Changes on the Structural Architecture and Growth Factor Release, and Degradation in Equine Platelet-Rich Fibrin Clots Cultured Over Time

Abstract

The aims of the present study were (1) to describe the microscopic and ultrastructural appearance of equine platelet-rich fibrin (PRF) clots and (2) to determine the release and degradation of transforming growth factor beta 1 (TGF-β₁) and insulin-like growth factor type I (IGF-I) from PRF clots incubated over 14 days. Whole blood from six horses was collected into plain tubes and centrifuged at 240 g for 8 minutes. Clots were evaluated by histology and by both transmission and scanning electronic microscopy (TEM and SEM). Growth factor concentrations were measured by ELISA at 48-hour intervals over 14 days and analyzed by one-way repeated-measures ANOVA. Histology showed a clot composed by a fibrin layer and a cellular layer with platelets and leukocytes. Scanning electron microscopy showed the cells trapped by an incipient fibrin network at 1 hour. At day 8, these cells were embedded by an incipient fibrin network. At day 14, the leukocytes and platelet aggregates from the clot were imbibed in an organized web of fibrin fibrils. TEM exhibited platelets with preserved cytoplasm and alpha granules randomly scattered at day 8, and damaged platelets with interrupted cytoplasm and organelle emigration to the periphery at day 14. TGF-β₁ and IGF-I concentrations showed a progressive increase until day 14. TGF-β₁ was released from PRF clots in a gradual and controlled manner, and increasing its concentration for two weeks, which supports TEM findings indicating that platelets began disintegrating by day 14. Furthermore, IGF-I production and release from PRF clots is sustained over time.

Keywords: Biomaterial; Horse; Platelet concentrate; Regenerative therapy; Ultrastructural assessment.