After pipetting the digestion solution, 50 μL of the digestion solution was collected every 20 min and was stored at −20 °C until protein measurement. Protein levels, which can be considered primarily as levels of digested fibrin fiber, were then determined by a BCA protein assay kit (Takara Bio, Kusatsu, Japan). The protein levels at the time point when the initial fibrin disks were completely digested overnight were evaluated at 100%.

The PRF clots that were compressed in a stainless-steel compressor, were fixed with 2.5% neutralized glutaraldehyde, dehydrated with a series of ethanol solutions and t-butanol, freeze-dried, and then were examined under a scanning electron microscope (TM-1000; Hitachi, Tokyo, Japan) with an accelerating voltage of 15 kV, as described previously [16].

The data were expressed as mean ± standard deviation (SD). For multi-group comparisons, statistical analyses were conducted to compare the mean values by one-way analysis of variance (ANOVA) followed by Dunn’s multiple-comparison test (SigmaPlot 12.5; Systat Software, Inc., San Jose, CA, USA). Differences with P values < 0.05 were considered significant.