Growth factor and cytokine characterization of canine platelet lysate with variable leukocyte concentration, plasma content, and heat-sensitive proteins

Abstract

Background: Platelet lysate is an acellular platelet product containing factors released from secretory granules, including cytokines and growth factors. This study aimed to evaluate different centrifugation methods used to prepare canine platelet lysate with variable content of leukocytes, plasma, and heat-sensitive proteins.

Methods: Whole blood was collected from six dogs and two double-spin preparation methods were used to generate the platelet-rich plasma with reduced (PRP) and high (L-PRP) concentration of leukocytes. A portion of both methods underwent plasma depletion via centrifugation and platelet lysate was generated via freeze-thaw cycles. A portion of the generated platelet lysate underwent complement inactivation via heat treatment. Growth factors (TGF-β1, VEGF, TNF-α, PDGF-BB, HGF) were quantified in all different platelet lysate preparations using ELISAs.

Results: Both platelet-rich plasma preparations had a 6.7-fold increase in platelet concentration. White blood cell (WBC) concentration compared to whole blood increased 1.2-fold times in PRP and 1.9-fold times in L-PRP. Negligible concentrations of platelets, WBC, and hematocrit were identified in all lysate groups. Statistically significant differences were identified for PDGF, VEGF, and TNF-α, and not for TGF-β or HGF. No growth factor differences were noted between centrifugation methods. PDGF was significantly higher in platelet lysate that was plasma depleted. VEGF was significantly higher in heat-treated lysate groups. TNF-α concentrations were overall very low, though were noted to significantly increase following plasma depletion.

Conclusion: These results support that growth factors and cytokine release can be affected by the platelet lysate preparation and processing.

Keywords: complement; growth factors; leukocyte concentration; plasma proteins; platelet lysate.

Figure 1

Overview of the study design. The image represents the experimental design and the procedures performed. (A) Generation of leukocyte-reduced platelet-rich plasma (PRP) and leukocyte-rich platelet-rich plasma (L-PRP). (B) Plasma depletion and generation of pure platelet pellet concentrate (PPC) and leukocyte rich platelet pellet concentrate (LPPC). (C) Generation of variable lysate formulations from PRP, L-PRP, PPC and LPPC (PL, LPL, PPL, LPPL). (D) Heat treatment to generate complement inactivated formulations from PL, LPL, PPL and LPPL (hPL, hLPL, hPPL, and hLPPL). ACD-A, acid citrate dextrose-A solution; PRP, leukocyte-reduced platelet-rich plasma; L-PRP, leukocyte-rich platelet-rich plasma; PL, leukocyte-reduced platelet lysate; LPL, leukocyte-rich platelet lysate; PPL, leukocyte-reduced platelet pellet lysate; LPPL, leukocyte-rich platelet pellet lysate; hPL, heat-treated PL; hLPL, heat-treated LPL; hPPL, heat-treated PPL; hLPPL, heat-treated LPPL. Created with BioRender.com.

Figure 2

Mean concentration of platelet-derived growth factor (PDGF) between different preparations methods of lysate from both leukocyte-reduced platelet-rich plasma (PRP) or leukocyte-rich platelet-rich plasma (L-PRP). (A) Between variable lysate formulations generated from PRP (B), or L-PRP (C) through ELISA (n = 2; mean ± SD). *p < 0.05. ELISA, enzyme-linked immunosorbent assay; PL, leukocyte-reduced platelet lysate; LPL, leukocyte-rich platelet lysate; PPL, leukocyte-reduced platelet pellet lysate; LPPL, leukocyte-rich platelet pellet lysate; hPL, heat-treated PL; hLPL, heat-treated LPL; hPPL, heat-treated PPL; hLPPL, heat-treated LPPL.

Figure 3

Quantification of vascular endothelial growth (VEGF) between different preparations methods of lysate from both leukocyte-reduced platelet-rich plasma (PRP) or leukocyte-rich platelet-rich plasma (L-PRP). (A) Between variable lysate formulations generated from PRP (B), or L-PRP (C) through ELISA (n = 2; mean ± SD). *p < 0.05. **p < 0.01. ELISA, enzyme-linked immunosorbent assay; PL, leukocyte-reduced platelet lysate; LPL, leukocyte-rich platelet lysate; PPL, leukocyte-reduced platelet pellet lysate; LPPL, leukocyte-rich platelet pellet lysate; hPL, heat-treated PL; hLPL, heat-treated LPL; hPPL, heat-treated PPL; hLPPL, heat-treated LPPL.

Figure 4

Quantification of tumor necrosis factor alpha (TNF-α) between different preparations methods of lysate from leukocyte-reduced platelet-rich plasma (PRP) or leukocyte-rich platelet-rich plasma (L-PRP). (A) Between variable lysate formulations generated from PRP (B), or L-PRP (C) through ELISA (n = 2; mean ± SD). *p < 0.05 compared to the rest of the groups. ELISA, enzyme-linked immunosorbent assay; PL, leukocyte-reduced platelet lysate; LPL, leukocyte-rich platelet lysate; PPL, leukocyte-reduced platelet pellet lysate; LPPL, leukocyte-rich platelet pellet lysate; hPL, heat-treated PL; hLPL, heat-treated LPL; hPPL, heat-treated PPL; hLPPL, heat-treated LPPL.

Figure 5

Quantification of transforming growth factor-β (TGF-β) (A) and hepatocyte growth factor (HGF) (B) from platelet rich plasma (PRP) or leukocyte and platelet rich plasma (L-PRP) as assessed through ELISA (n = 2; mean ± SD). ELISA, enzyme-linked immunosorbent assay; PL, leukocyte-reduced platelet lysate; LPL, leukocyte-rich platelet lysate; PPL, leukocyte-reduced platelet pellet lysate; LPPL, leukocyte-rich platelet pellet lysate; hPL, heat-treated PL; hLPL, heat-treated LPL; hPPL, heat-treated PPL; hLPPL, heat-treated LPPL.