The overall goal of this procedure is to generate and characterize leukocyte platelet rich fibrin or LPRF. This is accomplished by first collecting a peripheral whole blood sample into a red top tube containing no anticoagulants, followed by immediate centrifugation. The LPRF is then removed from the tube and gently compressed for 30 seconds.
The mechanical properties of leukocyte platelet-rich fibrin were assessed by UNI axial tensile testing and suture retention strength. Ultimately effects of genin crosslinking on the biological stability of LPRF were demonstrated. The main advantage of this technique over existing methods like using plate literature plasma, is that LPRF technique uses a natural 3D fibrin matrix generated from whole blood without altering its composition or coagulability.
A better understanding of the material properties of LPRF will expand its application beyond acute and chronic wound management to its use as scaffold for tissue engineering. After obtaining informed consent, set up a tray for the patient with red topped dry glass blood collection tubes containing no anticoagulants on a flat stable surface. Label the tube with the appropriate patient information.
Next, explain the procedure and that a small pinch will be felt and seat the patient comfortably. With the arm supported. Then wash hands and dong gloves.
Attach the needle to the adapter. Apply a tourniquet three to four inches above the puncture site, making sure that it is not too tight while still feeling the radial pulse. Using 70%isopropyl alcohol, clean the antecubital falsa for venipuncture in concentric circles from the center outward.
After 30 seconds of air drying, identify the appropriate vein by palpation and advise the patient to remain still throughout the procedure. Then insert the bevel of the needle 15 to 30 degrees into the skin in one smooth motion. When the needle is in place, push a blood collection tube through the bottom needle and collect the blood.
Once nine milliliters of blood have been obtained, apply a gauze square at the puncture site and withdraw the needle from the patient's arm. Remove the tourniquet from the patient's arm, discard the needles into the appropriate biohazard container. Ask the patient to maintain the pressure at the puncture site and check the puncture site to confirm that the bleeding has stopped.
If the patient is feeling well, offer thanks and discharge the patient immediately After venous blood collection, centrifuge the sample. The blood will separate into platelet poor plasma leukocyte, platelet rich fibrin, and red blood cell base layers. Using tweezers gently transfer the LPRF layer onto a sterile perforated metal mesh.
Next, use a surgical scalpel to scrape the bulk of the red blood cell layer away from the fibrin, taking care to leave the buffy coat intact. Then using a sterile metal plate weighing approximately 225 grams, gently compress the fibrin clot to squeeze out any remaining platelet poor plasma. After 30 seconds, remove the plate and gently transfer the LPRF membrane into a six well tissue culture plate.
After pre measuring and collecting six compressed platelet rich fibrin membranes, measure the thickness of each sample at three spots and use custom made metal dyes to punch them into dog bones. Then gently engage one platelet rich fibrin membrane mass within the center of the jaw grips of a UNI axial testing system, and carefully tear the filter paper support to further expose the membrane while the LPRF is still wet. Program the instrument so that the movable head operates at a constant rate of 10 millimeters per minute and start the experiment.
Then using the accompanying UNI axial testing system software record the elastic modulus energy to break and strain it.Break. To measure the suture retention strength, place the LPRF membrane onto a piece of filter paper and use a surgical scalpel to cut the membranes into 10 by 25 millimeter rectangles. Measure the thickness of each sample as just demonstrated.
Then use a 220 micron diameter stainless steel orthodontic ligature wire to make a pinhole in the center of each tissue. Next, pass the ligature wire through the pinhole of one of the samples to form a loop. Then fix the loop to the tensile testing machine and place the edge of the LPRF membrane against the lower jaw grip.
Set the instrument so that the movable head operates at a constant rate or 10 millimeters per minute, and start the experiment. Then using the accompanying UNI axial testing system software record the elastic modulus energy to break and strain it. Break as just demonstrated In these scanning electron microscope images, the top, middle and bottom layers of a leukocyte platelet-rich fibrin clot can be observed.
The top portion is composed predominantly of a fibrin network with no cells. The middle layer is enriched, activated, and degranulated platelets, and the lower layer is composed of a mixture of leukocytes and red blood cells entrapped within a fibrin matrix. Even though the elastic modulus of platelet-rich fibrins is low, the membrane is tough and capable of undergoing significant deformation.
Indeed, suture retention testing. An indicator of the ability of the membrane to be sutured to the tissues suggests a significantly tough and stretchable material. One of the limitations of using fibrin products in regenerative medicine is the short biological life of the tissue.
For example, in this experiment after treatment with 0.01%trypsin, the platelet rich fibrin was degraded in three days. Genin cross-linking of the LPRF membranes, however, decreased this degradation by almost 60%Further un cross-linked clots cultured with calvarial osteoblasts underwent various degrees of degradation. While genin cross-linking of the platelet rich fibrin preserved the structure of the membranes and supported the growth of the cells.
While attempting this procedure, it's important to remember to use freshly drawn whole blood, as well as red top tubes contain no anticoagulants or additives. After watching this video, you should have a good understanding of how to generate LPRF and how to perform detailed mechanical characterization of these biological membranes. Don't forget that working with freshly drawn whole blood is a prerequisite for generating LPRF.
Venipuncture on human subjects should be carried out by certified professionals, and that systematic research using humans is subject to institutional review board approval.
