Optimized Centrifugation and Activation Protocol for the Preparation of Plasma Rich in Growth Factors in Pigs

Background: Cartilage defects remain a clinical challenge due to the limited intrinsic repair capacity of hyaline cartilage, driving increasing interest in blood-derived products, including platelet-rich plasma (PRP). Variability in PRP preparation and activation protocols limits reproducibility and clinical translation, particularly in large animal models where species-specific differences are an additional cue. This study aimed to standardize and optimize in pigs a protocol for plasma rich in growth factors (PRGF), a leukocyte-poor PRP, aligned with current human clinical practice. Methods: Whole blood from six female pigs was processed via three centrifugation protocols and activated with varying CaCl₂ concentrations to evaluate gelation and morphology. PRGF was characterized through hematological analysis, ELISA-based quantification of soluble factors, and structural imaging of fibrin gel via histology and scanning electron microscopy. Data were further analyzed using protein-protein interaction networks, hierarchical clustering, and comparative human PRGF proteomic profiles. Results: Protocol with 400× g centrifugation followed by 13.3 mM CaCl₂ activation achieved the most favorable performance, yielding the highest platelet recovery, effective leukocyte clearance, and consistent formation of a well-organized fibrin network. Porcine activated PRGF showed substantial overlap in detected factors and concentration ranges with human activated PRGF prepared with the same protocol. Conclusions: These findings establish a robust, clinically aligned porcine PRGF protocol and support the pig as a relevant translational model for PRP-based regenerative strategies, providing a reliable platform for preclinical evaluation of cartilage therapies.