The Effect of Leukocyte- and Platelet-Rich Fibrin on Central and Peripheral Nervous System Neurons—Implications for Biomaterial Applicability

Leukocyte- and Platelet-Rich Fibrin (L-PRF) is a second-generation platelet concentrate that is prepared directly from the patient’s own blood. It is widely used in the field of regenerative medicine, and to better understand its clinical applicability we aimed to further explore the biological properties and effects of L-PRF on cells from the central and peripheral nervous system. To this end, L-PRF was prepared from healthy human donors, and confocal, transmission, and scanning electron microscopy as well as secretome analysis were performed on these clots. In addition, functional assays were completed to determine the effect of L-PRF on neural stem cells (NSCs), primary cortical neurons (pCNs), and peripheral dorsal root ganglion (DRG) neurons. We observed that L-PRF consists of a dense but porous fibrin network, containing leukocytes and aggregates of activated platelets that are distributed throughout the clot. Antibody array and ELISA confirmed that it is a reservoir for a plethora of growth factors. Key molecules that are known to have an effect on neuronal cell functions such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) were slowly released over time from the clots. Next, we found that the L-PRF secretome had no significant effect on the proliferative and metabolic activity of NSCs, but it did act as a chemoattractant and improved the migration of these CNS-derived stem cells. More importantly, L-PRF growth factors had a detrimental effect on the survival of pCNs, and consequently, also interfered with their neurite outgrowth. In contrast, we found a positive effect on peripheral DRG neurons, and L-PRF growth factors improved their survival and significantly stimulated the outgrowth and branching of their neurites. Taken together, our study demonstrates the positive effects of the L-PRF secretome on peripheral neurons and supports its use in regenerative medicine but care should be taken when using it for CNS applications.

[1]  Truong Nguyen Khanh Hung,et al.  Current Progress of Platelet-Rich Derivatives in Cartilage and Joint Repairs , 2023, International journal of molecular sciences.

[2]  Yeshun Zhang,et al.  Impact of a Novel Hydrogel with Injectable Platelet-Rich Fibrin in Diabetic Wound Healing , 2023, Journal of diabetes research.

[3]  G. Martino,et al.  Endogenous neural stem cells characterization using omics approaches: Current knowledge in health and disease , 2023, Frontiers in Cellular Neuroscience.

[4]  B. Shah,et al.  A Comparative Study on Therapeutic Efficacy of Autologous Platelet-rich Plasma, Autologous Platelet-rich Fibrin Matrix, Recombinant Human Epidermal Growth Factor, and Collagen Particles in Nonhealing Leg Ulcers , 2023, Journal of cutaneous and aesthetic surgery.

[5]  L. Nakachima,et al.  Peripheral nerve regeneration in rats using nerve graft in a vein conduit pre-filled with platelet-rich fibrin (PRF). , 2022, Hand surgery & rehabilitation.

[6]  P. D. de Groot,et al.  Reversible Platelet Integrin αIIbβ3 Activation and Thrombus Instability , 2022, International journal of molecular sciences.

[7]  M. Quirynen,et al.  The impact of gender and peripheral blood parameters on the characteristics of L-PRF membranes. , 2022, Journal of oral biology and craniofacial research.

[8]  P. Bajaj,et al.  Injectable Platelet-Rich Fibrin - A Revolution in Periodontal Regeneration , 2022, Cureus.

[9]  Anteneh Ayelign Kibret,et al.  Therapeutic Application of Stem Cells in the Repair of Traumatic Brain Injury , 2022, Stem cells and cloning : advances and applications.

[10]  M. Quirynen,et al.  Mechanical properties and cellular content of leukocyte- and platelet-rich fibrin membranes of patients on antithrombotic drugs. , 2022, Journal of periodontal research.

[11]  M. Quirynen,et al.  Effect of different platelet rich fibrin matrices for ridge preservation in multiple tooth extractions: a split-mouth randomized controlled clinical trial. , 2021, Journal of clinical periodontology.

[12]  W. Teughels,et al.  Impact of g force and timing on the characteristics of platelet-rich fibrin matrices , 2021, Scientific Reports.

[13]  A. Khojasteh,et al.  Effects of Platelet-Rich Fibrin/Collagen Membrane on Sciatic Nerve Regeneration. , 2020, The Journal of craniofacial surgery.

[14]  R. Barker,et al.  A blueprint for translational regenerative medicine , 2020, Science Translational Medicine.

[15]  S. Shao,et al.  Autologous Platelet-Rich Plasma for Diabetic Foot Ulcer , 2020, Trends in Endocrinology & Metabolism.

[16]  Bhushan Madke,et al.  Injectable Platelet‐Rich Fibrin (PRF): The newest biomaterial and its use in various dermatological conditions in our practice: A case series , 2020, Journal of cosmetic dermatology.

[17]  C. Valenzuela,et al.  The mouse-equivalent of the human BDNF VAL66MET polymorphism increases dorsal hippocampal volume and does not interact with developmental ethanol exposure. , 2020, Alcohol.

[18]  W. Teughels,et al.  Simultaneous sinus floor elevation and implant placement using leukocyte- and platelet-rich fibrin as a sole graft material. , 2019, The International journal of oral & maxillofacial implants.

[19]  P. Gervois,et al.  Preconditioning of Human Dental Pulp Stem Cells with Leukocyte- and Platelet-Rich Fibrin-Derived Factors Does Not Enhance Their Neuroregenerative Effect , 2019, Stem cells international.

[20]  W. Teughels,et al.  Characterization of the Leukocyte- and Platelet-Rich Fibrin Block: Release of Growth Factors, Cellular Content, and Structure. , 2019, The International journal of oral & maxillofacial implants.

[21]  K. Christman Biomaterials for tissue repair , 2019, Science.

[22]  M. Quirynen,et al.  Angiogenic Properties of ‘Leukocyte- and Platelet-Rich Fibrin’ , 2018, Scientific Reports.

[23]  Aditi Falnikar,et al.  Differential Response in Novel Stem Cell Niches of the Brain after Cervical Spinal Cord Injury and Traumatic Brain Injury. , 2018, Journal of neurotrauma.

[24]  M. Quirynen,et al.  Leucocyte- and platelet-rich fibrin (L-PRF) as a regenerative medicine strategy for the treatment of refractory leg ulcers: a prospective cohort study , 2018, Platelets.

[25]  M. Quirynen,et al.  Plasma-rich fibrin in neurosurgery: a feasibility study , 2018, Acta Neurochirurgica.

[26]  Paul M. George,et al.  Electrical preconditioning of stem cells with a conductive polymer scaffold enhances stroke recovery. , 2017, Biomaterials.

[27]  P. Gervois,et al.  The Angiogenic Potential of DPSCs and SCAPs in an In Vivo Model of Dental Pulp Regeneration , 2017, Stem cells international.

[28]  R. Miron,et al.  Platelet Rich Fibrin: A Second‐Generation Platelet Concentrate , 2017 .

[29]  Marcela Fernandes,et al.  Platelet-Rich Fibrin Conduits as an Alternative to Nerve Autografts for Peripheral Nerve Repair , 2017, Journal of Reconstructive Microsurgery.

[30]  Ji-xian Wang,et al.  Combining Injectable Plasma Scaffold with Mesenchymal Stem/Stromal Cells for Repairing Infarct Cavity after Ischemic Stroke , 2017, Aging and disease.

[31]  R. Jacobs,et al.  Platelet-rich plasma for regeneration of neural feedback pathways around dental implants: a concise review and outlook on future possibilities , 2017, International Journal of Oral Science.

[32]  G. Orive,et al.  Platelet-rich plasma, a source of autologous growth factors and biomimetic scaffold for peripheral nerve regeneration , 2017, Expert opinion on biological therapy.

[33]  D. Pizzo,et al.  Ionotropic glutamate receptors activate cell signaling in response to glutamate in Schwann cells , 2017, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[34]  Kohya Uematsu,et al.  Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma (PRP), plasma rich in growth factors (PRGF), advanced platelet-rich fibrin (A-PRF), and concentrated growth factors (CGF) , 2016, International journal of implant dentistry.

[35]  O. Detante,et al.  Biomaterial Applications in Cell-Based Therapy in Experimental Stroke , 2016, Stem cells international.

[36]  Xiaoping Zhou,et al.  Effect of platelet‐rich plasma (PRP) concentration on proliferation, neurotrophic function and migration of Schwann cells in vitro , 2016, Journal of tissue engineering and regenerative medicine.

[37]  Z. Haidar,et al.  Use of leukocyte and platelet-rich fibrin (L-PRF) in periodontally accelerated osteogenic orthodontics (PAOO): Clinical effects on edema and pain , 2016, Journal of clinical and experimental dentistry.

[38]  R. Carrau,et al.  Role of Leukocyte–Platelet-Rich Fibrin in Endoscopic Endonasal Skull Base Surgery Defect Reconstruction , 2016, Journal of Neurological Surgery Part B: Skull Base.

[39]  S. Hoerstrup,et al.  Synergistic interactions of blood-borne immune cells, fibroblasts and extracellular matrix drive repair in an in vitro peri-implant wound healing model , 2016, Scientific Reports.

[40]  J. Weisel,et al.  The Platelet Integrin αIIbβ3 Differentially Interacts with Fibrin Versus Fibrinogen* , 2016, The Journal of Biological Chemistry.

[41]  Kenneth M. Tichauer,et al.  Biomaterials with persistent growth factor gradients in vivo accelerate vascularized tissue formation. , 2015, Biomaterials.

[42]  David J. Mooney,et al.  Regenerative medicine: Current therapies and future directions , 2015, Proceedings of the National Academy of Sciences.

[43]  G. Gibbons Grafix®, a Cryopreserved Placental Membrane, for the Treatment of Chronic/Stalled Wounds , 2015, Advances in wound care.

[44]  F. Riccitiello,et al.  Influence of Leukocyte- and Platelet-Rich Fibrin (L-PRF) in the Healing of Simple Postextraction Sockets: A Split-Mouth Study , 2015, BioMed research international.

[45]  A. Cattaneo,et al.  A comparative analysis of the structural, functional and biological differences between Mouse and Human Nerve Growth Factor. , 2015, Biochimica et biophysica acta.

[46]  S. Kohl,et al.  Platelet-rich Concentrates Differentially Release Growth Factors and Induce Cell Migration In Vitro , 2015, Clinical orthopaedics and related research.

[47]  H. Yoon,et al.  The influence of platelet-rich fibrin on angiogenesis in guided bone regeneration using xenogenic bone substitutes: a study of rabbit cranial defects. , 2014, Journal of cranio-maxillo-facial surgery : official publication of the European Association for Cranio-Maxillo-Facial Surgery.

[48]  Christine E. Schmidt,et al.  Advanced biomaterials for repairing the nervous system: what can hydrogels do for the brain? , 2014 .

[49]  David F Williams,et al.  Neural tissue engineering options for peripheral nerve regeneration. , 2014, Biomaterials.

[50]  D. M. Dohan Ehrenfest,et al.  Classification of platelet concentrates (Platelet-Rich Plasma-PRP, Platelet-Rich Fibrin-PRF) for topical and infiltrative use in orthopedic and sports medicine: current consensus, clinical implications and perspectives. , 2014, Muscles, ligaments and tendons journal.

[51]  P. Formisano,et al.  Growth-promoting action and growth factor release by different platelet derivatives , 2014, Platelets.

[52]  D. A. Reed,et al.  Lyophilized Platelet-Rich Fibrin (PRF) Promotes Craniofacial Bone Regeneration through Runx2 , 2014, International journal of molecular sciences.

[53]  M. Ameloot,et al.  Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct in vitro , 2014, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[54]  A. Sebben,et al.  Effect of platelet rich plasma and platelet rich fibrin on sciatic nerve regeneration in a rat model , 2013, Microsurgery.

[55]  Huanxing Su,et al.  Nanofiber scaffolds facilitate functional regeneration of peripheral nerve injury. , 2013, Nanomedicine : nanotechnology, biology, and medicine.

[56]  A. Kolokythas,et al.  Platelet-Rich Fibrin Promotes Periodontal Regeneration and Enhances Alveolar Bone Augmentation , 2013, BioMed research international.

[57]  Hua-xiang Liu,et al.  Neuregulin-1β regulates tyrosine kinase receptor expression in cultured dorsal root ganglion neurons with excitotoxicity induced by glutamate , 2013, Regulatory Peptides.

[58]  N. Shubhashini,et al.  Platelet rich fibrin: a new paradigm in periodontal regeneration , 2012, Cell and Tissue Banking.

[59]  A. Rosenbaum,et al.  Bone grafts, bone substitutes and orthobiologics , 2012, Organogenesis.

[60]  Á. Dobolyi,et al.  The Neuroprotective Functions of Transforming Growth Factor Beta Proteins , 2012, International journal of molecular sciences.

[61]  D. M. Dohan Ehrenfest,et al.  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). , 2012, Current pharmaceutical biotechnology.

[62]  D. M. Dohan Ehrenfest,et al.  Current knowledge and perspectives for the use of platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) in oral and maxillofacial surgery part 1: Periodontal and dentoalveolar surgery. , 2012, Current pharmaceutical biotechnology.

[63]  P. Boileau,et al.  Leukocyte- and platelet-rich fibrin (L-PRF) for long-term delivery of growth factor in rotator cuff repair: review, preliminary results and future directions. , 2012, Current pharmaceutical biotechnology.

[64]  R. Borojevic,et al.  Enhancement of sciatic nerve regeneration after vascular endothelial growth factor (VEGF) gene therapy , 2011, Neuropathology and applied neurobiology.

[65]  Herman Goossens,et al.  Clinical Potential of Intravenous Neural Stem Cell Delivery for Treatment of Neuroinflammatory Disease in Mice? , 2011, Cell transplantation.

[66]  J. Chung,et al.  Platelet-rich fibrin is a Bioscaffold and reservoir of growth factors for tissue regeneration. , 2011, Tissue engineering. Part A.

[67]  S. Sakiyama-Elbert,et al.  Tissue-engineered fibrin scaffolds containing neural progenitors enhance functional recovery in a subacute model of SCI. , 2010, Soft matter.

[68]  Zhongyu Li,et al.  Effect of locally delivered IGF‐1 on nerve regeneration during aging: An experimental study in rats , 2010, Muscle & nerve.

[69]  M. Rota,et al.  Essential role of diastolic oscillatory potentials in adrenergic control of guinea pig sino-atrial node discharge , 2009, Journal of Biomedical Science.

[70]  W. Young,et al.  Postischemic IGF-1 Gene Transfer Promotes Neurovascular Regeneration after Experimental Stroke , 2009, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[71]  T. Albrektsson,et al.  Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF). , 2009, Trends in biotechnology.

[72]  E. Rosenzweig,et al.  Delivery of neurotrophin-3 from fibrin enhances neuronal fiber sprouting after spinal cord injury. , 2006, Journal of controlled release : official journal of the Controlled Release Society.

[73]  A. Dohan,et al.  Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part II: platelet-related biologic features. , 2006, Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics.

[74]  A. Dohan,et al.  Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution. , 2006, Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics.

[75]  J. Häggblad,et al.  Platelet-Derived Growth Factor (PDGF-BB) and Brain-Derived Neurotrophic Factor (BDNF) induce striatal neurogenesis in adult rats with 6-hydroxydopamine lesions , 2005, Neuroscience.

[76]  Austin G Smith,et al.  Niche-Independent Symmetrical Self-Renewal of a Mammalian Tissue Stem Cell , 2005, PLoS biology.

[77]  H D Li,et al.  Hyaluronic acid-poly-D-lysine-based three-dimensional hydrogel for traumatic brain injury. , 2005, Tissue engineering.

[78]  K. Jin,et al.  VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. , 2003, The Journal of clinical investigation.

[79]  T. Oya,et al.  Platelet‐derived growth factor‐b expression induced after rat peripheral nerve injuries , 2002, Glia.

[80]  James M. Anderson,et al.  Biological Responses to Materials , 2001 .

[81]  J. Hubbell,et al.  Controlled release of nerve growth factor from a heparin-containing fibrin-based cell ingrowth matrix. , 2000, Journal of controlled release : official journal of the Controlled Release Society.

[82]  Sunhee C. Lee,et al.  Neuronal death in cytokine‐activated primary human brain cell culture: role of tumor necrosis factor‐α , 1999, Glia.

[83]  E. Shooter,et al.  Interleukin‐6 Production by Schwann Cells and Induction in Sciatic Nerve Injury , 1995, Journal of neurochemistry.

[84]  Juliane Freud,et al.  Culturing Nerve Cells , 2016 .

[85]  C. Napoli,et al.  Platelet derivatives in regenerative medicine: an update. , 2015, Transfusion medicine reviews.

[86]  P. Boileau,et al.  Increased vascularization during early healing after biologic augmentation in repair of chronic rotator cuff tears using autologous leukocyte- and platelet-rich fibrin (L-PRF): a prospective randomized controlled pilot trial. , 2014, Journal of shoulder and elbow surgery.

[87]  C. Munaut,et al.  Transforming growth factor β as a neuronoglial signal during peripheral nervous system response to injury , 1993 .