L-PRF Scientific Literature

L-PRF Scientific Literature

The special texture of the L-PRF, thus, allows clinical use in the amorphous form but mainly in the membranous form, after a slight compression of the gel: this membranous form can be used to cover and protect like a tissue graft.  The biological properties of L-PRF shows clearly an interesting surgical versatility and all the characteristics that can support faster tissue regeneration and high-quality clinical outcomes. L-PRF is able to stimulate osteogenesis in bone environment, in addition to angiogenesis.  Furthermore, it provides a scaffold consisting of fibrin that allows cellular migration, and these are certainly the fundamental aspects for the process of bone regeneration.  All these features support the conclusion that the PRF has numerous advantages compared to other similar blood products, being able to better enhance the natural healing of soft and hard tissue.  The advantages of platelet concentrates are their autologous nature, simple collection, easy bedside preparation, and clinical application without the risks associated with allogenic products.  In oral surgery, it can replace xenografts.


References:

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. David M. Dohan EhrenfestByung-Soo Kang, Marco Del Corso, Mauricio Nally, Marc Quirynen, Hom-Lay Wang and Nelson R. Pinto.

Part 1: evaluation of the vibration shocks of 4 models of table centrifuges for L-PRF 

Abstract 

Background and Objectives. Platelet concentrates for surgical use (Platelet-Rich Plasma PRP or Platelet-rich fibrin PRF) are surgical adjuvants to improve healing and promote tissue regeneration. L-PRF (Leukocyte- and Platelet-Rich Fibrin) is one of the 4 families of platelet concentrates for surgical use and is widely used in oral and maxillofacial regenerative therapies. The objective of this first article was to evaluate the mechanical vibrations appearing during centrifugation in 4 models of commercially available table centrifuges frequently used to produce L-PRF. 

Materials and Methods. The 4 different tested centrifuges were the original L-PRF centrifuge (Intra-Spin, Intra-Lock, the only CE and FDA cleared system for the preparation of L-PRF) and 3 other laboratory centrifuges (not CE nor 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. Each centrifuge was tested in 2 configurations (full-load or half load with 9ml blood collection tubes filled with water) and at the following rotational speeds: 1500, 1800, 2100, 2400, 2700, 3000 and 3300 rpm. Extra rotational speeds were used on some centrifuges. One centrifuge (Salvin) had only one available rotational speed (3400 rpm). For each test, the software documented both radial and vertical vibration. 

Results. Very significant differences in the level of vibrations at each rotational speed were observed between the 4 tested machines. The original L-PRF centrifuge (Intra-Spin) was by far the most stable machine in all configurations. At the classical speed of production of L- PRF, the level of undesirable vibration on this centrifuge is between 4.5 and 6 times lower than with other centrifuges. Moreover, Intra-Spin always remains under the threshold of resonance, unlike the 3 other tested machines. 

Discussion and Conclusion. Each centrifuge has its clear own profile of vibrations depending on the rotational speed, and this may impact significantly the characteristics of the PRP or PRF produced with these devices. This result may reveal a considerable flaw in all the PRP/PRF literature, as this parameter was never considered. It is now necessary to evaluate the impact of the vibration parameter on the architecture and cell content of the L- PRF clots produced with these 4 different machines. 

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Part 2: macroscopic, photonic microscopy and Scanning Electron Microscopy analysis of 4 kinds of L-PRF clots and membranes 

Abstract 

Background and Objectives. Platelet concentrates for surgical use (Platelet-Rich Plasma PRP or Platelet-rich fibrin PRF) are surgical adjuvants to improve healing and promote tissue regeneration. L-PRF (Leukocyte- and Platelet-Rich Fibrin) is one of the 4 families of platelet concentrates for surgical use and is widely used in oral and maxillofacial regenerative therapies.

The objective of this second article was to evaluate the impact of the centrifuge characteristics (vibration intensity) on the cell and fibrin architecture of a L-PRF clot and membrane. 

Materials and Methods. Four different commercially available centrifuges were used to produce L-PRF, following the original L-PRF production method widely described in the literature (glass-coated plastic tubes, 400g 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 3 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). All clots and membranes were collected into a sterile adequate surgical box (Xpression kit). The exact macroscopic (weights, sizes) and microscopic (photonic and scanning electron microscopy SEM) characteristics and the cell composition of the L-PRF clots and membranes produced with these 4 different machines with 4 different vibration intensity levels were evaluated. 

Results. Intra-Spin showed the lowest temperature of the tubes. A-PRF and Salvin were both associated with a significant increase of temperature in the tube. Intra-Spin produced by far the heaviest clot and quantity of exudate among the 4 techniques. For clot and membrane length and width, Intra-Spin and Salvin presented similar sizes. A-PRF and LW produced much lighter, shorter and narrower clots and membranes than the 2 other centrifuges. Light microscopy analysis showed relatively similar features for all L-PRF types (concentration of cell bodies in the first half of the fibrin mesh). 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 all the visible cell bodies appeared destroyed (squashed or shrunk). 

Discussion and Conclusion. This study illustrated that the centrifuge characteristics (particularly the vibrations) are directly impacting the architecture and cell content of a L- PRF clot. The original L-PRF clot (Intra-Spin) used and validated since years 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 400g protocol developed initially for the L-PRF, and it is therefore impossible to classify these products in the L-PRF family. A-PRF, LW and Salvin centrifuges are not suitable for the production of original L-PRF clots and membranes at 400g. Further research would be interesting to evaluate how modifications of the protocol alone (for example reduction of the g forces) may influence the biological signature of the L-PRF clots and membranes, independently from the characteristics of the centrifuge. 

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Part 3: comparison of the growth factors content and slow release between the original L-PRF and the modified A-PRF (Advanced Platelet-Rich Fibrin) membranes

Abstract

Background and Objectives. L-PRF (Leukocyte- and Platelet-Rich Fibrin) is one of the 4 families of platelet concentrates for surgical use and is widely used in oral and maxillofacial regenerative therapies. The objective of this third article was to evaluate how the changes of the L-PRF protocol may influence its biological signature, independently from the characteristics of the centrifuge. 

Materials and Methods. In each volunteer donor, veinous blood was taken in 2 groups, respectively Intra-Spin 9ml glass-coated plastic tubes (Intra-Lock, Boca-Raton, FL, USA) and A-PRF 10ml glass tubes (Process, Nice, France). Tubes were immediately centrifuged at 2700rpm (around 400g) during 12 minutes to produce L-PRF clots, or at 1500 rpm during 14minutes to produce A-PRF clots. All centrifugations were done using the original L-PRF

centrifuge (Intra-Spin system, Intra-Lock), as recommended by the 2 manufacturers. All clots were collected into a sterile surgical box (Xpression kit) and compressed into membranes. Half of the membranes were placed individually in culture media and transferred in a new tube at 7 experimental times: 20 minutes, 1 hour, 4h, 24h, 72h, 120h and

168h. The releases of Transforming Growth Factor β-1 (TGFβ-1), Platelet Derived Growth Factor AB (PDGF-AB), Vascular Endothelial Growth Factor (VEGF) and BoneMorphogenetic Protein 2 (BMP-2) were quantified using ELISA kits at these 7experimental 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.

Results. The slow release of the 3 tested growth factors (TGFβ-1, PDGF-AB and VEGF) from original L-PRF membranes was significantly much 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 membrane. 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 9mL blood) were always significantly larger than the A-PRF clots and membranes (produced with 10mL 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.

Discussion and Conclusion. The cumulative curves are defining the biological signatures of the tested product. The original L-PRF signature is always more than twice stronger than the A-PRF signature. The same centrifuge was used for both products in this study; only the protocol (particularly the centrifugation forces) was different. The original L-PRF protocol allowed producing larger clots and membranes and a more intense release of growth factors than the modified A-PRF protocol. The exact impact of the tubes should also be investigated in the future. Both protocols are therefore very 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.

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Literature list

  • L-PRF membranes for increasing the width of keratinized mucosa around implants: A split-mouth, randomized, controlled pilot clinical trial.
    Temmerman Andy, Cleeren Gert-Jan, Castro Ana & Quirynen Marc.
    Journal of Periodontal Research (2018) Submitted.
  • L-PRF block for bone augmentation procedure: a proof-of-concept study.
    Cortellini Simone, Temmerman Andy, Ana Castro, Vandessel Jeroen, Jacobs Reinhilde, Wim Teughels & Quirynen Marc.
    Journal of Clinical Periodontology (2018) Accepted.
  • Regenerative potential of Leucocyte- and Platelet Rich Fibrin (L-PRF). Part A: intrabony defects, furcation defects, and periodontal plastic surgery. A systematic review and meta-analysis.
    Castro Ana, Meschi Nastaran, Temmerman Andy, Pinto Nelson, Lambrechts Paul, Teughels Wim, Quirynen Marc.
    Journal of Clinical Periodontology (2017) 44: 67-82.
  • Regenerative potential of Leucocyte- and Platelet Rich Fibrin (L-PRF). Part B: sinus floor elevation, alveolar ridge preservation, and implant therapy. A systematic review and meta-analysis.
    Castro Ana, Meschi Nastaran, Temmerman Andy, Pinto Nelson, Lambrechts Paul, Teughels Wim, Quirynen Marc.
    Journal of Clinical Periodontology (2017) 44: 225-234.
  • The use of leucocyte and platelet-rich fibrin in socket management and ridge preservation: a split-mouth, randomized, controlled clinical trial.
    Temmerman Andy, Vandessel Jeroen, Castro Ana, Jacobs Reinhilde, Teughels Wim, Pinto Nelson, Quirynen Marc.
    Journal of Clinical Periodontology (2016) 43: 990-999.
  • L-PRF in Periodontal Regeneration
    Nelson Pinto, Andy Temmerman, Simone Cortellini, Ana Castro, Wim Teughels & Marc Quirynen.
    Newman:  Carranza’s Clinical Periodontology 13e edition – Elsevier
    To be published in 2018.

    Weefselregeneratie door middel van L-PRF: ‘van mythe tot realiteit’.
    Andy Temmerman, Iris De Coster, Ana Castro Sarda, Nelson Pinto, Wim Teughels en Marc Quirynen
    Het tandheelkundig jaar 2017. Bohn Stafleu van Loghum
    ISBN 978-90-368-1029-6
  • Current misperceptions about protocols and systems
    New Biomaterials and Regenerative Medicine Strategies in Periodontology, Oral Surgery, Esthetic and Implant Dentistry 2016

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  • Dohan Ehrenfest, D. M., Bielecki, T., Jimbo, R., Barbe, G., Del Corso, M., Inchingolo, F. & Sammartino, G. (2012). Do the fibrin architecture and leukocyte content influence the growth factor release of platelet concentrates? An evidence-based answer comparing a pure platelet-rich plasma (P-PRP) gel and a leukocyte- and platelet-rich fibrin (L-PRF). Current Pharmaceutical Biotechnology, 13, 1145-1452.
  • Dohan Ehrenfest, D. M., De Peppo, G. M., Doglioli, P. & Sammartino, G. (2009). Slow release of growth factors and thrombospondin-1 in Choukroun's platelet-rich fibrin (PRF): a gold standard to achieve for all surgical platelet concentrates technologies. Growth Factors, 27, 63-69.
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  • Giannini, S., Cielo, A., Bonanome, L., Rastelli, C., Derla, C., Corpaci, F. & Falisi, G. (2015). Comparison between PRP, PRGF and PRF: lights and shadows in three similar but different protocols. European Review of Medical and Pharmacological Sciencies, 19, 927-930.
  • Hall, M. P., Band, P. A., Meislin, R. J., Jazrawi, L. M. & Cardone, D. A. (2009). Platelet-rich plasma: current concepts and application in sports medicine. Journal of the American Academy of Orthopaedic Surgeons, 17, 602-608.
  • Inchingolo, F., Tatullo, M., Marrelli, M., Inchingolo, A. M., Inchingolo, A. D., Dipalma, G., Flace, P., Girolamo, F., Tarullo, A., Laino, L., Sabatini, R., Abbinante, A. & Cagiano, R. (2012). Regenerative surgery performed with platelet-rich plasma used in sinus lift elevation before dental implant surgery: an useful aid in healing and regeneration of bone tissue. European Review for Medical and Pharmacological Sciences, 16, 1222-1226.
  • Intini, G. (2009). The use of platelet-rich plasma in bone reconstruction therapy. Biomaterials, 30, 4956-4966.
  • Lara, F. J., Serrano, A. M., Moreno, J. U., Carmona, J. H., Marquez, M. F., Perez, L. R., Del Rey Moreno, A. & Munoz, H. O. (2015). Platelet-rich fibrin sealant as a treatment for complex perianal fistulas: a multicentre study. Journal of Gastrointestinal Surgery, 19, 360-368.
  • Lingen, M. W. (2001). Role of leukocytes and endothelial cells in the development of angiogenesis in inflammation and wound healing. Archives of Pathololgy and Laboratory Medicine, 125, 67-71.
  • Lopez-Vidriero, E., Goulding, K. A., Simon, D. A., Sanchez, M. & Johnson, D. H. (2010). The use of platelet-rich plasma in arthroscopy and sports medicine: optimizing the healing environment. Arthroscopy, 26, 269-278.
  • Marrelli, M. & Tatullo, M. (2013). Influence of PRF in the healing of bone and gingival tissues. Clinical and histological evaluations. European Review of Medical and Pharmacological Sciences, 17, 1958-1962.
  • Marrelli, M., Paduano, F. & Tatullo, M. (2015). Human periapical cyst-mesenchymal stem cells differentiate into neuronal cells. J Dent Res, 94, 843-852.
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  • Pape, H. C., Marcucio, R., Humphrey, C., Colnot, C., Knobe, M. & Harvey, E. J. (2010). Trauma-induced inflammation and fracture healing. Journal of Orthopaedic Trauma, 24, 522-525
  • Randelli, P., Arrigoni, P., Ragone, V., Aliprandi, A. & Cabitza, P. (2011). Platelet rich plasma in arthroscopic rotator cuff repair: a prospective RCT study, 2-year follow-up. Journal of Shoulder and Elbow Surgery, 20, 518-528.
  • Reksodiputro, M., Widodo, D., Bashiruddin, J., Siregar, N. & Malik, S. (2014). PRFM enhance wound healing process in skin graft. Facial Plastic Surgery, 30, 670-675.
  • Rodeo, S. A., Delos, D., Weber, A., Ju, X., Cunningham, M. E., Fortier, L. & Maher, S. (2010). What's new in orthopaedic research. Journal of Bone and Joint Surgery. American volume, 92, 2491-2501.
  • Sanchez, M., Anitua, E., Azofra, J., Andia, I., Padilla, S. & Mujika, I. (2007). Comparison of surgically repaired Achilles tendon tears using platelet-rich fibrin matrices. American Journal of Sports Medicine, 35, 245-251.
  • Schnabel, L. V., Lynch M. E., van der Meulen, M. C., Yeager, A. E., Kornatowksi, M. A. & Nixon, A. J. (2009). Mesenchymal stem cells and insulin-like growth factor-I-gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons. Journal of Orthopaedics Research, 27, 1392-1398.
  • Schultz, G. S. & Wysocki, A. (2009). Interactions between extracellular matrix and growth factors in wound healing. Wound Repair Regeneration, 17, 153-162.
  • Simonpieri, A., Choukroun, J., Del Corso, M., Sammartino, G. & Dohan Ehrenfest, D. M. (2011). Simultaneous sinus-lift and implantation using microthreaded implants and leukocyte- and platelet-rich fibrin as sole grafting material: a six year experience. Implant Dentistry, 20, 2-12.
  • Tang, Y. Q., Yeaman, M. R. & Selsted, M. E. (2002). Antimicrobial peptides from human platelets. Infections and Immunity, 70, 6524-6533. 
  • Tsirogianni, A. K., Moutsopoulos, N. M. & Moutsopoulos, H. M. (2006). Wound healing: immunological aspects. Injury, 37 Suppl 1, S5-12.
  • Visser, L. C., Arnoczky, S. P., Caballero, O. & Egerbacher, M. (2010). Platelet-rich fibrin constructs elute higher concentrations of transforming growth factor-beta-1 and increase tendon cell proliferation over time when compared to blood clots: a comparative in vitro analysis. Veterinary Surgery, 39, 811-817.
  • Zimmermann, R., Arnold, D., Strasser, E., Ringwald, J., Schlegel, A., Wiltfang, J. & Eckstein, R. (2003). Sample preparation technique and white cell content influence the detectable levels of growth factors in platelet concentrates. Vox Sanguinis, 85, 283-289.
  • Zumstein, M. A., Bielecki, T. & Dohan Ehrenfest, D. M. (2011). The future of platelet concentrates in sport medicine: Platelet-Rich Plasma, Platelet-Rich Fibrin, and the impact of scaffolds and cells on teh lon-term delivery of growth factors. Operative techniques in Sports Medicine, 19, 190-197.
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L-PRF a dream or the future of bone regeneration?

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