Blood samples were collected from four non-smoking, healthy, male volunteers with ages ranging from 27 to 56 years. Although having lifestyle-related diseases and receiving medication, these donors had no hindrance in daily life. 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.

As described previously [6, 15, 16], blood samples (~9.0 mL) collected without anticoagulants using vacuum plain glass tubes (A-PRF+; Jiangxi Fenglin Medical Technology Co. Ltd., Fengcheng, China) or conventional vacuum plain glass tube (Plain BD Vacutainer Tube; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) from the same donors were immediately centrifuged by an A-PRF centrifugation system (A-PRF12; DRAGON LABORATORY Instruments Ltd., Beijing, China) or a Medifuge centrifugation system (Silfradent S. r. l., Santa Sofia, Italy). After eliminating the red blood cell (RBC) fractions, the resulting A-PRF and CGF clots were compressed using a stainless-steel compression device and preserved between wet gauze until mechanical testing (usually for a maximum of 3 h).

To prepare platelet-poor plasma (PPP), peripheral blood (~9.0 mL) was collected using syringes containing A-formulation of acid-citrate-dextrose (ACD-A) (1.0 mL; Terumo, Tokyo, Japan) and immediately fractionated by the conventional double-spin method [17, 18]. The supernatant was collected as the PPP fraction. To prepare fibrin clots, bovine thrombin (Liquid Thrombin MOCHIDA Softbottle, Mochida Pharmaceutical Co. Ltd., Tokyo, Japan) was added to the PPP at a final volume percentage of 2.5% (v/v) at ambient temperature in glass chambers. The resulting PPP clots, which is designated as platelet-poor, thrombin-activated fibrin (PPTF), were compressed and preserved between wet gauze until mechanical testing (usually for a maximum of 3 h).

After excess amounts of exudate were quickly absorbed by the dry gauze, wet weights of freshly prepared A-PRF, CGF, and PPTF clots were measured using an electric balance. After compression with the stainless compressor, their weights were measured again. The compressed clots were then dried by heating at 140 °C for 30 min and were weighed in a pre-heated moisture analyzer (MA35; Sartorius Corporate Administration GmbH, Goettingen, Germany).