Platelet-rich plasma (PRP) is gaining more and more attention in regenerative medicine as an innovative and efficient therapeutic approach. The regenerative properties of PRP rely on the numerous bioactive molecules released by the platelets: growth factors are involved in proliferation and differentiation of endothelial cells and fibroblasts, angiogenesis and extracellular matrix formation, while cytokines are mainly involved in immune cell recruitment and inflammation modulation. Attempts are ongoing to improve the therapeutic potential of PRP by combining it with agents able to promote regenerative processes. Two interesting candidates are ozone, administered at low doses as gaseous oxygen-ozone mixtures, and procaine. In the present study, we investigated the effects induced on platelets by the in vitro treatment of PRP with ozone or procaine, or both. We combined transmission electron microscopy to obtain information on platelet modifications and bioanalytical assays to quantify the secreted factors. The results demonstrate that, although platelets were already activated by the procedure to prepare PRP, both ozone and procaine induced differential morpho-functional modifications in platelets resulting in an increased release of factors. In detail, ozone induced an increase in surface protrusions and open canalicular system dilation suggestive of a marked α-granule release, while procaine caused a decrease in surface protrusions and open canalicular system dilation but a remarkable increase in microvesicle release suggestive of high secretory activity. Consistently, nine of the thirteen platelet-derived factors analysed in the PRP serum significantly increased after treatment with ozone and/or procaine. Therefore, ozone and procaine proved to have a remarkable stimulating potential without causing any damage to platelets, probably because they act through physiological, although different, secretory pathways.

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.