A proposed protocol for the standardized preparation of PRF membranes for clinical use
Highlights
► A novel compression device was developed for preparation of PRF membrane. ► The device minimizes the damage of platelets contained in the PRF membranes. ► The device minimizes the lost or degradation of growth factors in the PRF membranes. ► The protocol using the device could be a promising candidate for standardizing the preparation of PRF membrane for clinical use.
Introduction
Platelet-rich plasma (PRP) is a platelet-rich fraction of plasma and is clinically available as a source of growth factors to facilitate tissue repair and regeneration. To improve the handling efficiency, the retention at application sites, and the release of growth factors, bovine thrombin and/or calcium have been preferentially added to PRP to directly or indirectly facilitate the conversion from fibrinogen to fibrin. To avoid the use of a xenofactor, bovine thrombin has often been substituted with the autologous human thrombin. We have been using calcium [1] or an alginate hemostatic agent [2], [3] to improve the handling of PRP and to take advantage of PRP's beneficial effects in a clinical setting.
Recently, Choukroun and his coworkers developed a simple method to prepare fibrin gels without exogenously added supplements [4], [5]. This fibrin gel is designated as platelet-rich fibrin (PRF) and is widely recognized as a new generation of PRP. This preparation protocol is very simple: a blood sample is collected without an anticoagulant in 10-mL tubes and is immediately centrifuged at 3,000 rpm (800 g) for 10 min. Even when thrombin or calcium is not added to the blood sample, most platelets can be activated in a few minutes through contact with the tube walls to trigger the intrinsic coagulation cascades. Therefore, another characteristic of Choukroun's PRF is that the resulting fibrin gel is less stiff than that prepared by the addition of thrombin. As reported elsewhere [6], [7], the fiber density and the branch-point density of the fibrin networks mainly regulate the stiffness of the fibrin gel, and these parameters are increased by thrombin in a dose-dependent manner. However, PRF prepared without exogenous thrombin stimulation according to Choukroun is becoming more clinically accepted among dentists and oral surgeons.
For utilization in alveolar bone regeneration and plastic surgery, thick tube-like PRF clots should be shaped to fit the size of the implantation site. For compression of the PRF clot to make a PRF membrane, moist (or dry) gauze has been conventionally used. However, there has been concern whether this compression may damage the platelets and exude significant quantities of valuable growth factors. In support of this possibility, Su and Burnouf demonstrated that substantial amounts of growth factors, which are thought to be involved in tissue regeneration, are indeed removed by squeezing [8], [9]. Therefore, the squeezing process could influence the quality and clinical effectiveness of the PRF preparations as a grafting material.
The growth factor content in the PRF will largely vary with individual blood samples, and one reason for this observation can be attributed to how the PRF preparations are made. We believe that it is necessary to establish a standardized protocol for preparing PRF preparations to satisfy the following criteria: 1) growth factors stored in platelets should be retained to stimulate the cells of the surrounding host tissues; 2) platelets should be preserved in the fibrin meshwork with minimal damage or activation; and 3) the three-dimensional fibrin meshwork construction should be preserved as a scaffold for surrounding host cells. To prepare the most clinically effective PRF membrane, we have developed a compression device for the preparation of a standard PRF membrane, tested the performance of the device in retaining vital growth factors, and proposed a standardized protocol for PRF membrane preparation.
Section snippets
Preparation of PRF membranes with a compression device
The stainless steel PRF compression device developed for PRF membrane preparation is composed of two spoon shaped parts as illustrated in Fig. 1. The stage where the PRF clot was placed included many pinholes for securing the PRF clot and for draining excess fluid from the serum when the clot was compressed. The clearance of both spoon parts was adjusted to be 1 mm. Thus, when the PRF clots were compressed with this device, a standard 1-mm thick PRF membrane was consistently prepared.
Localization of platelets in PRF membranes
In comparing the two compression methods, it was observed that the PRF compressor reduced the wet weight of the PRF clots (the original PRF: 2.180 ± 0.545 g, n = 3) by 84% (0.352 ± 0.030 g), while the dry gauze method reduced the clot wet weight by 98% (0.040 ± 0.013 g) (Fig. 2). The C-PRF was divided into three regions, which were equal in length (Fig. 3A), and the presence of platelets was observed by SEM in each region (Fig. 3B–D). In Region 1 (Fig. 3B), numerous platelets aggregated on the
Discussion
When preparing a standardized protocol proposal, the most important point to consider is the type of application. In the field of tissue engineering and regenerative medicine, there are three major applications of PRF preparations. These applications include 1) biodegradable barrier membranes for guided tissue regeneration, including alveolar ridge augmentation [15], 2) a source (or reservoir) of growth factors as a gel form of PRP for tissue regeneration, such as bone induction [4], and 3)
Acknowledgments
This project was funded through support by Grants-in-Aid for Scientific Research from the Ministry of Education, Sports, Science, and Technology, Japan (Contract grant numbers: 23592881) and by Research for Promoting Technological Seeds 2011 from the Japan Science and Technology Agency.
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2020, Tissue and CellCitation Excerpt :The present study analyzed the three-dimensional architecture and elemental composition of different regions of PRF clot samples obtained from patients for use in dental clinical procedures, in order to provide new insight into this biomaterial used in clinical procedures in various biomedical areas (Choukroun et al., 2006; Singh et al., 2013; Montanari et al., 2013; Sharma and Pradeep, 2011; Mazor et al., 2009; Tabrizi et al., 2018). The three-dimensional architecture of the PRF clot was analyzed using two different sample preparation protocols for scanning electron microscopy: traditional glutaraldehyde fixation (2.5%), employed in other studies (Dohan et al., 2010; Kobayashi et al., 2012; Isobe et al., 2017; Dohan Ehrenfest et al., 2018; Bootkrajang et al., 2020; Miron et al., 2020); and a second method of analysis after partial removal of extracellular elements (Watanabe and Konig, 1984; de Morais et al., 1994d; Evan et al., 1976), in order to enrich the analysis of biomaterial morphology. Using the traditional fixation method, the samples revealed characteristics similar to other studies: a red zone in which erythrocytes were concentrated, surrounded by non-dense or immature fibrin networks; and a yellow fibrin zone with a very dense, compact fibrin network of amorphous appearance containing a few erythrocytes (Dohan Ehrenfest et al., 2010, Kobayashi et al., 2012; Bootkrajang et al., 2020).
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