Because of its simple operation, platelet-rich fibrin (PRF) is becoming more popular than the original form, platelet-rich plasma (PRP), in regenerative dentistry. PRF preparation requires plain glass blood-collection tubes, but not either anticoagulants or coagulation factors. However, such glass tubes designed for laboratory testing are no longer commercially available. Although several glass tubes specifically designed for PRF preparation are available, many clinicians prefer to obtain stably supplied substitutes, such as silica-coated plastic tubes produced by major medical device companies. The quality of PRF prepared by silica-coated tubes has not been assessed and we previously reported significant contamination of silica microparticles in the resulting PRF matrix and alerted clinicians against the use for PRF preparation. To further assess the biosafety of the silica microparticles, we presently examined their effects on human normal periosteal cells derived from alveolar bone. The periosteal cells were obtained from explant cultures of small periosteal tissues obtained from healthy donors. Silica microparticles were obtained from silica-coated tubes and added to cell cultures. Cellular responses were monitored using a tetrazolium assay, phase-contract inverted microscopy, an immunofluorescence method, and scanning electron microscopy. Silica microparticles adsorbed onto the cell surface with seemingly high affinity and induced apoptosis, resulting in significant reduction of cell proliferation and viability. These findings suggest that silica microparticles contained in plastic tubes for the purpose of blood coagulation are hazardous for various cell types around sites where silicacontaminated PRF matrices are implanted.Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Compared with platelet-rich plasma, the preparation of platelet-rich fibrin (PRF) is simple and has not been overly modified. However, it was recently demonstrated that centrifugation conditions influence the composition of PRF and that silica microparticles from silica-coated plastic tubes can enter the PRF matrix. These factors may also modify platelet distribution. To examine these possibilities, we prepared PRF matrices using various types of blood-collection tubes (plain glass tubes and silica-containing plastic tubes) and different centrifugation speeds. The protocols of concentrated growth factors and advanced-PRF represented high- and low-speed centrifugation, respectively. Platelet distribution in the PRF matrix was examined immunohistochemically. Using low-speed centrifugation, platelets were distributed homogeneously within the PRF matrix regardless of tube types. In high-speed centrifugation, platelets were distributed mainly on one surface region of the PRF matrix in glass tubes, whereas in silica-coated tubes, platelet distribution was commonly more diffusive than in glass tubes. Therefore, both blood-collection tube types and centrifugal conditions appeared to influence platelet distribution in the PRF matrix. Platelets distributed in the deep regions of the PRF matrix may contribute to better growth factor retention and release. However, clinicians should be careful in using silica-coated tubes because their silica microparticles may be a health hazard.
Polyphosphate (polyP), a biopolymer of inorganic phosphate, is widely distributed in living organisms. In platelets, polyP is released upon activation and plays important roles in coagulation and tissue regeneration. However, the lack of a specific quantification method has delayed the in-depth study of polyP. The fluorescent dye 4′,6-diamidine-2-phenylindole dihydrochloride (DAPI) has recently received attention as a promising probe for the visualization and quantification of cellular polyP levels. In this study, we further optimized quantification conditions and applied this protocol in quantification of platelet polyP levels in a Japanese population. Blood samples were collected from non-smoking, healthy Japanese subjects (23 males, 23 females). Washed platelets were fixed and probed with DAPI for fluorometric determination. PolyP levels per platelet count were significantly higher in women than that in men. A moderate negative correlation between age and polyP levels was found in women. Responsiveness to CaCl2 stimulation was also significantly higher in women than that in men. Overall, our optimized protocol requires neither purification nor degradation steps, reducing both the time and bias for reproducible quantification. Thus, we suggest that despite its low specificity, this DAPI-based protocol would be useful in routine laboratory testing to quantify platelet polyP levels efficiently and economically.
Platelet-rich fibrin (PRF) is a fibrin matrix enriched with platelets. The PRF matrix is thought to form a steep gradient of platelet density around the region corresponding to the buffy coat in anticoagulated blood samples. However, this phenomenon has not yet been proven. To visualize platelet distribution in PRF in a non-invasive manner, we utilized near-infrared (NIR) imaging technology. In this study, four types of PRF matrices, bio-PRF, advanced-PRF (A-PRF), leukocyte-rich PRF (L-PRF), and concentrated growth factors (CGF) were compared. Blood samples collected from healthy, non-smoking volunteers were immediately centrifuged using four different protocols in glass tubes. The fixed PRF matrices were sagittally divided into two equal parts, and subjected to modified immunohistochemical examination. After probing with NIR dye-conjugated secondary antibody, the CD41+ platelets were visualized using an NIR imager. In L-PRF and CGF, platelets were distributed mainly on and below the distal surface, while in bio-PRF and A-PRF, platelet distribution was widespread and homogenous. Among three regions of the PRF matrices (upper, middle, and lower), no significant differences were observed. These findings suggest that platelets aggregate on polymerizing fibrin fibers and float up as a PRF matrix into the plasma fraction, amending the current “gradient” theory of platelet distribution.
Polyphosphate (polyP) is released from activated platelets and activates the intrinsic coagulation pathway. However, polyP may also be involved in various pathophysiological functions related to platelets. To clarify these functions, we established a cytochemical method to reproducibly visualize polyP in platelets. Platelets obtained from healthy non-smoking donors were suspended in phosphate-buffered saline and quickly immobilized on glass slides using a Cytospin. After fixation and membrane permeabilization, platelets were treated with 4′,6- diamidino-2-phenylindole (DAPI) and examined using a fluorescence microscope with a blue-violet excitation filter block (BV-2A). Fixed platelets were also subjected to immunocytochemical examination to visualize serotonin distribution. Under the optimized conditions for polyP visualization, immobilized platelets were fixed with 10% neutral-buffered formalin for 4 h or longer and treated with DAPI at a concentration of 10 µg/mL in 0.02% saponin- or 0.1% Tween-20-containing Hanks balanced salt solution as a permeabilization buffer for 30 min at room temperature (22–25 °C). Based on the results obtained by using activated platelets, treatment with alkaline phosphatases, and serotonin release, the DAPI+ targets were identified as polyP. Therefore, this cytochemical method is useful for determining the amount and distribution of polyP in platelets.
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