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.

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
<ol>
	<li>Identify the patient/ participant and confirm with existing records. Explain the study in detail and get an informed consent.</li>
	<li>Have a tray set up for individual patient with tubes marked on a flat stable surface.</li>
	<li>Explain the procedure and make the patient seat comfortably with the arm supported. Inform the patient that he or she will feel a small pinch and should remain still throughout the procedure.</li>
	<li>Attach the needle to the adapter.</li>
	<li>Wash hands and wear gloves.</li>
	<li>Prepare the antecubital fossa for venipuncture by cleaning with 70% isopropyl alcohol in concentric circles from center outward. Allow the site to air-dry for 30 sec.</li>
	<li>Identify the appropriate vein by palpation.</li>
	<li>Apply the tourniquet 3-4 in&#160;above the puncture site making sure that it is not too tight (while still feeling the radial pulse).</li>
	<li>Perform venipuncture by inserting the bevel of the needle 15-30 degrees to the skin in one smooth motion. Push the blood collection tube through the needle and collect 9 ml&#160;of whole blood.</li>
	<li>Remove the tourniquet. Remove the tube from the needle.</li>
	<li>Withdraw the needle and apply the gauze square at the puncture site prior to needle removal. Dispose the needle in an appropriate biohazard container.</li>
	<li>Ask patient to maintain pressure at the puncture site.</li>
	<li>Label the tubes</li>
	<li>Check the puncture site to be sure bleeding has stopped.</li>
	<li>Apply adhesive bandage or tape over the gauze square, ask if the patient is feeling alright (no pain, swelling or light headedness)</li>
	<li>Thank the patient/participant prior to discharge.</li>
</ol>
2. L-PRF Preparation
<ol>
	<li>Immediately after venous blood collection in the red-topped dry glass tube, place it in the Centrifuge.</li>
	<li>Centrifuge at 400 x g for 12 min&#160;at RT&#160;after placing an appropriate counter balance.</li>
	<li>Remove the tube at the end of the cycle. Notice the three layers: platelet-poor plasma (PPP), platelet-rich fibrin (L-PRF) and RBC base (Figure 1).</li>
	<li>Aspirate the PPP using a pipette. Using tweezers gently pull the L-PRF out and place it in a sterile, perforated metal mesh.</li>
	<li>Using surgical scalpel, scrape the bulk of RBC layer carefully leaving the buffy coat intact.</li>
	<li>Gently compress the L-PRF clot (using the sterile metal plate, approximate weight 225 g) for 30 sec. Platelet poor plasma will be squeezed out.</li>
	<li>Remove the plate and gently lift the L-PRF membrane. The L-PRF membrane is ready for use in experiments12.</li>
</ol>
3. Uniaxial&#160;Tensile&#160;Testing
<ol>
	<li>Place L-PRF membranes (n=6) on a filter paper for ease of handling and punch into &#8220;dog bones&#8221; using custom-made metal dies (2.75 mm wide at their narrowest point with a gauge length of 7.5 mm).</li>
	<li>Measure the thickness of each sample at three spots and take the average.</li>
	<li>Carefully engage the L-PRF membrane in the center of the jaw grips of the uniaxial testing system.</li>
	<li>Carefully tear the filer paper support to expose the L-PRF membrane.</li>
	<li>Program the instrument so that the movable head is operating at a constant rate (10.0 mm/min) and start the experiment when the L-PRF is still wet.</li>
	<li>Record the elastic modulus, energy to break, and strain at break from the software accompanying the uniaxial testing system. These values are calculated automatically and no user defined input is required. Please see Figure 3.</li>
</ol>
4. Suture&#160;Retention&#160;Strength
<ol>
	<li>Place L-PRF membranes (n=3) on a filter paper for ease of handling and cut into rectangular samples measuring (10 mm x 25 mm) using a surgical scalpel.</li>
	<li>Measure the thickness of each sample (average of 3).</li>
	<li>Make a pinhole in the center of the sample using the stainless steel orthodontic ligature wire (220 &#181;m in diameter).</li>
	<li>Pass the ligature wire through the pinhole to form a loop and fix it to the tensile testing machine. Place the edge of the L-PRF membrane to the lower jaw grip13.</li>
	<li>Program the instrument so that the movable head is operating at a constant rate (10 mm/min) and start the experiment.</li>
	<li>Record the elastic modulus, energy to break, and strain at break from the software accompanying the uniaxial testing system. These values are calculated automatically and no user defined input is required. Please see Figure 3.</li>
</ol>
5. Morphological&#160;Examination
<ol>
	<li>Prepare the L-PRF samples for SEM examination using a 10 mm dermal biopsy punch and place them in a 24-well plate.</li>
	<li>Wash the samples with PBS and fix with 2.5% glutaraldehyde (in PBS) for 20 min.</li>
	<li>Dehydrate the specimens by immersion in sequentially increasing concentrations of ethanol (50%, 70%, 80%, 90% and 100%) for 5 min&#160;each.</li>
	<li>Treat with 0.5 ml&#160;of 100% HMDS (Hexamethyldisilazane) for 5 min. Aerate O/N to remove excess HMDS14.</li>
	<li>Mount samples on stubs using a double-sided tape, sputter-coat platinum for 70 sec&#160;and examine in a scanning electron microscope operating at an acceleration voltage of 20 kV (or appropriate setting).</li>
</ol>
6. Genipin&#160;Crosslinking&#160;of L-PRF, Trypsin&#160;Susceptibility&#160;and&#160;Ninhydrin&#160;Assay
<ol>
	<li>To prepare genipin cross-linked L-PRF, rinse membranes with PBS and soak in 4 ml&#160;of 1% genipin solution (in 70% ethanol) for 48 hr.&#160;Rinse with PBS prior to experiments to remove excess genipin15, 16.</li>
	<li>Assess the stability of genipin crosslinking of L-PRF by its resistance to degradation by trypsin. Place L-PRF membrane (n=3) and genipin crosslinked L-PRF in 500 &#181;l of 0.01% trypsin and incubated at 37 &#176;C for 3 days with a daily change of trypsin.
	<ol>
		<li>Weigh samples at day 1 prior to enzyme exposure and at day 3. The difference in start and end weight represents enzymatic degradation17.</li>
	</ol>
	</li>
	<li>Quantify the amount of cross-linking in genipin treated L-PRF (G-PRF) by ninhydrin assay. First prepare the standard curve using glycine (1 mM-0.031 mM) curve to establish the relationship between free amino acid concentration (FAA) and absorbance.
	<ol>
		<li>Heat PRF samples with 1 ml&#160;of 2% (w/v) ninhydrin for 15 min&#160;at 100 &#176;C.</li>
		<li>Allow the solution to cool to RT and add 1.5 ml&#160;of 50% ethanol.</li>
		<li>Analyze the absorbance at 570 nm using a suitable spectrophotometer.</li>
		<li>Determine cross-linking percentage using the formula below15</li>
	</ol>
	</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/53221/53221eq1.jpg" />
7. MTS Cell&#160;Proliferation&#160;Assay
<ol>
	<li>Grow MC3T3 (mouse calvarial preosteoblasts) in Minimum Essential Medium &#8211;alpha modification (&#945;MEM) in T-75 flasks until an 80% confluent monolayer is obtained.</li>
	<li>Prepare fresh, sterile L-PRF membrane (open the L-PRF tube inside the cell culture hood) and transfer the membrane onto a new cell culture dish.</li>
	<li>Aspirate media from the flask and rinse the monolayer with PBS, add 5 ml&#160;of 0.05% trypsin and place the flask in the 37 oC incubator for 5 min, pipette the contents of the flask into a centrifuge tube and centrifuge at 400 x g for 5 min.</li>
	<li>Decant the supernatant, gently tap the tube to break the cell pellet and re-suspend with 4 ml&#160;of fresh &#945; MEM, dispense a mixture of cell suspension (50 &#181;l) and trypan blue (50 &#181;l) into the hemocytometer and count the number of cells</li>
	<li>Seed 4x105 cells within 10 mm glass cloning rings placed on top of L-PRF membranes to retain the cells within the membranes (rings can be removed after 24 hr).</li>
	<li>At day 4, rinse constructs with PBS thrice for 10 min.</li>
	<li>Add 1 ml&#160;of serum free media and 200 &#181;l&#160;MTS reagent to each well and incubate for 2 hr at 37 &#176;C.</li>
	<li>Measure absorbance from 200 &#181;l&#160;aliquots at 490 nm.</li>
</ol>

<ol>
	<li>Marx, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasma+Growth+factor+enhancement+for+bone+grafts.">Platelet-rich plasma Growth factor enhancement for bone grafts.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 85 (6), 638-646 (1998).</li><li>Rodriguez, I. A., Growney Kalaf, E. A., Bowlin, G. L., Sell, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasma+in+bone+regeneration:+Engineering+the+delivery+for+improved+clinical+efficacy.">Platelet-rich plasma in bone regeneration: Engineering the delivery for improved clinical efficacy.</a> BioMed Res In. 2014, (2014).</li><li>Del Corso, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Knowledge+and+Perspectives+for+the+Use+of+Platelet-Rich+Plasma+(PRP)+and+Platelet-Rich+Fibrin+(PRF).">Current Knowledge and Perspectives for the Use of Platelet-Rich Plasma (PRP) and Platelet-Rich Fibrin (PRF).</a> in Oral and Maxillofacial Surgery Part 1: Periodontal and Dentoalveolar Surgery. Curr Pharm Biotechno. 13 (7), 1207-1230 (2012).</li><li>Mazzucco, L., Balbo, V., Cattana, E., Guaschino, R., Borzini, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+every+PRP-gel+is+born+equal+Evaluation+of+growth+factor+availability+for+tissues+through+four+PRP-gel+preparations:+Fibrinet,+RegenPRP-Kit,+Plateltex+and+one+manual+procedure.">Not every PRP-gel is born equal Evaluation of growth factor availability for tissues through four PRP-gel preparations: Fibrinet, RegenPRP-Kit, Plateltex and one manual procedure.</a> Vox San. 97 (2), 110-118 (2009).</li><li>Fern&#225;ndez-Barbero, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+and+morphological+characterization+of+platelet-rich+plasma+gel.">Flow cytometric and morphological characterization of platelet-rich plasma gel.</a> Clin Oral Implants Re. 17 (6), 687-693 (2006).</li><li>Li, Z., Guan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+for+cardiac+tissue+engineering.">Hydrogels for cardiac tissue engineering.</a> Polymer. 3 (2), 740-761 (2011).</li><li>Zhu, J., Cai, B., Ma, Q., Chen, F., Wu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+bricks-enriched+platelet-rich+plasma+gel+for+injectable+cartilage+engineering+&#8211;+an+in+vivo+experiment+in+nude+mice.">Cell bricks-enriched platelet-rich plasma gel for injectable cartilage engineering &#8211; an in vivo experiment in nude mice.</a> J Tissue Eng Regen Med. 7 (10), 819-830 (2013).</li><li>Dohan, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+fibrin+(PRF):+a+second-generation+platelet+concentrate.+Part+I:+technological+concepts+and+evolution.">Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101 (3), e37-e44 (2006).</li><li>Dohan, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+fibrin+(PRF):+a+second-generation+platelet+concentrate.+Part+II:+platelet-related+biologic+features.">Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part II: platelet-related biologic features.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101 (3), e45-e50 (2006).</li><li>Choukroun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+fibrin+(PRF):+a+second-generation+platelet+concentrate.+Part+V:+histologic+evaluations+of+PRF+effects+on+bone+allograft+maturation+in+sinus+lift.">Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part V: histologic evaluations of PRF effects on bone allograft maturation in sinus lift.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101 (3), 299-303 (2006).</li><li>Dohan Ehrenfest, D. M., Bielecki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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).">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).</a> Curr Pharma Biotechno. 13 (7), 1145-1152 (2012).</li><li>Dohan Ehrenfest, D. M., Rasmusson, L., Albrektsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+platelet+concentrates:+from+pure+platelet-rich+plasma+(P-PRP)+to+leucocyte-+and+platelet-rich+fibrin+(L-PRF).">Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF).</a> Trends Biotechno. 27 (3), 158-167 (2009).</li><li>Mine, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suture+Retention+Strength+of+Expanded+Polytetrafluoroethylene+(ePTFE)+Graft.">Suture Retention Strength of Expanded Polytetrafluoroethylene (ePTFE) Graft.</a> Acta Med Okayam. 64 (2), 121-128 (2010).</li><li>Braet, F., De Zanger, R., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drying+cells+for+SEM+,+AFM+and+TEM+by+hexamethyldisilazane:+a+study+on+hepatic+endothelial+cells.">Drying cells for SEM , AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells.</a> J Micros. 186 (1), 84-87 (1997).</li><li>Yuan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cross-linking+of+chitosan+microspheres+with+genipin+on+protein+release.">The effect of cross-linking of chitosan microspheres with genipin on protein release.</a> Carbohydr Poly. 68 (3), 561-567 (2007).</li><li>Sell, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+methods+of+electrospun+fibrinogen+scaffolds+for+tissue+engineering+applications.">Cross-linking methods of electrospun fibrinogen scaffolds for tissue engineering applications.</a> Biomed Mater. 3 (4), (2008).</li><li>Gorczyca, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+genipin+cross-linked+porous+chitosan-collagen-gelatin+scaffolds+using+chitosan-CO2+solution.">Preparation and characterization of genipin cross-linked porous chitosan-collagen-gelatin scaffolds using chitosan-CO2 solution.</a> Carbohydr Poly. 102, 901-911 (2014).</li><li>Rozman, P., Semenic, D., Wislet-Gendebien, S. a. b. i. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+15.+The+Role+of+Platelet+Gel+in+Regenerative+Medicine.">Chapter 15. The Role of Platelet Gel in Regenerative Medicine.</a> Advances In Regenerative Medicine. , 319-349 (2011).</li><li>Dohan Ehrenfest, ., Lemo, D. M., Jimbo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selecting+a+relevant+animal+model+for+testing+the+in+vivo+effects+of+Choukroun&#8217;s+platelet-rich+fibrin+(PRF):+Rabbit+tricks+and+traps.">Selecting a relevant animal model for testing the in vivo effects of Choukroun&#8217;s platelet-rich fibrin (PRF): Rabbit tricks and traps.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 110 (4), 413-416 (2010).</li><li>Guilak, F., Baaijens, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tissue+engineering:+Ten+more+years+of+progress.">Functional tissue engineering: Ten more years of progress.</a> J Biomec. 47 (9), 1931-1932 (2014).</li><li>Guilak, F., Butler, D. L., Goldstein, S. A., Baaijens, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+and+mechanobiology+in+functional+tissue+engineering.">Biomechanics and mechanobiology in functional tissue engineering.</a> J Biomec. 47 (9), 1933-1940 (2014).</li><li>Collet, J. P., Shuman, H., Ledger, R. E., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+an+individual+fibrin+fiber+in+a+clot.">The elasticity of an individual fibrin fiber in a clot.</a> Proc Natl Acad Sci. 102 (26), 9133-9137 (2005).</li><li>Liu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+fibers+have+extraordinary+extensibility+and+elasticity.">Fibrin fibers have extraordinary extensibility and elasticity.</a> Science. 313 (5787), 634 (2006).</li><li>American National Standard. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+8:+Test+methods+for+vascular+prostheses.">Chapter 8: Test methods for vascular prostheses.</a> Association for the Advancement of Medica lnstrumentation Guidance document: Cardiovascular Implants - Tubular vascular prostheses. , 33-34 (2001).</li></ol>

The authors have nothing to disclose and confirm that there are no known conflicts of interest associated with this publication.

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...

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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Needle 19G</td>
			<td>BD</td>
			<td>305186</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Disposal Container</td>
			<td>Fisherbrand</td>
			<td>14-827-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Red-Topped Glass Collection Tube</td>
			<td>BD</td>
			<td>8020129</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze Pads</td>
			<td>Tyco</td>
			<td>5750</td>
			<td></td>
		</tr>
		<tr>
			<td>Bandage</td>
			<td>Johnson &#38; Johnson</td>
			<td>5005989</td>
			<td></td>
		</tr>
		<tr>
			<td>Surshield</td>
			<td>Terumo</td>
			<td>SV*S19BL</td>
			<td>Safety winged infusion set</td>
		</tr>
		<tr>
			<td>Blood Collection Assembly</td>
			<td>BD</td>
			<td>303380</td>
			<td></td>
		</tr>
		<tr>
			<td>Tourniquets</td>
			<td>BD</td>
			<td>367203</td>
			<td></td>
		</tr>
		<tr>
			<td>Brand Luer Adapter</td>
			<td>Vacutainer</td>
			<td>L42179</td>
			<td></td>
		</tr>
		<tr>
			<td>Intra-Spin System&#160;</td>
			<td>Intra-Lock International</td>
			<td>ISS110</td>
			<td>Centrifuge and Xpression L-PRF FabricationKit&#160;</td>
		</tr>
		<tr>
			<td>Pipettes (Serological &#38; Micro)</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Exelint</td>
			<td>29552</td>
			<td></td>
		</tr>
		<tr>
			<td>MTS Bionix 200</td>
			<td>MTS Systems Corporation</td>
			<td></td>
			<td>Material testing systems</td>
		</tr>
		<tr>
			<td>MTS Test Works 4</td>
			<td>MTS Systems Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Filter Paper</td>
			<td>Whatman</td>
			<td>1004 070</td>
			<td></td>
		</tr>
		<tr>
			<td>SS Orthodontic ligature wire</td>
			<td>Patterson Dental</td>
			<td>628-4228</td>
			<td></td>
		</tr>
		<tr>
			<td>200 Proof Ethanol</td>
			<td>Koptec</td>
			<td>V1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Aldrich</td>
			<td>440191</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium Mounting Stubs</td>
			<td>Ted Pella</td>
			<td>16324</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Sided Carbon Tape</td>
			<td>PELCO Tabs</td>
			<td>16084-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>JEOL</td>
			<td>LV 5610</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Incubator</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td>51026282</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimicotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Genipin</td>
			<td>Wako</td>
			<td>078-03021</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Media</td>
			<td>Gibco</td>
			<td>12000-022</td>
			<td>Minimum Essential Medium-Alpha</td>
		</tr>
		<tr>
			<td>MTS Reagent</td>
			<td>Promega</td>
			<td>G1118</td>
			<td></td>
		</tr>
		<tr>
			<td>PMS Reagent</td>
			<td>Sigma</td>
			<td>P9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>BioTek</td>
			<td></td>
			<td>Epoch Spectrophotometer</td>
		</tr>
		<tr>
			<td>10mm Glass Cloning Rings</td>
			<td>Corning</td>
			<td>3166-10</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 Flask</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Corning</td>
			<td>55-031-PB</td>
			<td></td>
		</tr>
		<tr>
			<td>Ninhydrin 98%</td>
			<td>Aldrich</td>
			<td>454044</td>
			<td></td>
		</tr>
		<tr>
			<td>24 Well Plate</td>
			<td>Corning</td>
			<td>3987</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch</td>
			<td>Acu Punch</td>
			<td>P1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Micrometer</td>
			<td>Pittsburgh</td>
			<td>68305</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>G6257</td>
			<td></td>
		</tr>
		<tr>
			<td>12 Well Plate</td>
			<td>Corning</td>
			<td>3336</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Plate</td>
			<td>Corning</td>
			<td>3596</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization of leukocyte-platelet rich fibrin, a novel biomaterial

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 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...

Watch this Scientific Journal Video about Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial at JoVE.com

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

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 ...

characterization-of-leukocyte-platelet-rich-fibrin-a-novel-biomaterial

Research

JoVE Journal

Bioengineering

12.8K Views.  School of Dentistry, Virginia Commonwealth University. 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. 