Montano, Chiara (2025) Platelet-rich fibrin for treatment of equine wounds: an in vitro and in vivo study. [Tesi di dottorato]

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Abstract

The advanced platelet-rich fibrin plus (A-PRF+) represents a second-generation platelet concentrate which has shown remarkable regenerative abilities in human medicine. It was developed in 2014 by Ghanaati and colleagues and, since then, it has been used in different fields of human medicine, from cosmetic to orthodontic surgery. A-PRF+ has a higher concentration of growth factors (GFs) and a looser fibrin structure compared to the leukocyte PRF, allowing a GFs release for a longer period. All these features make A-PRF+ a product that promotes haemostasis, enhances cell migration and proliferation, and supports the immune system. Compared to other platelet concentrates, such as platelet-rich plasma (PRP), studies have shown that A-PRF+ clots release growth factors over a longer period, up to seven days, post-application. Wounds are very common in equine patients and, among many therapeutic options, platelet concentrates, like PRP, have demonstrated to modulate tissue healing in horses. However, no studies in the literature investigate the effectiveness of A-PRF+ in equine wounds. Therefore, the aims of this doctoral thesis were to evaluate: 1) the correlation between macroscopic and microscopic features of A-PRF+ produced with two different centrifuges and the feasibility to produce A-PRF+ in the field; 2) in vitro effects of equine A-PRF+ on primary equine fibroblast cultures, particularly proliferation, migration, metabolic activity, cell cycle re-entry, reactive oxygen species production and proteomic profile of the secretome; 3) the efficacy of A-PRF+ in accelerating healing, reducing healing complications and facilitating the regeneration of acute full-thickness traumatic wounds in horses. This study has provided a comprehensive evaluation of the effects of A-PRF+ in both in vitro and in vivo situations, contributing valuable insights into its regenerative ability. Haematological parameters had minimal influence on the size of A-PRF+ clots and membranes, and the production process was practical, even in field conditions, with clot formation achievable at temperatures below 21°C. The molecular analysis of A-PRF+ revealed a detailed understanding of its effects on equine fibroblasts, with proteomic profiling identifying a broad range of proteins involved in extracellular matrix remodelling, metabolic regulation, and cytoskeletal organization. These findings suggest that A-PRF+ supports long-term regenerative processes, promoting fibroblast migration, and proliferation. The study also highlighted the metabolic reprogramming and cellular stress adaptation mechanisms triggered by A-PRF+, offering an explanation for its regenerative capacity at the molecular level. The application of A-PRF+ to equine traumatic wounds led to rapid healing, proper vascularization, along with a low incidence of complications. These clinical results underscore the potential of A-PRF+ in equine wound management. However, further studies with larger sample sizes are needed to validate these findings and explore its antimicrobial properties. The results of this doctoral thesis highlight the remarkable regenerative capacity of A-PRF+, showing encouraging effects on both tissue repair and cellular activity. Additional studies are needed to thoroughly clarify its molecular mechanisms and refine its clinical use, opening the way for a more standardized and effective approach in equine regenerative medicine.

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