Cytokine and Growth Factor Analysis of L-PRF Subfractions

For screening, a Human Cytokine Antibody Array (ab133998, ABCAm, Cambridge, UK) was performed on L-PRF CM (collected after 96 h total) and EX of four different healthy donors at a protein concentration of 10 mg/mL according to the manufacturer’s instructions (Thermo Scientific, Erembodegem, Belgium). Protein concentrations of the samples were determined with a bicinchoninic acid assay (BCA, Thermo Fisher Scientific, Waltham, MA, USA) following the user manual. Relative pixel density was measured using ImageJ/Fiji software 1.53 (NIH, Bethesda, MD, USA) to compare relative protein levels between L-PRF EX and CM. This analysis was previously performed by our group [50 (link),83 (link)]. This study reinterpreted the data of these arrays focusing on the neurotrophic factors and other growth factors present in both L-PRF subfractions that may affect neuronal cell functions. In addition, an Enzyme-Linked Immunosorbent Assay (ELISA) (Raybiotech, Peachtree Corners, GA, USA) was performed to quantify and verify vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and platelet-derived growth factor (PDGF) protein levels in L-PRF EX and CM collected at 24, 48, 96, and 144 h to evaluate the release of these growth factors from the clot over time.