Stored WB samples were then mixed intermittently with 200 μL (20 μL × 10 times) of 10% CaCl₂ solution and centrifuged by a Medifuge centrifugation system to prepare PRF. When lower amounts of CaCl₂ were added, PRF clots were less reproducibly prepared. When higher amounts of CaCl₂ were added intermittently, or when the optimal amount of CaCl₂ were added at once, PRF clots were never prepared (Kawase, unpublished observations).

The supernatant serum fractions were subjected to determine calcium and glucose levels as described above and using a commercial kit based on GOD method (Glucose CII-test WAKO; Wako Pure Chemicals). The serum fractions were also used to determine pH levels by pH indicators (MColorHast; EMD Millipore Corp., Billerica, MA, USA).

The frozen PRF samples were minced with scissors and homogenized using a disposable homogenizer (BioMasher II, Nippi, Tokyo, Japan). After high-speed centrifugation (7340g), supernatants (PRF extracts) were collected and used for the bioassay described below and for measurement of growth factor levels.

Because alveolar periosteum strongly contributes to regeneration of periodontal skeletal tissue [11], we used human alveolar bone-derived periosteal cells for evaluation of the potency and efficacy of PRF preparations. The periosteal cells were obtained and expanded as described elsewhere [8, 12]. With informed consent, human periosteum tissue segments were aseptically excised from the periodontal tissue on the healthy buccal side of the retromolar region of the mandibles of two non-smoking female volunteers (age = 19 and 29). Small periosteum pieces were expanded to form multilayered cellular periosteal sheets (∅ 30–40 mm), and then these sheets were enzymatically digested with 0.05% trypsin plus 0.52 mM EDTA (Invitrogen, Carlsbad, CA, USA) to release single cells. After expansion in monolayer cultures, the cells were seeded at a density of 0.4 × 10⁴ per well in 24-well plates and treated with PRF extracts (0, 0.5, 1, 2, or 4%) for 72 h in DMEM containing 1% of fetal bovine serum (Invitrogen, Carlsbad, CA, USA). Six different lots of PRF extracts were used for each experiment. At the end of the incubation periods, the cells were harvested using 0.05% trypsin plus 0.53 mM EDTA and immediately counted on an automated cell counter (Moxi-z; ORLFO Technologies, Ketchum, ID, USA) (N = 6) [13].