Does platelet-rich plasma freeze-thawing influence growth factor release and their effects on chondrocytes and synoviocytes?

Abstract

PRP cryopreservation remains a controversial point. Our purpose was to investigate the effect of freezing/thawing on PRP molecule release, and its effects on the metabolism of chondrocytes and synoviocytes. PRP was prepared from 10 volunteers, and a half volume underwent one freezing/thawing cycle. IL-1β, HGF, PDGF AB/BB, TGF-β1, and VEGF were assayed 1 hour and 7 days after activation. Culture media of chondrocytes and synoviocytes were supplemented with fresh or frozen PRP, and, at 7 days, proliferation, gene expression, and secreted proteins levels were evaluated. Results showed that in the freeze-thawed PRP the immediate and delayed molecule releases were similar or slightly lower than those in fresh PRP. TGF-β1 and PDGF AB/BB concentrations were significantly reduced after freezing both at 1 hour and at 7 days, whereas HGF concentration was significantly lower in frozen PRP at 7 days. In fresh PRP IL-1β and HGF concentrations underwent a significant further increase after 7 days. Similar gene expression was found in chondrocytes cultured with both PRPs, whereas in synoviocytes HGF gene expression was higher in frozen PRP. PRP cryopreservation is a safe procedure, which sufficiently preserves PRP quality and its ability to induce proliferation and the production of ECM components in chondrocytes and synoviocytes.

Figure 1

RT-PCR analysis: messenger RNAs expression in chondrocytes grown in presence of 10% fresh or frozen PRP at 7 days. Data were normalized to GAPDH and expressed as a percentage of the reference gene. Boxes indicate the 25% and 75% percentiles, whiskers indicate the minimum to maximum values, and bars indicate the median.

Figure 2

Expression analysis of factors involved in joint physiopathology: messenger RNAs expression in synoviocytes grown in presence of 10% fresh or frozen PRP at 7 days. Data were expressed as n°mol mRNA ×100000 GAPDH. Hyaluronic acid production was evaluated in culture supernatants and protein production was normalized per number of cells. Boxes indicate the 25% and 75% percentiles, whiskers indicate the minimum to maximum values, and bars indicate the median.

Figure 3

Hyaluronan levels in the culture media of human chondrocytes grown in presence of 10% fresh or frozen PRP at 7 days. Mean values are expressed as ng/mL, boxes indicate the 25% and 75% percentiles, whiskers indicate the minimum to maximum values, and bars indicate the median.