Blood collection, preservation, and PRF preparation
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) , 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%.
Determination of glucose, calcium, and pH
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).
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).
A bioassay on human periosteal cells
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 , 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) .
Quantification of a growth factor by an enzyme-linked immunosorbent assay (ELISA)
PRF extracts prepared as described above were subjected to measurement of PDGF-BB levels using the Human PDGF-BB Quantikine ELISA Kit (R&D Systems, Inc., Minneapolis, MN, USA) as described previously .
Scanning electron microscopy (SEM)
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 examined under a scanning electron microscope (TM-1000, Hitachi, Tokyo, Japan) with an accelerating voltage of 15 kV, as described elsewhere [7, 14].
The data were expressed as mean ± standard deviation (SD). For multigroup comparisons, statistical analyses were conducted to compare the mean values by one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test (SigmaPlot 12.5; Systat Software, Inc., San Jose, CA, USA). Differences with P values <0.05 were considered significant.