Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.
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Method Article
Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial
In This Article
Summary
Abstract
Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.
Introduction
The use of blood and blood-derived products to seal wounds and improve healing in different clinical situations started with fibrin glues, which are mainly fibrinogen concentrates. Addition of platelets to fibrin glue not only improved their strength but also promoted neoangiogenesis and regeneration. These benefits are attributed to the release of a variety of peptide growth factors from the alpha-granules of platelets upon activation1. Platelet concentrates (PC) were seen as a practical way to deliver growth factors2 and its use was driven by commercial interests rather than research characterization3. In fact, PCs are difficult to c....
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Protocol
All blood-drawing procedures should be done by licensed and certified professionals. Use of human subjects for research involves approval from the Institutional Review Board or other appropriate authority. Special precautions regarding informed consent and protecting participant identification need to be followed. All experiments listed in this protocol involve handling of human blood and/or blood products and appropriate personal protective equipment need to be worn at all times. The waste should be considered as biohazard and disposed of according to regulations.
1. Venipuncture
- Identify the patient/ participant and confirm wi....
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Results
The scanning electron microscope image of the L-PRF clot at different sections (top, middle and bottom) layer is illustrated in Figure 2. As can be seen, the top portion is composed predominantly of fibrin network with no cells. The middle layer is enriched with platelets with evidence of their activation and degranulation. The lower layer has a mixture of leukocytes and red blood cells entrapped within a fibrin matrix.
The mechanical properties were evaluated in two modes: un.......
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Discussion
Autologous platelet concentrates are promising in the field of regenerative medicine18 because of the abundance of growth factors. However, these preparations often lacked a defined structure that makes surgical manipulation very difficult. Many times, the suspensions and gels are not retained effectively at the site of delivery, resulting in unpredictable outcomes. L-PRF represents a huge advance in the evolution of platelet concentrates in that it is essentially a firm fibrin membrane with entrapped platelet.......
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Disclosures
The authors have nothing to disclose and confirm that there are no known conflicts of interest associated with this publication.
Acknowledgements
The project was supported by CTSA (UL1TR000058) from the National Center for Advancing Translational Sciences) and the CCTR Endowment Fund of Virginia Commonwealth University. The contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the National Institutes of Health.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Needle 19G | BD | 305186 | |
| Needle Disposal Container | Fisherbrand | 14-827-122 | |
| Red-Topped Glass Collection Tube | BD | 8020129 | |
| Gauze Pads | Tyco | 5750 | |
| Bandage | Johnson & Johnson | 5005989 | |
| Surshield | Terumo | SV*S19BL | Safety winged infusion set |
| Blood Collection Assembly | BD | 303380 | |
| Tourniquets | BD | 367203 | |
| Brand Luer Adapter | Vacutainer | L42179 | |
| Intra-Spin System | Intra-Lock International | ISS110 | Centrifuge and Xpression L-PRF FabricationKit |
| Pipettes (Serological & Micro) | Corning | ||
| Scalpel | Exelint | 29552 | |
| MTS Bionix 200 | MTS Systems Corporation | Material testing systems | |
| MTS Test Works 4 | MTS Systems Corporation | ||
| Whatman Filter Paper | Whatman | 1004 070 | |
| SS Orthodontic ligature wire | Patterson Dental | 628-4228 | |
| 200 Proof Ethanol | Koptec | V1001 | |
| Hexamethyldisilazane (HMDS) | Aldrich | 440191 | |
| Aluminium Mounting Stubs | Ted Pella | 16324 | |
| Double Sided Carbon Tape | PELCO Tabs | 16084-1 | |
| Scanning Electron Microscope | JEOL | LV 5610 | |
| Trypsin | HyClone | SH30042.01 | |
| Cell Culture Incubator | Thermo Fisher Scientific Inc | 51026282 | |
| Antibiotic-Antimicotic | Gibco | 15240-062 | |
| Genipin | Wako | 078-03021 | |
| Cell Culture Media | Gibco | 12000-022 | Minimum Essential Medium-Alpha |
| MTS Reagent | Promega | G1118 | |
| PMS Reagent | Sigma | P9625 | |
| Spectrophotometer | BioTek | Epoch Spectrophotometer | |
| 10mm Glass Cloning Rings | Corning | 3166-10 | |
| T-75 Flask | Corning | 430641 | |
| DPBS | Corning | 55-031-PB | |
| Ninhydrin 98% | Aldrich | 454044 | |
| 24 Well Plate | Corning | 3987 | |
| Biopsy Punch | Acu Punch | P1025 | |
| Digital Micrometer | Pittsburgh | 68305 | |
| Glutaraldehyde | Sigma | G6257 | |
| 12 Well Plate | Corning | 3336 | |
| 96 Well Plate | Corning | 3596 |
References
- Marx, R. E., et al. Platelet-rich plasma Growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 85 (6), 638-646 (1998).
- Rodriguez, I. A., Growney Kalaf, E. A., Bowlin, G. L., Sell, S. A.
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