Novel Platelet Rich Plasma/ Hyaluronic acid lyophilized formulations for wound healing applications

Antonella D'Agostino¹Maria d'Agostino¹Marta Nardini²Anita Muraglia²CELESTE DI MEO¹Maddalena Mastrogiacomo^2*Chiara Schiraldi^1*

• ¹Department of Experimental Medicine, University of Campania Luigi Vanvitelli, Naples, Italy
• ²Department of Internal Medicine and Medical Specialties, School of Medical and Pharmaceutical Sciences, University of Genoa, Genova, Italy

Platelet Rich Plasma (PRP) is a well-known biological product used for regenerative medicine. One of the limitations of its clinical use is related to the need for the surgeons to obtain ready to use preparation. A lyophilized formulation with specific and reproducible levels of growth factors can thus be considered a significant improvement for tissue regeneration. Therefore, it is of great interest to develop a formulation that enables the prolonged release of the growth factors and bioactive components present in PRP whilst improving the stability of these biomolecules during storage. To this aim, specific preparations, obtained by mixing hyaluronic acid (HA) of low-medium molecular weight (56 or 200 kDa) with PRP, were lyophilized to achieve an "off the shelf" product. These formulations were characterized from a biophysical and biological viewpoint. Primary human dermal fibroblasts proliferation and time lapse scratch assays were accomplished on freshly lyophilized formulations and during storage at different temperatures (4°C, 25°C) to assess their biological activity upon resuspension for up to 6 months. Gene and protein expression of collagen type 1 and elastin at time zero were evaluated. PDGF-BB and VEGF release were accomplished by ELISA assay. All HA/PRP formulations were able to induce cell proliferation comparably to PRP alone. HA/PRP formulations exhibited a superior repair rate respect to PRP in the scratch assay, confirming the HA ability to improve fibroblast migration. In the presence of HA, 80% of wound closure was achieved within 24 hours, whereas PRP-treated samples reached approximately 60% of the repaired area. These data were supported by collagen and elastin expression. In in vitro wound healing assays, lyophilized HA/PRP products exhibited a superior effect compared to PRP alone at 3 months, but no significant improvement was found after 6 months. Prolonged storage needs very low temperatures to preserve PRP features (-20°C). In conclusion, here we propose novel lyophilized HA/PRP formulations, as promising products for topical and intra-dermic applications, and especially for wound healing. The combination of HA as a biopolymer facilitates the slow release of growth factors contained in PRP while also allowing for a prolonged shelf life under cold-controlled conditions (4°C).