Aim:The hypothesis is that the production of leukocyte and platelet-rich fibrin (L-PRF) is easy in horses without modifying human protocol, thus allowing better standardization of human protocol. In this study, the aim is to standardize the production of L-PRF in horses to direct it to human production. Methods: The authors took 9 mL of blood from the jugular vein of 6 horses to produce L-PRF membranes at a temperature > 21 °C by measuring the size of the membranes (height, length, thickness, weight, surface) and clot. Therefore we analyzed their microbiological characteristics. Results: The production of leukocyte and platelet-rich fibrin (L-PRF) is easy in horses without modifying human protocol, thus allowing better standardization of human protocol. The parameters found in the horse are clearly similar to the parameters found in humans. In optical microscopy, most of the cellular bodies were found and concentrated in the proximal portion of each membrane, with the last 1/4 observed in the center; the distal part only had residual traces of cellular bodies. The L-PRF is composed constantly when the production process below described is strictly respected. The success of L-PRF depends entirely on blood collection, on the quick transfer to a centrifuge (within 1 min) and on centrifugation and squeezing temperatures (between 21 and 30 °C) of the clot. Conclusion:The experiments on horses for the standardization of L-PRF production will improve our understanding about wound healing, particularly in the regenerative therapy of chronic skin lesions in humans. The data collected show that the best preparation method is the 2 min compression of the clot after 0 min of blood sampling, using the 9 mL vacutainer system and not a 9 mL syringe. Key words:Autologous, buffy coat, growth factor level, platelet-rich fibrin, thrombocyte concentrate ABSTRACTArticle history:

In this study, the use of fibrin rich in leukocytes and platelets (L-PRF) was explored to heal osteomyelitis ulcers in a diabetic foot. The goal was to standardize the utilization of L-PRF in patients with osteomyelitis to direct it for healing. L-PRF was obtained autologously from the peripheral blood of the diabetic patients (n = 3) having osteomyelitis and skin lesions for at least six months. The L-PRF and supernatant serum were inserted into the skin lesion to the bone after a surgical debridement. The evolution of lesions over time was analyzed. All three patients showed positivity to the Probe-to-Bone test and Nuclear Magnetic Resonance detected cortico-periosteal thickening and/or outbreaks of spongy cortical osteolysis in adjacency of the ulcer. The infections were caused by Cocci Gram-positive bacteria, such as S. Aureus, S. β-hemolytic, S. Viridans and Bacilli; and Gram-negative such as Pseudomonas, Proteus, Enterobacter; and yeast, Candida. The blood count did not show any significant alterations. To date, all three patients have healed skin lesions (in a patient for about two years) with no evidence of infection. These preliminary results showed that L-PRF membranes could be a new method of therapy in such problematic diseases. Overall, the L-PRF treatment in osteomyelitis of a diabetic foot seems to be easy and cost-effective by regenerative therapy of chronic skin lesions. In addition, it will improve our understanding of wound healing.

2 production of L-PRF in horse directing it to human production. Our hypothesis is that the L-PRF is easy to produce in the horse, without modifications of the human protocol, thus allowing a better standardization of the human protocol. A new device for the preparation and the standardization of L-PRF clots and membranes is the L-PRF Wound Box ® . The optical microscopy, most cell bodies were highlighted concentrated in the proximal portion of each membrane, the last 1/4 was observed at the center; the distal part had only residual traces of cell bodies. The L-PRF will form constantly when the phases described above are strictly adhered to. The success of the art L-PRF depends entirely on the speed of blood collection and transfer in centrifuge within a minute and by a temperature of centrifugation and compression is higher than 21°C (between 21 and 30°C). Our experiments on the horse will no doubt be able to improve our understanding on wound healing, in particularly in chronic skin lesions therapy.