This study investigates the effects of activated platelet-rich plasma (PRP) on tendon repair in a rat model. The methodology includes surgical procedures to create a tendon lesion and the subsequent application of activated PRP to promote healing.
Place an anesthetized recipient rat with a shaved hind limb on a warm pad under a dissecting microscope.
Incise the skin and dissect the fascia, exposing the Achilles tendon complex.
Remove the plantaris tendon and excise a small segment from the Achilles tendon, creating a lesion.
Suture the incision and allow the rat to recover.
Collect whole blood from a genetically different donor rat, mixed with an anticoagulant.
Centrifuge to obtain a lower phase of red blood cells and an upper phase of platelet-rich plasma, or PRP.
Transfer the PRP to a tube and spin down the platelets. Partially remove the supernatant and resuspend the platelets to obtain concentrated PRP.
Add calcium chloride and incubate to induce platelet activation. Then, inject the activated platelets into the sutured site of the recipient.
Activated platelets release specific cytokines and growth factors that induce tendon repair at the lesion site.
Begin by weighing and ear-tagging the rat. After confirming the appropriate level of sedation by toe pinch, apply water drops onto the animal's corneas and remove the hair from the left hind limb. Disinfect the exposed skin with a 1-to-10 dilution of Iso-betadine solution, and place the animal on a 20 degrees Celsius heating pad, under a dissecting microscope.
In the lateral debits position with the left hind limb in the superior position, grasp the left hind limb paw with forceps, and make a 20 to 25-millimeter lateral skin incision around the Achilles tendon, using fine scissors. Dissect the fascia to expose the Achilles tendon complex, and remove the plantaris tendon.
Cut the Achilles tendon transversally 5 millimeters proximal to its calcaneal insertion, and remove a 5-millimeter long portion. Then, use resorbable yarn in continuous sutures to close the fascia and the skin, and place the rat under a heat source with monitoring until full recovery. While the recipient animals are recovering, collect 20 milliliters of whole blood from one donor rat heart, per experimental animal into individual tubes containing 3.2% buffered sodium citrate.
To obtain the platelet-rich plasma, centrifuge the blood, and use a plastic transfer pipette to carefully harvest the upper PRP-containing phase into a second plastic tube. Determine the volume of collected PRP, and measure the platelet count on a hematology analyzer.
Centrifuge the PRP again, and carefully transfer the supernatant into a new plastic tube. The remaining volume in the PRP tube should be around 2/3 of the volume required to achieve 2.5 x 106 platelets per microliter. Next, gently resuspend the platelet pellet with pipetting. Measure the platelet count of this concentrated platelet-rich plasma, adding back the appropriate volume of autologous platelet-poor plasma to reach the target concentration as necessary.
Activate the platelets with 50 microliters of calcium chloride per milliliter of PRP for about 1 hour at room temperature. Then load the platelets into a 1-milliliter syringe equipped with a 21-gauge needle, and inject 50 microliters of the platelets directly into the suture site of each animal.
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