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

With informed consent, blood samples (~9.0 mL per tube) were collected from six non-smoking, healthy, male volunteers (27 to 67 years old) using 21-gauge needles equipped with conventional vacuum plain glass tube (Plain BD Vacutainer Tube; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) as described previously [6–8]. For preparation of control PRF by the conventional method, the anticoagulant was not added. Blood samples were immediately centrifuged or stored by gentle mixing using a tube rotator at ambient temperature (18–22 °C).

The blood samples collected with the anticoagulant and stored for up to 2 days were centrifuged by means of a Medifuge centrifugation system (Silfradent S.r.l., Santa Sofia, Italy). After elimination of the red blood cell fractions, the resulting PRF clots, more specifically termed as concentrated growth factors (CGF) [9], were stored at −80 °C until measurement of growth factor concentration.

For preparation of platelet-poor plasma (PPP), blood samples (~9.0 mL) were collected from the same volunteers by means of plastic vacuum blood collection tubes (Neotube®; NIPRO, Osaka, Japan) equipped with 21-gauge needles, in the presence of 1.0 mL acid citrate dextrose solution-A formulation (ACD-A; Terumo, Tokyo, Japan), an anticoagulant [8, 10]. The blood samples were centrifuged on a KS-5000 centrifuge (Kubota, Tokyo, Japan) equipped with a swing rotor at 1700 rpm (530g) and 3000 rpm (1660g) for the first and second spin (8 min), respectively. The resulting supernatant fractions were collected as PPP preparations. To form fibrin clots, bovine thrombin (Liquid Thrombin MOCHIDA softbottle, Mochida Pharmaceutical Co., Ltd., Tokyo, Japan) was added to PPP at a final volume percentage of 2.5%.

WB samples were quickly centrifuged at 1500 rpm for 3 min to prepare plasma fraction, which were subjected to determine total free calcium levels using a commercial kit based on MXB method (Calcium E-test WAKO; Wako Pure Chemicals, Osaka, Japan).