Tissue Engineering by Exosomes From the Blood

Objectives: Platelet-rich plasma (PRP) has been used in wound healing and tissue regeneration without clear understanding of its mechanisms of action. PRP consists of cytokines, growth factors, and chemokines. We recently discovered that ~100 nm, CD63-positive vehicles, likely exosomes, play crucial roles in the crosstalk of dental epithelium and mesenchyme. However, whether PRP harbors exosomes is elusive. The present study was designed to explore whether PRP harbors exosomes and whether PRP-derived exosomes promote angiogenesis as a pivotal process of tissue regeneration.
Methods: Upon IRB approval, whole blood was drawn from eight human donors, followed by clinically compatible PRP preparation. PRP was degranulated by 10% CaCl₂ and thrombin (1U/mL) and evaluated by ELISA. Exosomes were extracted and purified from activated PRP by multiple ultracentrifuges and confirmed by western blot (CD63). Migration assay was performed by Transwell system. Exosomes were separately applied to keratinocytes (5 and 10 ug/ml) and dermal fibroblasts (25 and 50 ug/ml) with exosome-free medium. Cell proliferation was tested by CCK-8. Tube formation assay was performed by applying exosomes to HUVECs to detect its potential for angiogenesis. All quantitative data were statistically treated with α≤0.05.
Results: ELISA showed ~4-fold higher concentration of enriched proteins in PRP than donor-matched platelet-poor plasma (PPP). CD63 positive PRP exosomes were confirmed by western blot and induced robust migration of dermal fibroblasts in a dose-dependent manner, but not for keratinocytes. Exosomes increased the proliferation of both fibroblasts and keratinocytes for up to the tested 7 days. PRP-derived exosomes further promoted tubular formation of HUVEC cells. Dermal fibroblasts increased Collagen 1, MMP9 and keratinocytes increased ZO-1, MMP1 and E-cadherin at both mRNA and protein levels upon exosome exposure.
Conclusions: PRP-derived exosomes promote matrix synthesis and angiogenesis, two key processes of tissue regeneration. Exosomes related from PRP may account for some of its unexplained therapeutic efficacy.