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 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 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><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 &amp;amp; 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&amp;nbsp;</td><td>Intra-Lock International</td><td>ISS110</td><td>Centrifuge and Xpression L-PRF FabricationKit&amp;nbsp;</td></tr><tr><td>Pipettes (Serological &amp;amp; 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: 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&#183;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).
<img alt="Figure 1" src="/files/ftp_upload/53221/53221fig1.jpg" /> 
Figure 1.&#160;Steps in the generation of L-PRF.&#160;(A)&#160;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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53221/53221fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53221/53221fig2.jpg" /> 
Figure 2.&#160;SEM image of different layers of fresh L-PRF.&#160;(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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53221/53221fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53221/53221fig3.jpg" /> 
Figure 3.&#160;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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53221/53221fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53221/53221fig4.jpg" /> 
Figure 4.&#160;Photographs of actual failure in suture retention strength testing. A 220 &#181;m 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53221/53221fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53221/53221fig5.jpg" /> 
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.
<img alt="Figure 6" src="/files/ftp_upload/53221/53221fig6.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53221/53221fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.9K 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. 

Video: Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Immunology and Infection

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Medicine

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

Biology

Mechanical Stromal Vascular Fraction in Fibrin Hydrogel for Regenerative Medicine

The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the past, the extraction of these cells from adipose tissue required a multistep enzyme-based process, resulting in a heterogenous cell mix consisting of ACSs and other cells, which are jointly termed the stromal vascular fraction (SVF). More recently introduced mechanical SVF (mSVF) isolation protocols are less time-consuming and bypass regulatory concerns. We recently proposed a protocol that generates mSVF rich in stromal cells based on a combination of emulsification and centrifugation. One current issue in mSVF application for wound therapy application is the lack of a scaffold providing protection from mechanical manipulation and desiccation. Fibrin hydrogels have been shown to be a useful adjunct in cell transfer for wound healing purposes in the past. In the work herein, we delineate the preparation steps of an mSVF-fibrin hydrogel construct as a novel approach for translational research and clinical application.

Author Spotlight: Exploring the Potential of Fat-Derived Stromal Vascular Fraction for Wound Healing

Mechanically isolated stromal vascular fraction (SVF) in combination with a fibrin hydrogel offers an easy and efficient carrier for viable adipose-derived stromal cells for various indications, including tissue engineering and or wound healing purposes. Here, we present the preparation of a mechanical SVF (mSVF)-fibrin hydrogel construct for translational research and clinical application.

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the ...

Platform for Assessing Platelet Function in Trauma and Transfusion

Microfluidics in Assessing Platelet Function

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying aspects of thrombosis and hemostasis. At high-shear environments simulating arterial flow, a microfluidic assay facilitates the study of platelet function, as platelet-rich thrombi form in a localized stenotic region of a flow channel. Utilizing devices that allow for small sample volume can additionally aid in evaluating platelet function under flow from volume-limited patient samples or animal models. Studying trauma patient samples or samples following platelet product transfusion may aid in directing therapeutic strategies for patient populations in which platelet function is critical. Effects of platelet inhibition via pharmacological agents can also be studied in this model. The objective of this protocol is to establish a microfluidic platform that incorporates physiologic flow, biological surfaces, and relevant hemostatic mechanisms to assess platelet function with implications for the study of trauma induced coagulopathy and transfusion medicine.

Platelet function under flow can be assessed and simulated hemostatic resuscitation can be modeled using a microfluidic device, which has applications in trauma and transfusion medicine.

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

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