Standardized Platelet-Rich Fibrin (PRF) from canine and feline origin: An analysis on its secretome pattern and architectural structure.

Platelet-rich fibrin (PRF) has been incorporated in surgical procedures to promote tissue and bone healing, particularly in human medicine. The rationale for the use of platelet-based products stems from the fact that platelets, after being activated, release growth factors (GFs) and other active molecules such as cytokines, that modulate inflammation and tissue repair. Although PRF has been advanced as a therapeutic treatment for veterinary use, namely in canine and feline patients (following human medicine developments), to our knowledge a full characterization of PRF therapeutic effectors has never been performed. Herein, we studied the biological properties and release profile of GFs and other cytokines throughout ten days in in vitro culture conditions, in order to investigate the potential therapeutic ability of PRF for canine and feline practice. A protocol for obtaining PRF from whole blood without anti-coagulant from both species was optimized, originating large and homogenous PRF clots. Then, PRF clots obtained from four dogs and four cats were incubated in culture medium to assess the temporal release of platelet-derived growth factor-BB (PDGF-BB), vascular endothelial factor-A (VEGF-A), transforming growth factor ß-1 (TGF-ß1), and interleukin-8 (IL-8). Furthermore, morphological characterization of PRF clots, fresh and after 10 days of incubation, was performed by histology and high-resolution field emission electron scanning microscopy. In standard culture conditions, PRF clots from both species released PDGF-BB, TGF- ß1 and VEGF-A, in a sustained manner, up to day 10. Moreover, PRF presents an initial burst release of IL-8, a mediator of inflammatory response which plays a key role in neutrophil recruitment and degranulation. Overall, our findings show that PRF clots may be an efficient therapeutic strategy in canine and feline clinical practice, accelerating the local healing mechanism, through the sustained delivery of signalling molecules involved in the healing cascade.

Ultrafiltration and Injection of Islet Regenerative Stimuli Secreted by Pancreatic Mesenchymal Stromal Cells.

The secretome of mesenchymal stromal cells (MSCs) is enriched for biotherapeutic effectors contained within and independent of extracellular vesicles (EVs) that may support tissue regeneration as an injectable agent. We have demonstrated that the intrapancreatic injection of concentrated conditioned media (CM) produced by bone marrow MSC supports islet regeneration and restored glycemic control in hyperglycemic mice, ultimately providing a platform to elucidate components of the MSC secretome. Herein, we extend these findings using human pancreas-derived MSC (Panc-MSC) as "biofactories" to enrich for tissue regenerative stimuli housed within distinct compartments of the secretome. Specifically, we utilized 100 kDa ultrafiltration as a simple method to debulk protein mass and to enrich for EVs while concentrating the MSC secretome into an injectable volume for preclinical assessments in murine models of blood vessel and islet regeneration. EV enrichment (EV+) was validated using nanoscale flow cytometry and atomic force microscopy, in addition to the detection of classical EV markers CD9, CD81, and CD63 using label-free mass spectrometry. EV+ CM was predominately enriched with mediators of wound healing and epithelial-to-mesenchymal transition that supported functional regeneration in mesenchymal and nonmesenchymal tissues. For example, EV+ CM supported human microvascular endothelial cell tubule formation in vitro and enhanced the recovery of blood perfusion following intramuscular injection in nonobese diabetic/severe combined immunodeficiency mice with unilateral hind limb ischemia. Furthermore, EV+ CM increased islet number and ß cell mass, elevated circulating insulin, and improved glycemic control following intrapancreatic injection in streptozotocin-treated mice. Collectively, this study provides foundational evidence that Panc-MSC, readily propagated from the subculture of human islets, may be utilized for regenerative medicine applications.