Fig. 4. Bioactivities and PDGF-BB concentrations in PRF extracts and the supernatant serum fraction. a PRF extracts were added to periosteal cell cultures and incubated for 3 days to evaluate their effects on cell proliferation. No significant differences were observed among three groups. b PRF extracts were subjected to measurement of PDGF-BB levels using an ELISA kit. No significant differenc...
Fig. 3. SEM examination of fibrin fibers formed in self-clotted PRF and thrombin-stimulated PPP clots. PRF was prepared from fresh and 2-day-old WB samples. Similar observations were obtained from WB samples collected from three other donors. Scale bars = 10 μm
Fig. 3. SEM examination of fibrin fibers formed in self-clotted PRF and thrombin-stimulated PPP clots. PRF was prepared from fresh ...
Fig. 2. Appearance of PRF clots prepared from WB samples stored for 2 days. These observations are representative of WB samples obtained from four donors
Fig. 2. Appearance of PRF clots prepared from WB samples stored for 2 days. These observations are representative of WB samples obtained from four donors
Fig. 1. Glucose levels (a), calcium levels (b), and pH (c) of stored WB samples. Supernatant serum fractions were examined. Plasma fractions prepared by quick centrifugation were used to determine calcium levels in fresh and stored WB samples that were not added CaCl2. N = 6
Fig. 1. Glucose levels (a), calcium levels (b), and pH (c) of stored WB samples. Supernatant serum fractions were...
Isobe, K., Suzuki, M., Watanabe, T. et al. Platelet-rich fibrin prepared from stored whole-blood samples. Int J Implant Dent 3, 6 (2017). https://doi.org/10.1186/s40729-017-0068-4
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Received: 21 December 2016
Accepted: 15 February 2017
Published: 01 March 2017
DOI: https://doi.org/10.1186/s40729-017-0068-4
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were...
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Correspondence to Tomo...
Tokyo Plastic Dental Society, Kita-ku, Tokyo, Japan
Kazushige Isobe, Masashi Suzuki, Taisuke Watanabe, Yutaka Kitamura, Taiji Suzuki, Hideo Kawabata, Masayuki Nakamura, Toshimitsu Okudera & Hajime Okudera
Division of Oral Implantology, Niigata University Medical and Dental Hospital, Niigata, Japan
Kohya Uematsu
Bioscience Medical Research Center, Niigata University Medical and Dental ...
Duan X, Bradbury SR, Olsen BR, Berendsen AD. Matrix Biol. 2016;52-54:127–40.
Kawase T, Okuda K, Kogami H, Nakayama H, Nagata M, Nakata K, Yoshie H. Characterization of human cultured periosteal sheets expressing bone-forming potential: in vitro and in vivo animal studies. J Tissue Eng Regen Med. 2009;3:218–29.
Kawase T, Hayama K, Tsuchimochi M, Nagata M, Okuda K, Yoshie H, Burns DM, Nakata K...
Hess J. Conventional blood banking and blood component storage regulation: opportunities for improvement. Blood Transfus. 2010;8 Suppl 3:s9–s15.
World Health Organization. Manual on the management, maintenance and use of blood cold chain equipment. http://www.who.int/bloodsafety/Manual_on_Management,Maintenance_and_Use_of_Blood_Cold_Chain_Equipment.pdf. Accessed 26 Nov 2016.
van der Meer PF, d...
Acid citrate dextrose solution
Concentrated growth factors
Citrate phosphate and dextrose
Ethylenediaminetetraacetic acid
Platelet-derived growth factor
Platelet-poor plasma
Platelet-rich fibrin
Platelet-rich plasma
Scanning electron microscope
Whole blood
The self-clotted types of platelet concentrates (PRF) can be prepared from ACD-containing stored WB by addition of CaCl2 without a significant reduction in their bioactivity and without other specific reagents or devices. This approach should contribute to dissemination of PRF therapy.
Considering the current status of clinical use of platelet concentrates in the fields of periodontology and oral surgery, in this study, we used 10-mL glass tubes that are not oxygen-permeable instead of oxygen-permeable plastic bags for storage of large volumes of WB or platelets. We advanced a working hypothesis that the storage of WB samples in glass tubes would result in a more rapid and subst...
Platelet preservation is restricted to 3 and 5 days in Japan and worldwide, respectively. This limit is based on the fact that platelets are sensitive to changes in temperature and pH: when samples are stored at 2 to 6 °C, platelets become unsuitable for production of platelet concentrates [3]. Preservation of platelet concentrates results in a drop of pH below 6.0 depending on the platelet cou...
Glucose and calcium contents and pH of WB or serum samples after centrifugation are shown in Fig. 1. Because glucose is contained in the ACD-A solution, glucose levels in the stored WB and serum samples (see Fig. 4c) after centrifugation were significantly greater than those of freshly collected WB samples. Total free calcium levels, including calcium chelated by citrate, in WB samples decreased...
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 [8].
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...
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 prepa...
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) us...
Blood preservation is generally and widely used in the fields of blood transfusion and surgery for either autologous or allogeneic blood [1–3]. In case of small lots of blood-derived materials used in regenerative therapy, such as platelet concentrates, it is generally accepted that autologous blood samples should be collected on-site and immediately centrifuged for processing [4]. Accordingly, ...
In regenerative therapy, self-clotted platelet concentrates, such as platelet-rich fibrin (PRF), are generally prepared on-site and are immediately used for treatment. If blood samples or prepared clots can be preserved for several days, their clinical applicability will expand. Here, we prepared PRF from stored whole-blood samples and examined their characteristics.
Blood samples were collected ...
Isobe, K., Suzuki, M., Watanabe, T. et al. Platelet-rich fibrin prepared from stored whole-blood samples.
Int J Implant Dent 3, 6 (2017). https://doi.org/10.1186/s40729-017-0068-4
Download citation
Received: 21 December 2016
Accepted: 15 February 2017
Published: 01 March 2017
DOI: https://doi.org/10.1186/s40729-017-0068-4
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were...
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Tokyo Plastic Dental Society, Kita-ku, Tokyo, Japan
Kazushige Isobe, Masashi Suzuki, Taisuke Watanabe, Yutaka Kitamura, Taiji Suzuki, Hideo Kawabata, Masayuki Nakamura, Toshimitsu Okudera & Hajime Okudera
Division of Oral Implantology, Niigata University Medical and Dental Hospital, Niigata, Japan
Kohya Uematsu
Bioscience Medical Research Center, Niigata University Medical and Dental ...
Duan X, Bradbury SR, Olsen BR, Berendsen AD. Matrix Biol. 2016;52-54:127–40.
Kawase T, Okuda K, Kogami H, Nakayama H, Nagata M, Nakata K, Yoshie H. Characterization of human cultured periosteal sheets expressing bone-forming potential: in vitro and in vivo animal studies. J Tissue Eng Regen Med. 2009;3:218–29.
Kawase T, Hayama K, Tsuchimochi M, Nagata M, Okuda K, Yoshie H, Burns DM, Nakata K...
Hess J. Conventional blood banking and blood component storage regulation: opportunities for improvement. Blood Transfus. 2010;8 Suppl 3:s9–s15.
World Health Organization. Manual on the management, maintenance and use of blood cold chain equipment. http://www.who.int/bloodsafety/Manual_on_Management,Maintenance_and_Use_of_Blood_Cold_Chain_Equipment.pdf. Accessed 26 Nov 2016.
van der Meer PF, d...
Acid citrate dextrose solution
Concentrated growth factors
Citrate phosphate and dextrose
Ethylenediaminetetraacetic acid
Platelet-derived growth factor
Platelet-poor plasma
Platelet-rich fibrin
Platelet-rich plasma
Scanning electron microscope
Whole blood
The self-clotted types of platelet concentrates (PRF) can be prepared from ACD-containing stored WB by addition of CaCl2 without a significant reduction in their bioactivity and without other specific reagents or devices. This approach should contribute to dissemination of PRF therapy.
Considering the current status of clinical use of platelet concentrates in the fields of periodontology and oral surgery, in this study, we used 10-mL glass tubes that are not oxygen-permeable instead of oxygen-permeable plastic bags for storage of large volumes of WB or platelets. We advanced a working hypothesis that the storage of WB samples in glass tubes would result in a more rapid and subst...
Platelet preservation is restricted to 3 and 5 days in Japan and worldwide, respectively. This limit is based on the fact that platelets are sensitive to changes in temperature and pH: when samples are stored at 2 to 6 °C, platelets become unsuitable for production of platelet concentrates [3]. Preservation of platelet concentrates results in a drop of pH below 6.0 depending on the platelet cou...
Glucose and calcium contents and pH of WB or serum samples after centrifugation are shown in Fig. 1. Because glucose is contained in the ACD-A solution, glucose levels in the stored WB and serum samples (see Fig. 4c) after centrifugation were significantly greater than those of freshly collected WB samples. Total free calcium levels, including calcium chelated by citrate, in WB samples decreased...
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 [8].
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...
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 prepa...
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) us...
Blood preservation is generally and widely used in the fields of blood transfusion and surgery for either autologous or allogeneic blood [1–3]. In case of small lots of blood-derived materials used in regenerative therapy, such as platelet concentrates, it is generally accepted that autologous blood samples should be collected on-site and immediately centrifuged for processing [4]. Accordingly, ...
In regenerative therapy, self-clotted platelet concentrates, such as platelet-rich fibrin (PRF), are generally prepared on-site and are immediately used for treatment. If blood samples or prepared clots can be preserved for several days, their clinical applicability will expand. Here, we prepared PRF from stored whole-blood samples and examined their characteristics.
Blood samples were collected ...
Toyoda, T., Isobe, K., Tsujino, T. et al. Direct activation of platelets by addition of CaCl2 leads coagulation of platelet-rich plasma.
Int J Implant Dent 4, 23 (2018). https://doi.org/10.1186/s40729-018-0134-6
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Received: 13 March 2018
Accepted: 11 May 2018
Published: 01 August 2018
DOI: https://doi.org/10.1186/s40729-018-0134-6
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were ...
The study design and consent forms of all the procedures performed were approved by the ethics committee for human participants of the Niigata University School of Medicine (Niigata, Japan) in accordance with the Helsinki Declaration of 1964 as revised in 2013. Written informed consents to participate in the study were obtained from all the participants.
Written informed consent for the publicati...
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TT, KI, TT, and TK conceived and designed the study, performed the experiments and data analysis, and wrote the manuscript. YK, FO, TW, ...
Tokyo Plastic Dental Society, Kita-ku, Tokyo, Japan
Toshihisa Toyoda, Kazushige Isobe, Tetsuhiro Tsujino, Yasuo Koyata, Fumitaka Ohyagi, Taisuke Watanabe, Masayuki Nakamura, Yutaka Kitamura & Hajime Okudera
Bioscience Medical Research Center, Niigata University Medical and Dental Hospital, Niigata, Japan
Koh Nakata
Division of Oral Bioengineering, Institute of Medicine and Dentistry, ...
The authors appreciate Shoshin EM (Okazaki, Japan) for giving an opportunity to use DHM.
Because an article performed and prepared in parallel is now submitted elsewhere for publication, the authors do not wish to share their data at present time.
Blitz A. Pump thrombosis—a riddle wrapped in a mystery inside an enigma. Ann Cardiothor Surg. 2014;3:450–71.
Kawase T, Watanabe T, Okuda K. Platelet-rich plasma and its derived platelet concentrates: what dentists involved in cell-based regenerative therapy should know. Nihon Shishubyou Gakkai Kaishi. 2017;59:68–76. (in Japanese)
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Kobayashi M, Kawase T, Horimizu M, Okuda K, Wolff LF, Yoshie H. A proposed protocol for the standardized preparation of PRF membranes for clinical use. Biologicals. 2012;40:323–9.
Kawase T, Okuda K, Nagata M, Tsuchimochi M, Yoshie H, Nakata K. Non-invasive, quantitative assessment of the morphology of gamma-irradiated human mesenchymal stem cells and periosteal cells using digital holographic m...
Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR, Strauss JE, Georgeff KR. Platelet-rich plasma: growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1998;85:638–46.
Anitua E. Plasma rich in growth factors: preliminary results of use in the preparation of future sites for implants. Int J Oral Maxillofac Implants. 1999;14:529–35.
Masuki H, Okudera T, Wa...
Acid citrate dextrose solution
Adenosine diphosphate
Calcium-sensing receptor
Digital holographic microscopy
Phosphate-buffered saline
Platelet-poor plasma
Platelet-rich fibrin
Platelet-rich plasma
Scanning electron microscope
Tris-buffered saline
Thromboxane A2
In addition to the well-known intrinsic coagulation pathway, which activates platelets via thrombin conversion, added Ca2+ may directly activate washed platelets and promote clot formation alone and in cooperation with the coagulation pathway as illustrated in Fig. 8. Therefore, it is possible to control platelet activation levels and the consequent growth factor release by modulating not only in...
On the other hand, in PRF prepared from fresh whole-blood without anticoagulants, it is thought that the centrifugal force increases the contact of factor XII with a glass surface, thereby primarily forming fibrin clots. Although platelets can also be activated by a glass surface, the centrifugal force accumulates platelets at and just below the interface between the red blood cell fraction and pl...
An alternative possibility may be related to the status of platelets stored in a low-Ca2+ environment: platelets constantly increase thromboxane A2 (TXA2) production and tend to easily aggregate under the influence of the TXA2 autocrine loop [19]. Therefore, it is thought that platelets prepared from citrated whole-blood samples continuously repeat Ca2+ discharge from intracellular Ca2+ stores and...
It is well known that platelets are activated by adenosine diphosphate (ADP), thrombin, epinephrine, thromboxane A2, collagen, and many other compounds and thus aggregate through binding of fibrinogen and glycoprotein IIb/IIIa receptors and upregulate surface antigens known as “platelet activation markers”: CD62P and CD63 [13, 14]. During activation, platelet morphology generally changes from ...
The surface microstructures of the formed fibrin clots are depicted in Fig. 6. The loop-like substances that initially formed in PRP on watch glasses were composed of abundant aggregated platelets, and relatively smaller amounts of fibrin fibers were deposited around platelet aggregates (Fig. 6a). By contrast, in the final version of fibrin clots, platelet aggregates were hardly detectable and m...
Morphological changes of (and microparticle release and fibrin formation by) Ca2+-stimulated platelets are shown in Fig. 1. When washed platelets were treated with 0.1% CaCl2 in sample tubes and plated in culture dishes for the last 5 min of treatment (control, 2 min), platelets’ ability to adhere to the dish bottom surface increased with the duration of Ca2+ treatment (Fig. 1a). On the othe...
Imaging by DHM (HoloMonitor M4; Phase Holographic Imaging AB, Lund, Sweden) was performed as described elsewhere [12]. The data were analyzed using specialized software, HoloStudio M4 (Phase Holographic Imaging AB). For surface roughness and area analysis, after a series of images were captured, the grayscale images were converted to the black-and-white format by the Otsu method, and the cell iden...
As described in the subsection above, the platelet fractions were prepared and treated with CaCl2 in polypropylene sample tubes. At the end of incubation, the platelets were fixed with an equal volume of a commercial fixative, ThromboFix (Beckman-Coulter, Brea, CA, USA), for 30 min, washed twice with PBS, and probed simultaneously with phycoerythrin (PE)-conjugated mouse monoclonal anti-CD41 and ...
Blood samples were collected from eight nonsmoking healthy male volunteers at ages from 32 to 68 years. The study design and consent forms of all the procedures performed were approved by the ethics committee for human participants of the Niigata University School of Medicine (Niigata, Japan) in accordance with the Helsinki Declaration of 1964 as revised in 2013.
Peripheral blood (~ 9 mL) was...
In this study, we attempted to dissociate platelets from the coagulation pathway and to evaluate possible direct action of Ca2+ on platelet functions in citrated whole-blood samples. In addition, in response to recent increasingly frequent requests for scheduled or outsourced, but not immediate on-site, preparation of various platelet concentrate types [4, 5], we examined time course changes in pl...
Since Marx’s report [1], platelet-rich plasma (PRP) and subsequently modified PRP derivatives have been widely applied in regenerative dentistry. Unlike self-clotted platelet-rich fibrin (PRF), for better handling efficiency and minimizing the loss of growth factors to diffusion, PRP and some other derivatives in liquid form are usually clotted by addition of exogenous coagulation factors, such ...
Based on the notion that full activation of platelets is required for a growth factor release, in regenerative dentistry, platelet-rich plasma (PRP) in liquid form is usually clotted by addition of CaCl2 in glassware before topical implantation. However, there has been no evidence as to which is better, full or partial activation of platelets, for minimizing the loss of growth factors and improvin...