New frontiers in regenerative medicine: Protocol standardization and morphological assessment of leukocyte-platelet rich fibrin in cows, with a longitudinal study on growth factor release dynamics

• This study provides the first in-depth investigation into autologous L-PRF membranes derived from bovine blood. • This study provides the standardisation of the L-PRF production protocol. • Our results highlight the membranes’ promising macroscopic, histological, and biological characteristics, alongside a dynamic release of growth factors over time. • The study opens exciting possibilities for regenerative medicine in dairy cattle, particularly in supporting wound healing and reducing antimicrobial use, aligning with the One Health approach. Leukocyte-Platelets Rich Fibrin (L-PRF) is a platelet-derived autologous agent. It is a powerful wound-sealing biological scaffold that promotes the healing process through the release of growth factors (GFs) and matrix proteins. To date, the L-PRF in bovines has not yet been investigated and a standardized production protocol has not yet been described. Therefore, the aim of the study was to standardise the L-PRF production protocol and to evaluate macroscopically and histologically the L-PRF membranes obtained and observe the GFs release over time and lactation phases: at 1h (T0), 4h (T1), 24h (T2), and 168h (T3) after extraction. Eighty cows were divided into four groups of 20 animals, based on their lactation phase. A whole blood sample of 20 ml was taken and divided for L-PRF clot production and haemato-biochemical investigations. The membranes presented the following overall mean characteristics: a length of 34.4 ± 7.3 cm, a width of 12.5 ± 2.6 cm, and a weight of 4.9 ± 0.17 cm. Histological analysis showed a well-defined histoarchitecture consisting of a first layer of erythrocytes, a layer of leukocytes, erythrocytes, and platelets, a layer of leukocytes, a layer of platelets, and finally a layer of slightly eosinophilic fibrin. The GFs significantly increased with upward trends in all phases of lactation and at each time control, overlapping the behavior of GF in other species. Based on the encouraging findings of this study, further clinical trials for applicative potential of the L-PRF membrane in bovine medicine should be assessed.