Based on their characteristics and fractionation, the differences among PRP and PRP derivatives are concisely described in our previous article [1].

As previously described [5, 6], blood samples (11.5 mL) were collected using syringes or vacuum blood collection tubes equipped with 18G needles from seven non-smoking, healthy, middle-aged, male volunteers (37 to 68 years old) three times with a 2-week interval. Even though they are suffering from lifestyle-related diseases and receiving medication, these donors had no hindrance in daily life.

To quantify each blood cell component, peripheral blood (~12.0 mL) was collected using syringes containing acid citrate dextrose solution (ACD-A) (1.5 mL; Terumo, Tokyo, Japan). Because of its high efficiency, PRP was prepared using FDA-approved Ycellbio PRP preparation tubes (Ycellbio Medical Co., Ltd., Seoul, South Korea). As previously described [7], freshly collected blood samples were subjected to blood cell count and simultaneously transferred to funnel-shaped tubes and centrifuged at 3200 rpm (1800g) for 4 min (Table 1). After adjusting the level of the buffy coat, the tubes were centrifuged for an additional 4 min. The resulting PRP fractions (buffy coat) were collected using syringes equipped with long needles and stored at −80 °C until determination of growth factor levels and the in vitro bioassay using periosteal cells (n = 20). Small aliquots of the freshly prepared PRP were subjected to blood cell count.

The study design and consent forms for all procedures performed with the study subjects were approved by the ethical committee for human subjects at Niigata University School of Medicine in accordance with the Helsinki Declaration of 1975 as revised in 2008.

According to the manufacturer’s instructions, blood samples (~9.6 mL) were collected from the same volunteers using 18G needles and PRGF-Endoret^® tubes (BTI Biotechnology Institute, S.L., Miñano, Spain), which contained 0.2 mL sodium citrate. The tubes were centrifuged at 1850 rpm (580g) for 8 min (Table 1) [8]. Fraction 2 (a fraction above the buffy coat) was collected and stored at −80 °C until use. Small aliquots of the freshly prepared PRP were subjected to blood cell count.