to these properties, in the last decade interest has arisen in the fields of orthopedics and regenerative medicine on the potential of O3 to counteract joint diseases involving cartilage degeneration. In this pilot study, we have explored the anti-degenerative potential of O3 on knee articular cartilage explanted from a healthy adult rabbit and maintained in vitro. Light and transmission electron microscopy were used to monitor chondrocyte and extracellular matrix features of cartilage samples undergoing O3 treatment every three days for two weeks. Results demonstrated that low O3 concentrations act on chondrocytes and the molecular components of the extracellular matrix of articular cartilage explants, significantly improving their preservation under in vitro conditions, likely by promoting both protective and pro-regenerative pathways. This opens promising perspectives for further investigations on the therapeutic potential of O3 for the treatment of cartilage degeneration not only as painkilling and anti-inflammatory agent but also as a cartilage regenerative agent.

Procaine is a local anesthetic used at low concentrations in a complementary medical approach, neural therapy, due to its ability to promote antioxidant and anti-inflammatory response. Previous biochemical studies showed that low concentrations of procaine may also stimulate mitochondrial function. In the present study, low procaine concentrations were used to investigate in vitro the effects on mitochondria of human fibroblasts and mouse myoblasts. Histochemical, ultrastructural, and biomolecular techniques were employed. After 24 h from procaine treatment, mitochondrial membrane potential was unaltered in both cell types, but some mitochondrial features showed cell-specific modifications. In detail, in fibroblasts, procaine induced decreased mitochondrial cristae length and cellular adenosine triphosphate (ATP) levels. In myoblasts, procaine induced modifications of mitochondrial shape and oxidative phosphorylation subunit amount, together with increased cellular ATP levels. Most modifications were restored at 48 h. To explain the differential response of the two cell lines, we hypothesize a species-specific sensitivity to procaine or a cell-specific sensitivity related to the different extent of the endoplasmic reticulum, which is especially susceptible to procaine. Taken together, the results of this pilot study show that procaine at low concentration exerts a modulatory effect on mitochondrial features, opening the way to further studies aimed at elucidating the cellular pathways involved in the beneficial effects observed in procaine-based neural therapy.