Comparison of bioactive substances in novel-developed freeze-dried platelet-rich plasma (PRP) and activated normal PRP, and investigation of bioactive substance levels after long-term storage.

INTRODUCTION: In recent years, biotherapy in orthopedics has become widespread, and platelet-rich plasma (PRP) has been readily used to treat sports injuries and osteoarthritis. Production of freeze-dried PRP (PRP-FD) results in PRP that is in powder form, allowing it to be stored for long periods at room temperature. Using this technology, we have developed Valuable Platelet-Derived Factor Concentrate Freeze Dry (VFD). However, whether VFD contains sufficient levels of bioactive substances (BS) remains unknown and retains the same levels of BS during long-term storage. In this study, we examined whether VFD contains sufficient amounts of BS and whether they retain these BS levels during long-term storage.

METHODS: Peripheral blood was collected from 10 healthy men (mean ± SD: 46.5 ± 15 years old) and various BS, including transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), tissue inhibitors of metalloproteinases-1 (TIMP-1), interleukin-1 receptor antagonist (IL-1ra), matrix metallopeptidase-9 (MMP-9), and interleukin-6 (IL-6), were compared between VFD and normal PRP samples, including both leukocyte-rich PRP (LR-PRP) and leukocyte-poor PRP (LP-PRP). VFD was prepared using two rounds of centrifugation. LP-PRP and LR-PRP were activated by freezing and thawing before measurement. To evaluate the effects of long-term storage, the BS of VFD purified from five professional football players was compared between baseline and 1 year after storage.

RESULTS: In terms of the growth factors, the TGF-β and EGF levels were higher in LR-PRP than in VFD and LP-PRP ( p  < 0.05), while the bFGF levels were higher in VFD than in the LR-PRP and LP-PRP groups ( p  < 0.01). In terms of anti-inflammatory cytokines, the TIMP-1 level was lower in VFD than that in the other groups ( p  < 0.01), whereas the IL-1ra levels were higher in VFD than those in LP-PRP ( p  < 0.05) and lower than those in LR-PRP ( p  < 0.01). In terms of inflammatory enzymes and cytokines, the IL-1ra level was higher in VFD than that in LP-PRP (p < 0.05) and lower than that in LR-PRP (p < 0.01), whereas the IL-6 levels did not differ among the groups. Furthermore, the TGF-β, bFGF, TIMP-1, and IL-1ra levels were 5.61 → 3.38 (x103  pg/μL), 61.0 → 63.0 (pg/μL), 3.4 → 2.7 (x105  pg/μL), and 14.9 → 14.5 (x103  pg/μL) at baseline and 1 year later, respectively. No significant differences in the BS levels were observed between baseline and 1 year after storage.

CONCLUSIONS: The VFD samples prepared in this study exhibited higher levels of anti-inflammatory cytokines than LP-PRP and contained growth factor levels similar to LP-PRP and LR-PRP. In addition, the BS levels in VFD samples were maintained after one year of storage. These results suggest that VFD can be prepared and stored and may serve as a novel treatment strategy for sports injuries in high-risk groups, such as athletes.