What is the Best Degree of Hyaluronic Acid Crosslinking in Increasing Growth Factors Level of Platelet-Rich Fibrin Lysate? Ariyati Nora,Handono Kusworini,Nurdiana Nurdiana,Wirohadidjojo Yohanes Widodo Journal of stem cells & regenerative medicine Various therapeutic materials such as hyaluronic acid (HA) and platelet-rich fibrin lysate (PRF-L) have been represented to improve chronic fibroblast ulcers and premature aging. HA crosslinking is the most popular dermal filler presently in the therapy of aging skin. With the PRF-L properties that are rich in growth factors (GF), the combination of these materials is expected to produce synergistic and potentiation effects. This study aimed to determine the effect of various degrees of HA crosslinking on GF levels in PRF-L. PRF-L was obtained from PRF of healthy adult venous blood with 72 hours of incubation. HA was taken from preparations with 3 crosslinking degrees of 3%, 4%, and 10%. Measurement of GF levels was performed using sandwich ELISA method. The GF levels released in PRF-L increased with the addition of HA crosslinking. The lower HA crosslinking degree (3%) triggered greater release of GF by PRF-L compared to higher HA crosslinking degree (4% & 10%). Low degree HA crosslinking elevated all measured GF levels in PRF-L. The lower HA crosslinking degree provoked higher release of GF.

Growth-promoting action and growth factor release by different platelet derivatives. Passaretti F,Tia M,D'Esposito V,De Pascale M,Del Corso M,Sepulveres R,Liguoro D,Valentino R,Beguinot F,Formisano P,Sammartino G Platelets Abstract Platelet derivatives are commonly used in wound healing and tissue regeneration. Different procedures of platelet preparation may differentially affect growth factor release and cell growth. Preparation of platelet-rich fibrin (PRF) is accompanied by release of growth factors, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGFβ1), and several cytokines. When compared with the standard procedure for platelet-rich plasma (PRP), PRF released 2-fold less PDGF, but >15-fold and >2-fold VEGF and TGFβ1, respectively. Also, the release of several cytokines (IL-4, IL-6, IL-8, IL-10, IFNγ, MIP-1α, MIP-1β and TNFα) was significantly increased in PRF-conditioned medium (CM), compared to PRP-CM. Incubation of both human skin fibroblasts and human umbilical vein endothelial cells (HUVECs) with PRF-derived membrane (mPRF) or with PRF-CM enhanced cell proliferation by >2-fold (p<0.05). Interestingly, PRP elicited fibroblast growth at a higher extent compared to PRF. At variance, PRF effect on HUVEC growth was significantly greater than that of PRP, consistent with a higher concentration of VEGF in the PRF-CM. Thus, the procedure of PRP preparation leads to a larger release of PDGF, as a possible result of platelet degranulation, while PRF enhances the release of proangiogenic factors. 10.3109/09537104.2013.809060

An Injectable Fibrin Scaffold Rich in Growth Factors for Skin Repair. Shao Zhengwei,Lyu Chengqi,Teng Lin,Xie Xuetao,Sun Jiayue,Zou Derong,Lu Jiayu BioMed research international Platelet aggregates, such as PRP, PRF, and CGF, have been used alone or in combination with other grafting materials to enhance restoration outcomes. The process for preparing these autografting materials requires two-step centrifugation or specific centrifuges. In this study, we obtained an injectable fibrin scaffold (IFS) rich in growth factors by one-step centrifugation of whole blood from rabbits. The purpose of this study is to introduce some characteristics of IFS. This scaffold was characterized using various techniques, including Masson's trichrome staining, scanning electron microscopy, porosity measurements, and cell counting. The sustained release of growth factors, including PDGF, VEGF, TGF-1, IGF, FGF, and EGF, was quantified using ELISA assay. The obtained IFS was tested for its effects on cell proliferation, extracellular matrix deposition, and full-thickness skin defect repair. The prepared IFS is characterized by a loose fibrin network structure with white blood cells and platelets that slowly release growth factors and can promote the healing of skin defects via the promotion of cell proliferation, collagen deposition, and tissue revascularization. In addition, its liquid properties and porous structure are conducive to its application as a therapeutic component in tissue engineering. 10.1155/2021/8094932

Platelet-Rich Fibrin, Preparation and Use in Dermatology. Dashore Shuken,Chouhan Kavish,Nanda Soni,Sharma Aseem Indian dermatology online journal The goal of these recommendations is to provide a framework to practitioners for implementing useful, evidence-based recommendations for the preparation of platelet-rich fibrin (PRF) and its use in various dermatological indications. The Indian Association of Dermatologists, Venereologists and Leprologists (IADVL) assigned the task of preparing these recommendations to its taskforce on platelet-rich plasma. A comprehensive literature search was done in the English language on the PRF across multiple databases. The grade of evidence and strength of recommendation was evaluated on the GRADE framework (Grading of Recommendation, Assessment, Development and Evaluation). A draft of clinical recommendations was developed on the best available evidence which was also scrutinized and critically evaluated by the IADVL Academy of Dermatology. Based on the inputs received, this final consensus statement was prepared. A total of 40 articles (meta-analyses, prospective and retrospective studies, reviews [including chapters in books] and case series) were critically evaluated and the evidence thus gathered was used in the preparation of these recommendations. This expert group recommends use of A-PRF+ protocol, that is (200 g for 8 min) for preparation of solid PRF and C-PRF protocol (700 g for 8 min) for liquid PRF. Swing out bucket model of centrifuge or the horizontal centrifuge is recommended for preparation of both PRF, and liquid PRF. Centrifugation must begin within 90-120 s of drawing of blood. PRF can be used in various indications for skin rejuvenation and nonhealing ulcers as either monotherapy or in combination with other therapies. 10.4103/idoj.idoj_282_21

The impact of the centrifuge characteristics and centrifugation protocols on the cells, growth factors, and fibrin architecture of a leukocyte- and platelet-rich fibrin (L-PRF) clot and membrane. Dohan Ehrenfest David M,Pinto Nelson R,Pereda Andrea,Jiménez Paula,Corso Marco Del,Kang Byung-Soo,Nally Mauricio,Lanata Nicole,Wang Hom-Lay,Quirynen Marc Platelets L-PRF (leukocyte- and platelet-rich fibrin) is one of the four families of platelet concentrates for surgical use and is widely used in oral and maxillofacial regenerative therapies. The first objective of this article was to evaluate the mechanical vibrations appearing during centrifugation in four models of commercially available table-top centrifuges used to produce L-PRF and the impact of the centrifuge characteristics on the cell and fibrin architecture of a L-PRF clot and membrane. The second objective of this article was to evaluate how changing some parameters of the L-PRF protocol may influence its biological signature, independently from the characteristics of the centrifuge. In the first part, four different commercially available centrifuges were used to produce L-PRF, following the original L-PRF production method (glass-coated plastic tubes, 400 g force, 12 minutes). The tested systems were the original L-PRF centrifuge (Intra-Spin, Intra-Lock, the only CE and FDA cleared system for the preparation of L-PRF) and three other laboratory centrifuges (not CE/FDA cleared for L-PRF): A-PRF 12 (Advanced PRF, Process), LW-UPD8 (LW Scientific) and Salvin 1310 (Salvin Dental). Each centrifuge was opened for inspection, two accelerometers were installed (one radial, one vertical), and data were collected with a spectrum analyzer in two configurations (full-load or half load). All clots and membranes were collected into a sterile surgical box (Xpression kit, Intra-Lock). The exact macroscopic (weights, sizes) and microscopic (photonic and scanning electron microscopy SEM) characteristics of the L-PRF produced with these four different machines were evaluated. In the second part, venous blood was taken in two groups, respectively, Intra-Spin 9 ml glass-coated plastic tubes (Intra-Lock) and A-PRF 10 ml glass tubes (Process). Tubes were immediately centrifuged at 2700 rpm (around 400 g) during 12 minutes to produce L-PRF or at 1500 rpm during 14 minutes to produce A-PRF. All centrifugations were done using the original L-PRF centrifuge (Intra-Spin), as recommended by the two manufacturers. Half of the membranes were placed individually in culture media and transferred in a new tube at seven experimental times (up to 7 days). The releases of transforming growth factor β-1 (TGFβ-1), platelet derived growth factor AB (PDGF-AB), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2) were quantified using ELISA kits at these seven experimental times. The remaining membranes were used to evaluate the initial quantity of growth factors of the L-PRF and A-PRF membranes, through forcible extraction. Very significant differences in the level of vibrations at each rotational speed were observed between the four tested centrifuges. The original L-PRF centrifuge (Intra-Spin) was by far the most stable machine in all configurations and always remained under the threshold of resonance, unlike the three other tested machines. At the classical speed of production of L-PRF, the level of undesirable vibrations on the original centrifuge was between 4.5 and 6 times lower than with other centrifuges. Intra-Spin showed the lowest temperature of the tubes. A-PRF and Salvin were both associated with a significant increase in temperature in the tube. Intra-Spin produced the heaviest clot and quantity of exudate among the four techniques. A-PRF and LW produced much lighter, shorter and narrower clots and membranes than the two other centrifuges. Light microscopy analysis showed relatively similar features for all L-PRF types (concentration of cell bodies in the first half). However, SEM illustrated considerable differences between samples. The original Intra-Spin L-PRF showed a strongly polymerized thick fibrin matrix and all cells appeared alive with a normal shape, including the textured surface aspect of activated lymphocytes. The A-PRF, Salvin and LW PRF-like membranes presented a lightly polymerized slim fibrin gel and most of the visible cell bodies appeared destroyed (squashed or shrunk). In the second part of this study, the slow release of the three tested growth factors from original L-PRF membranes was significantly stronger (more than twice stronger, p<0.001) at all experimental times than the release from A-PRF membranes. No trace of BMP2 could be detected in the A-PRF. A slow release of BMP2 was detected during at least 7 days in the original L-PRF. Moreover, the original L-PRF clots and membranes (produced with 9 mL blood) were always significantly larger than the A-PRF (produced with 10 mL blood). The A-PRF membranes dissolved in vitro after less than 3 days, while the L-PRF membrane remained in good shape during at least 7 days. Each centrifuge has its clear own profile of vibrations depending on the rotational speed, and the centrifuge characteristics are directly impacting the architecture and cell content of a L-PRF clot. This result may reveal a considerable flaw in all the PRP/PRF literature, as this parameter was never considered. The original L-PRF clot (Intra-Spin) presented very specific characteristics, which appeared distorted when using centrifuges with a higher vibration level. A-PRF, LW and Salvin centrifuges produced PRF-like materials with a damaged and almost destroyed cell population through the standard protocol, and it is therefore impossible to classify these products in the L-PRF family. Moreover, when using the same centrifuge, the original L-PRF protocol allowed producing larger clots/membranes and a more intense release of growth factors (biological signature at least twice stronger) than the modified A-PRF protocol. Both protocols are therefore significantly different, and the clinical and experimental results from the original L-PRF shall not be extrapolated to the A-PRF. Finally, the comparison between the total released amounts and the initial content of the membrane (after forcible extraction) highlighted that the leukocytes living in the fibrin matrix are involved in the production of significant amounts of growth factors. The centrifuge characteristics and centrifugation protocols impact significantly and dramatically the cells, growth factors and fibrin architecture of L-PRF. 10.1080/09537104.2017.1293812