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.
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.
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 characterize unlike homogenous and defined pharmacological preparations, they are a potpourri of signaling molecules and blood cells (platelet and leukocytes) entrapped within a fibrin matrix. Different commercial and proprietary preparations yield a variety of PC that are different in cellular composition, growth factor recovery and kinetics of release4.
It is important to realize that in most oral surgeries, platelet-rich plasma (PRP) preparations are used as a gel in open surgical wounds and not as platelet suspensions. In these situations, the gelation is induced by the addition of thrombin, calcium chloride, batroxobin or other agents and directly placed in the site of injury5. Due to rapid activation, fibrinogen polymerization is often incomplete and results in friable fibrin gels with very little mechanical strength. In addition, injectable PRP gels undergo rapid fibrinolysis6,7.
In contrast, the processes of blood coagulation (fibrinogen polymerization), platelet enrichment and activation occur simultaneously in the preparation of L-PRF8. The coagulation cascade is triggered when whole blood contacts the walls of a dry glass tube and continues throughout the centrifugation process. This results in the formation of a mechanically-strong blood clot (L-PRF) that can be surgically handled and used.
Even though L-PRF has been investigated in terms of optimal methods of preparation, growth factor release and cell distribution9-11, detailed mechanical characterization of these membranes are lacking. This is significant gap in knowledge, given the popularity of these membranes in clinical practice as well as its potential to be used as a biomaterial. Current study focusses on the protocol for deriving L-PRF as well as methods that can be employed to study its mechanical properties. This data is intended to serve as baseline for ongoing studies investigating the viscoelastic properties of this interesting natural biomaterial.
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
2. L-PRF Preparation
3. Uniaxial Tensile Testing
4. Suture Retention Strength
5. Morphological Examination
6. Genipin Crosslinking of L-PRF, Trypsin Susceptibility and Ninhydrin Assay
7. MTS Cell Proliferation Assay
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: uniaxial tensile testing and suture retention strength test. The results demonstrate viscoelastic behavior of L-PRF. Even though the elastic modulus is low (0.47 MPa), the membrane is tough (energy to break, 5 N·mm) and is capable of undergoing significant deformation (217%, Figure 3). Data from suture retention testing, an indicator of the ability of the membrane to be sutured to the tissues, suggested a significantly tough and deformable material (modulus-0.2 MPa, strain-140% and energy to break-3.2 N.mm) in L-PRF (Figure 4).
One of the limitations of fibrin products in regenerative medicine is its short biological life. Made from endogenous fibrin, L-PRF is susceptible to enzyme degradation and undergoes fibrinolysis. In order to evaluate the resistance of L-PRF to enzyme-mediated degradation, fresh L-PRF was subjected to trypsin treatment (0.01%) and incubated at 37 oC. We observed complete degradation of L-PRF within three days. Genipin crosslinking of L-PRF membranes decreased degradation by almost 60% (Figure 5).
The ability of L-PRF membranes to support cell growth was evaluated by culturing mouse calvarial osteoblasts on crosslinked and uncrosslinked membranes. Uncrosslinked clots underwent degradation to various levels while the genipin crosslinked membranes retained their structure and supported cell growth (Figure 6).
Figure 1. Steps in the generation of L-PRF. (A) After centrifugation of whole blood in a glass tube, three layers will be visible. (B) After decanting the PPP, the L-PRF is being removed using a sterile tweezers. (C) The red blood cell base is being scraped off using a scalpel and laid on a perforated metal tray (D). After gentle compression, the PPP is squeezed out and a firm L-PRF membrane is formed. Please click here to view a larger version of this figure.
Figure 2. SEM image of different layers of fresh L-PRF. (A) represents the fibrin-rich layer; (B) is a zone of enriched platelets with various degree of activation; (C) is the buffy coat with numerous leukocytes and (D) is the red blood cell base. Please click here to view a larger version of this figure.
Figure 3. Stress-strain curves following mechanical loading of L-PRF in uniaxial tensile testing mode (A) and Suture retention strength (B). The loading pattern of each sample is represented in different color. The uniaxial tensile testing data (A) indicate a low modulus, a large elastic deformation and a rapid failure. Upon distension by a suture (B), L-PRF represents a membrane that is tough (area under the curve) as well as distensible. Good clustering of data suggests minimal variation between samples. Please click here to view a larger version of this figure.
Figure 4. Photographs of actual failure in suture retention strength testing. A 220 µm thick stainless steel orthodontic ligature wire was passed through the middle of L-PRF and tied to the upper jaw member of the tensile testing machine. The other end was attached to the lower grip and stretched at a constant rate. Notice the elongation of the membrane and its resistance to tear, suggesting excellent resilience of L-PRF. Please click here to view a larger version of this figure.
Figure 5. Degradation of L-PRF membranes following incubation in 0.01% trypsin. All L-PRF membranes disintegrated completely in trypsin within 3 days while genipin-crosslinked L-PRF were 60% more stable. This shows that chemical crosslinking can be a viable strategy to improve the longevity of L-PRF membranes when placed in vivo.
Figure 6. Effect of L-PRF crosslinking on cell viability. Representative images of 4 day culture of MC3T3 cells on uncrosslinked L-PRF (A), genipin-crosslinked L-PRF (B) and tissue culture plastic (C). Uncrosslinked L-PRF degraded in culture to variable extent and showed cell activity similar to plastic. Genipin crosslinked L-PRF maintained their structure and supported robust cell survival. To the right is quantified data (+SD) from independent experiments with three replicates. Please click here to view a larger version of this figure.
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 platelets. These solid membranes possess excellent handling characteristics, and can be securely sutured at an anatomically desired location during open surgeries. However, its physical and biological properties are relatively unknown.
The L-PRF will form consistently when steps described above are strictly adhered to (Figure 1). One of the important considerations in generating a good L-PRF membrane is the time delay between blood draw and centrifugation. The success of L-PRF technique entirely depends on the speed of blood collection and immediate transfer to the centrifuge19, usually within a minute. It is impossible to generate a well-structured L-PRF clot (with its specific cell content, matrix architecture and growth factor release profile), if blood harvesting is prolonged and not homogenous; a small incoherent, friable mass of fibrin with unknown content is formed instead.
It has been accepted that mechanobiological interactions between cells and extracellular matrix (ECM) have a critical influence in all aspects of cell behavior including migration, proliferation and differentiation20,21. L-PRF, a unique type of blood clot, is formed under specific circumstances and is comprised of complex, branched network of fibrin. L-PRF functions as a provisional ECM that is turned over into functional tissue during healing. Being subjected to mechanical forces, successful healing outcomes are dependent on the structural integrity of L-PRF and hence elucidating their physical properties is important. We performed uniaxial tensile testing (to identify the intrinsic material properties) and suture retention testing (to identify the failure characteristics) on fresh L-PRF. Unlike PRP gel or clotted blood that does not have a defined structures, L-PRF resembles dense connective tissue with superior handling characteristics. We report an elastic modulus of 0.470 MPa (SD=0.107) for L-PRF membranes and stretch twice its initial length before failure (strain of 215%). These data match with published literature22,23 who reported low stiffness (1-10 MPa) and high strain (up to 150%) before breaking. The difference in the values can be due to the use of fibrin network compared to the use of AFM analysis of single fibrin fiber in the above mentioned studies.
Suture retention strength is a surgically important parameter of graft materials and it is defined as the force necessary to pull a suture from the graft or cause the wall of the graft to fail. Our experiments used straight-across procedure (as defined by ANSI24). The force required to the pull the ligature wire through the L-PRF of 3.23 N.mm (SD=0.329). Overall, we found the L-PRF to be mechanically tough, capable of supporting loads and the ability to stretch twice as much on tension and retains sutures quite well (deforms significantly before tearing).
The lack of stability and structural integrity of L-PRF in biological environments is a major limitation in its use in tissue engineering. We sought to address this issue by chemically crosslinking L-PRF using genipin. Unlike gluteraldehyde which is associated with toxicity, genipin is a naturally occurring biodegradable molecule with low cytotoxicity. After genipin treatment, the membranes were significantly stable in trypsin and supported cell proliferation over 4 days. However, only 20% of L-PRF was crosslinked with genipin (determined by ninhydrin assay). This data suggests that while chemical crosslinking is a viable strategy, other alternatives need to be explored.
Based on these findings, it is clear that L-PRF is a novel biomaterial with unique attributes: predictable preparation from autologous blood, simplicity of protocol, defined architecture, impressive mechanical properties and abundance of growth factors from activated platelets. The blood is allowed to clot under physiological conditions with no exposure to anti-coagulants, exogenous thrombin and calcium chloride. All of these characteristics make L-PRF promising biomaterial for applications in regenerative medicine.
One of the clinical issues to deal with in the application of L-PRF is the heterogeneity in the quality of platelets and blood components. At present, very little is understood about L-PRF generated from patients with coagulation disorders or patients on medications that affect blood clotting (heparin, warfarin or platelet inhibitors). Answers to these questions will undoubtedly improve our understanding of healing as well as contribute to advance the field of personalized medicine.
The authors have nothing to disclose.
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.
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 |