Advertisement

Clinical Oral Investigations

, Volume 23, Issue 3, pp 1309–1318 | Cite as

Injectable platelet rich fibrin: cell content, morphological, and protein characterization

  • Hugo Almeida VarelaEmail author
  • Júlio C. M. Souza
  • Rubens M. Nascimento
  • Raimundo F. AraújoJr
  • Roseane C. Vasconcelos
  • Rômulo S. Cavalcante
  • Paulo M. Guedes
  • Aurigena A. Araújo
Original Article

Abstract

Objectives

The aim of the present study was to evaluate the blood cell content, morphological aspects, gene expression of type I collagen, and release of growth factors on an injectable platelet rich fibrin (i-PRF).

Materials and methods

Blood samples were collected from 15 volunteers to prepare i-PRF samples. Peripheral blood was used as a control group. Blood clot and i-PRF samples were cultured for 10 days. The supernatant of the samples was collected for ELISA immunoassay quantification of PDGF and VEGF growth factors over periods of 1, 8, 24, 72, and 240 h. I-PRF and blood clot samples were biologically characterized using histological and immunohistochemistry analysis for IL-10, osteocalcin, and TGF-β. Scanning electron microscopy (SEM) was used to inspect the fibrin network and distribution of blood platelets and leukocytes. Reverse transcriptase polymerase chain reaction (RT-PCR) method was used to evaluate gene expression for type I collagen.

Results

A higher concentration of platelets and lymphocytes was recorded in i-PRF than in peripheral blood (p < 0.05). The release of VEGF was higher in blood clot samples (1933 ± 704) than that for i-PRF (852 ± 376; p < 0.001). Immunohistochemistry showed upregulation of TGF-B, IL-10, and osteocalcin in the i-PRF group. RT-PCR showed increased type I collagen gene expression in i-PRF (p < 0.05). SEM images revealed agglomeration of platelets in some regions, while a fibrin networking was noticeable in the entire i-PRF sample.

Conclusions

Injectable platelet rich fibrin becomes a good approach for soft and mineralized tissue healing considering the formation of a three-dimensional fibrin network embedding platelets, leukocytes, type I collagen, osteocalcin, and growth factors. Indeed, the injectable platelet rich fibrin can be indicated in several medical applications regarding bioactivity, simplied technique, and flowable mixing with other biomaterials.

Clinical relevance

Morphological, cell, and protein characterization of platelet rich fibrin provides a better understanding of the clinical effects and improvement of clinical guidelines for several medical applications. Once well physicochemical and biologically characterized, the use of an injectable platelet rich fibrin can be extended to other applications in the field of orthopedics, periodontics, and implant dentistry on the repairing process of both soft and mineralized tissues.

Keywords

Platelet rich fibrin Morphology Histopathology Growth factors 

Notes

Funding

This work was supported by the Conselho Nacional de Pesquisa (CNPq) from Brazil in the subject of the project 443840/2014-8.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed/All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

References

  1. 1.
    Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJJ, Mouhyi J, Gogly B (2006) Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 101(3):e37–e44.  https://doi.org/10.1016/j.tripleo.2005.07.008 CrossRefGoogle Scholar
  2. 2.
    Marcazzan S, Weinstein RL, Del Fabbro M (2017) Efficacy of platelets in bone healing: a systematic review on animal studies. Platelets 29:1–12.  https://doi.org/10.1080/09537104.2017.1327652 Google Scholar
  3. 3.
    Abraham CM (2014) A brief historical perspective on dental implants, their surface coatings and treatments. Open Dent J 8(1):50–55.  https://doi.org/10.2174/1874210601408010050 CrossRefGoogle Scholar
  4. 4.
    Sood V, Masamatti SS, Khatri M, Kumar A, Jindal V (2012) Platelet concentrates–part I. Indian J Dent Sci 4(4):123–126Google Scholar
  5. 5.
    Wu C-L, Lee S-S, Tsai C-H, Lu K-H, Zhao J-H, Chang Y-C (2012) Platelet-rich fibrin increases cell attachment, proliferation and collagen-related protein expression of human osteoblasts. Aust Dent J 57(2):207–212.  https://doi.org/10.1111/j.1834-7819.2012.01686.x CrossRefGoogle Scholar
  6. 6.
    Castro AB, Meschi N, Temmerman A, Pinto N, Lambrechts P, Teughels W, Quirynen M (2017) Regenerative potential of leucocyte- and platelet-rich fibrin. Part A: intra-bony defects, furcation defects and periodontal plastic surgery. A systematic review and meta-analysis. J Clin Periodontol 44(1):67–82.  https://doi.org/10.1111/jcpe.12643 CrossRefGoogle Scholar
  7. 7.
    Castro AB, Meschi N, Temmerman A, Pinto N, Lambrechts P, Teughels W, Quirynen M (2017) Regenerative potential of leucocyte- and platelet-rich fibrin. Part B: sinus floor elevation, alveolar ridge preservation and implant therapy. A systematic review. J Clin Periodontol 44(2):225–234.  https://doi.org/10.1111/jcpe.12658 CrossRefGoogle Scholar
  8. 8.
    Del Corso M, Dohan Ehrenfest DM (2013) Immediate implantation and peri-implant natural bone regeneration (NBR) in the severely resorbed posterior mandible using leukocyte-and platelet-rich fibrin (L-PRF): a 4-year follow-up. http://asnanportal.com/images/dental_report/periodontics-oral-surgery-implants/poseido-201312109-16-delcor-copia.pdf
  9. 9.
    Simonpieri A, Del Corso M, Sammartino G, Dohan Ehrenfest DM (2009) The relevance of Choukrounʼs platelet-rich fibrin and metronidazole during complex maxillary rehabilitations using bone allograft. Part II: implant surgery, prosthodontics, and survival. Implant Dent 18(3):220–229.  https://doi.org/10.1097/ID.0b013e31819b5e3f CrossRefGoogle Scholar
  10. 10.
    Mosesson MW, Siebenlist KR, Meh DA (2006) The structure and biological features of fibrinogen and fibrin. Ann N Y Acad Sci 936(1):11–30.  https://doi.org/10.1111/j.1749-6632.2001.tb03491.x CrossRefGoogle Scholar
  11. 11.
    Guthold M, Liu W, Sparks EA, Jawerth LM, Peng L, Falvo M, Superfine R, Hantgan RR, Lord ST (2007) A comparison of the mechanical and structural properties of fibrin fibers with other protein fibers. Cell Biochem Biophys 49(3):165–181.  https://doi.org/10.1007/s12013-007-9001-4 CrossRefGoogle Scholar
  12. 12.
    Wang X, Zhang Y, Choukroun J, Ghanaati S, Miron RJ (2017) Effects of an injectable platelet-rich fibrin on osteoblast behavior and bone tissue formation in comparison to platelet-rich plasma. Platelets 29:1–8.  https://doi.org/10.1080/09537104.2017.1293807. Google Scholar
  13. 13.
    Mourão CF de AB, Valiense H, Melo ER, Mourão NBMF, Maia MD-C (2015) Obtention of injectable platelets rich-fibrin (i-PRF) and its polymerization with bone graft. Rev Col Bras Cir 42(6):421–423CrossRefGoogle Scholar
  14. 14.
    Wang X, Zhang Y, Choukroun J, Ghanaati S, Miron RJ (2017) Behavior of gingival fibroblasts on titanium implant surfaces in combination with either injectable-PRF or PRP. Int J Mol Sci 18(2):E331.  https://doi.org/10.3390/ijms18020331 CrossRefGoogle Scholar
  15. 15.
    Choukroun J, Ghanaati S (2017) Reduction of relative centrifugation force within injectable platelet-rich-fibrin (PRF) concentrates advances patients??? Own inflammatory cells, platelets and growth factors: the first introduction to the low speed centrifugation concept. Eur J Trauma Emerg Surg 44:1–9Google Scholar
  16. 16.
    Miron RJ, Fujioka-Kobayashi M, Hernandez M et al (2017) Injectable platelet rich fibrin (i-PRF): opportunities in regenerative dentistry? Clin Oral Investig 21:1–9CrossRefGoogle Scholar
  17. 17.
    Chomczynski P, Sacchi N (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate – phenol – chloroform extraction: twenty-something years on. Nat Protoc 1(2):581–585.  https://doi.org/10.1038/nprot.2006.83 CrossRefGoogle Scholar
  18. 18.
    Ota-Tsuzuki C, Datte CE, Nomura KA, Gouvea Cardoso LA, Shibli JA (2011) Influence of titanium surface treatments on formation of the blood clot extension. J Oral Implantol 37(6):641–647.  https://doi.org/10.1563/AAID-JOI-D-09-00125.1 CrossRefGoogle Scholar
  19. 19.
    Fujioka-Kobayashi M, Miron RJ, Hernandez M, Kandalam U, Zhang Y, Choukroun J (2016) Optimized platelet rich fibrin with the low speed concept: growth factor release, biocompatibility and cellular response. J Periodontol 88:1–17.  https://doi.org/10.1902/jop.2016.160443 Google Scholar
  20. 20.
    Weibrich G, Kleis WKG, Hafner G, Hitzler WE (2002) Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count. J Craniomaxillofac Surg 30(2):97–102.  https://doi.org/10.1054/jcms.2002.0285 CrossRefGoogle Scholar
  21. 21.
    El-Sharkawy H, Kantarci A, Deady J et al (2007) Platelet-rich plasma: growth factors and pro- and anti-inflammatory properties. J Periodontol 78(4):661–669.  https://doi.org/10.1902/jop.2007.060302 CrossRefGoogle Scholar
  22. 22.
    Dohan Ehrenfest DM, Del Corso M, Diss A, Mouhyi J, Charrier J-B (2010) Three-dimensional architecture and cell composition of a Choukroun’s platelet-rich fibrin clot and membrane. J Periodontol 81(4):546–555.  https://doi.org/10.1902/jop.2009.090531 CrossRefGoogle Scholar
  23. 23.
    Nishimoto S, Fujita K, Sotsuka Y, Kinoshita M, Fujiwara T, Kawai K, Kakibuchi M (2015) Growth factor measurement and histological analysis in platelet rich fibrin: a pilot study. J Maxillofac Oral Surg 14(4):907–913.  https://doi.org/10.1007/s12663-015-0768-3 CrossRefGoogle Scholar
  24. 24.
    Schär MO, Diaz-Romero J, Kohl S, Zumstein MA, Nesic D (2015) Platelet-rich concentrates differentially release growth factors and induce cell migration in vitro. Clin Orthop Relat Res 473(5):1635–1643.  https://doi.org/10.1007/s11999-015-4192-2 CrossRefGoogle Scholar
  25. 25.
    Andrade CX, Orrego A, Pinto N (2016) Hematologic profile of four autologous fibrinogen based binder obtained with differents protocols. In :2–3. doi: https://doi.org/10.13140/RG.2.2.27106.12485
  26. 26.
    Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJJ, Mouhyi J, Gogly B (2006) Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part III: leucocyte activation: a new feature for platelet concentrates. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 101(3):e51–e55.  https://doi.org/10.1016/j.tripleo.2005.07.010. CrossRefGoogle Scholar
  27. 27.
    Dohan Ehrenfest DM, Doglioli P, de Peppo GM, Del Corso M, Charrier JB (2010) Choukroun’s platelet-rich fibrin (PRF) stimulates in vitro proliferation and differentiation of human oral bone mesenchymal stem cell in a dose-dependent way. Arch Oral Biol 55(3):185–194.  https://doi.org/10.1016/j.archoralbio.2010.01.004 CrossRefGoogle Scholar
  28. 28.
    Bielecki T, Dohan Ehrenfest DM, Everts PA, Wiczkowski A (2012) The role of leukocytes from L-PRP/L-PRF in wound healing and immune defense: new perspectives. Curr Pharm Biotechnol 13(7):1153–1162.  https://doi.org/10.2174/138920112800624373. CrossRefGoogle Scholar
  29. 29.
    Dohan Ehrenfest DM, de Peppo GM, 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(1):63–69.  https://doi.org/10.1080/08977190802636713. CrossRefGoogle Scholar
  30. 30.
    Choukroun J, Diss A, Simonpieri A, Girard MO, Schoeffler C, Dohan SL, Dohan AJJ, Mouhyi J, Dohan DM (2006) Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part V: histologic evaluations of PRF effects on bone allograft maturation in sinus lift. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 101(3):299–303.  https://doi.org/10.1016/j.tripleo.2005.07.012 CrossRefGoogle Scholar
  31. 31.
    Yajamanya SR, Chatterjee A, Babu CN, Karunanithi D (2016) Fibrin network pattern changes of platelet-rich fibrin in young versus old age group of individuals: a cell block cytology study. J Indian Soc Periodontol 20(2):151–156Google Scholar
  32. 32.
    Dohan Ehrenfest DM, Pinto NR, Pereda A et al (2017) 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. Platelets 29:1–14Google Scholar
  33. 33.
    Clark RAF (2006) Fibrin and wound healing. Ann N Y Acad Sci 936(1):355–367.  https://doi.org/10.1111/j.1749-6632.2001.tb03522.x CrossRefGoogle Scholar
  34. 34.
    Xie X, Zhang C, Tuan RS (2014) Biology of platelet-rich plasma and its clinical application in cartilage repair. Arthritis Res Ther 16(1):204.  https://doi.org/10.1186/ar4493 CrossRefGoogle Scholar
  35. 35.
    Kawase T, Okuda K, Wolff LF, Yoshie H (2003) Platelet-rich plasma-derived fibrin clot formation stimulates collagen synthesis in periodontal ligament and osteoblastic cells in vitro. J Periodontol 74(6):858–864.  https://doi.org/10.1902/jop.2003.74.6.858 CrossRefGoogle Scholar
  36. 36.
    Janmey PA, Winer JP, Weisel JW (2009) Fibrin gels and their clinical and bioengineering applications. J R Soc Interface 6(30):1–10.  https://doi.org/10.1098/rsif.2008.0327 CrossRefGoogle Scholar
  37. 37.
    Kobayashi M, Kawase T, Horimizu M, Okuda K, Wolff LF, Yoshie H (2012) A proposed protocol for the standardized preparation of PRF membranes for clinical use. Biologicals 40(5):323–329.  https://doi.org/10.1016/j.biologicals.2012.07.004 CrossRefGoogle Scholar
  38. 38.
    Kang Y-H, Jeon SH, Park J-Y, Chung JH, Choung YH, Choung HW, Kim ES, Choung PH (2011) Platelet-rich fibrin is a bioscaffold and reservoir of growth factors for tissue regeneration. Tissue Eng A 17(3–4):349–359.  https://doi.org/10.1089/ten.tea.2010.0327 CrossRefGoogle Scholar
  39. 39.
    Cieslik-Bielecka A, Bielecki T, Gazdzik TS, Arendt J, Król W, Szczepanski T (2009) Autologous platelets and leukocytes can improve healing of infected high-energy soft tissue injury. Transfus Apher Sci 41(1):9–12.  https://doi.org/10.1016/j.transci.2009.05.006 CrossRefGoogle Scholar
  40. 40.
    Shi S, Kirk M, Kahn AJ (2009) The role of type I collagen in the regulation of the osteoblast phenotype. J Bone Miner Res 11(8):1139–1145.  https://doi.org/10.1002/jbmr.5650110813 CrossRefGoogle Scholar
  41. 41.
    Franceschi RT (1999) The developmental control of osteoblast-specific gene expression: role of specific transcription factors and the extracellular matrix environment. Crit Rev Oral Biol Med 10(1):40–57.  https://doi.org/10.1177/10454411990100010201 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Hugo Almeida Varela
    • 1
    Email author
  • Júlio C. M. Souza
    • 2
  • Rubens M. Nascimento
    • 3
  • Raimundo F. AraújoJr
    • 4
  • Roseane C. Vasconcelos
    • 5
  • Rômulo S. Cavalcante
    • 6
  • Paulo M. Guedes
    • 7
  • Aurigena A. Araújo
    • 1
  1. 1.Post-graduate program in Public Health, Department of Biophysics and PharmacologyFederal University of Rio Grande Norte (UFRN)NatalBrazil
  2. 2.Center for MicroElectroMechanical Systems (CMEMS-UMINHO)University of MinhoGuimarãesPortugal
  3. 3.Post-graduate Program in Materials Science and Engineering, Department of Materials Science and EngineeringFederal University of Rio Grande Norte (UFRN)NatalBrazil
  4. 4.Post-graduate program in Functional and Structural Biology and Health Science, Department of MorphologyFederal University of Rio Grande Norte (UFRN)NatalBrazil
  5. 5.Post-graduate program in Public Health, Department of DentistryFederal University of Rio Grande Norte (UFRN)NatalBrazil
  6. 6.Post-graduate Program in Health Science, Department of MorphologyFederal University of Rio Grande Norte (UFRN)NatalBrazil
  7. 7.Post-graduate program in Parasite Biology, Department of Microbiology and ParasitologyFederal University of Rio Grande Norte (UFRN)NatalBrazil

Personalised recommendations