Name: effects of allogeneic platelet-rich plasma (prp) on the healing process of sectioned achilles tendons of rats: a methodological description
Uploaded: 2018-03-19T12:30:00
Description: Watch this Scientific Journal Video about Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description at JoVE.com

This study was supported by Standard de Li&#232;ge and Lejeune- Lechien grants of the Leon Fr&#233;d&#233;ricq Funds.

Care and handling of the animals were performed in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (USA). European and national legislation were followed carefully.
1. Animal Preparation
<ol>
	<li>Use 132 2-month old male Sprague-Dawley rats weighing 320-450 g (120 rats for experimentation and 12 rats for blood sampling, Figure 1). Based on Dell<s...

<ol>
	<li>Kaux, J., Degrave, N., Crielaard, J. <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+traitement+des+tendinopathies+chroniques?+Revue+de+la+litt&#233;rature.+Platelet+rich+plasma+treatment+of+chronic+tendinopathies?+Review+of+literature.">Platelet rich plasma traitement des tendinopathies chroniques? Revue de la litt&#233;rature. Platelet rich plasma treatment of chronic tendinopathies? Review of literature.</a> J. Traumatol. du Sport. 24, 99-102 (2007).</li><li>Anitua, 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=Autologous+preparations+rich+in+growth+factors+promote+proliferation+and+induce+VEGF+and+HGF+production+by+human+tendon+cells+in+culture.">Autologous preparations rich in growth factors promote proliferation and induce VEGF and HGF production by human tendon cells in culture.</a> J. Orthop. Res. 23, 281-286 (2005).</li><li>Bosch, 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=Effects+of+platelet-rich+plasma+on+the+quality+of+repair+of+mechanically+induced+core+lesions+in+equine+superficial+digital+flexor+tendons:+A+placebo-controlled+experimental+study.">Effects of platelet-rich plasma on the quality of repair of mechanically induced core lesions in equine superficial digital flexor tendons: A placebo-controlled experimental study.</a> J. Orthop. Res. 28, 211-217 (2010).</li><li>Kaux, J. F., Drion, P., Croisier, J. L., Crielaard, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendinopathies+and+platelet-rich+plasma+(PRP):+From+pre-clinical+experiments+to+therapeutic+use.">Tendinopathies and platelet-rich plasma (PRP): From pre-clinical experiments to therapeutic use.</a> J. Stem Cells Regen. Med. 11, P7-P17 (2015).</li><li>Maffulli, N., Wong, J., Almekinders, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Types+and+epidemiology+of+tendinopathy.">Types and epidemiology of tendinopathy.</a> Clin. Sports Med. 22, 675-692 (2003).</li><li>Molloy, T., Wang, Y., Murrell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+growth+factors+in+tendon+and+ligament+healing.">The roles of growth factors in tendon and ligament healing.</a> Sports Med. 33, 381-394 (2003).</li><li>Lyras, D. N., 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+platelet-rich+plasma+gel+in+the+early+phase+of+patellar+tendon+healing.">The effect of platelet-rich plasma gel in the early phase of patellar tendon healing.</a> Arch. Orthop. Trauma Surg. 129, 1577-1582 (2009).</li><li>Aspenberg, P., Virchenko, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+concentrate+injection+improves+Achilles+tendon+repair+in+rats.">Platelet concentrate injection improves Achilles tendon repair in rats.</a> Acta Orthop. Scand. 75, 93-99 (2004).</li><li>Visser, L. C., 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=Growth+Factor-Rich+Plasma+Increases+Tendon+Cell+Proliferation+and+Matrix+Synthesis+on+a+Synthetic+Scaffold:+An+In+Vitro+Study.">Growth Factor-Rich Plasma Increases Tendon Cell Proliferation and Matrix Synthesis on a Synthetic Scaffold: An In Vitro Study.</a> Tissue Eng. Part A. 16, 1021-1029 (2010).</li><li>Zhang, J., Wang, J. H. -. C. <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+Releasate+Promotes+Differentiation+of+Tendon+Stem+Cells+Into+Active+Tenocytes.">Platelet-Rich Plasma Releasate Promotes Differentiation of Tendon Stem Cells Into Active Tenocytes.</a> Am. J. Sports Med. 38, 2477-2486 (2010).</li><li>Kajikawa, 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=Platelet-rich+plasma+enhances+the+initial+mobilization+of+circulation-derived+cells+for+tendon+healing.">Platelet-rich plasma enhances the initial mobilization of circulation-derived cells for tendon healing.</a> J. Cell. Physiol. 215, 837-845 (2008).</li><li>Pasternak, B., Aspenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metalloproteinases+and+their+inhibitors-diagnostic+and+therapeutic+opportunities+in+orthopedics.">Metalloproteinases and their inhibitors-diagnostic and therapeutic opportunities in orthopedics.</a> Acta Orthop. 80, 693-703 (2009).</li><li>Dell, R. B., Holleran, S., Ramakrishnan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+size+determination.">Sample size determination.</a> ILAR J. 43, 207-213 (2002).</li><li>M&#228;hler Convenor, 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=FELASA+recommendations+for+the+health+monitoring+of+mouse,+rat,+hamster,+guinea+pig+and+rabbit+colonies+in+breeding+and+experimental+units.">FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units.</a> Lab. Anim. 48, 178-192 (2014).</li><li>Kaux, J. -. F., 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=&#201;tude+comparative+de+cinq+techniques+de+pr&#233;paration+plaquettaire+(platelet-rich+plasma).">&#201;tude comparative de cinq techniques de pr&#233;paration plaquettaire (platelet-rich plasma).</a> Pathol. Biol. 59, 157-160 (2011).</li><li>Wieloch, P., Buchmann, G., Roth, W., Rickert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cryo-jaw+designed+for+in+vitro+tensile+testing+of+the+healing+Achilles+tendons+in+rats.">A cryo-jaw designed for in vitro tensile testing of the healing Achilles tendons in rats.</a> J. Biomech. 37, 1719-1722 (2004).</li><li>Kaux, J. -. F., 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=Vascular+Endothelial+Growth+Factor-111+(VEGF-111)+and+tendon+healing:+preliminary+results+in+a+rat+model+of+tendon+injury.">Vascular Endothelial Growth Factor-111 (VEGF-111) and tendon healing: preliminary results in a rat model of tendon injury.</a> Muscles. Ligaments Tendons J. 4, 24-28 (2014).</li><li>Docheva, D., Hunziker, E. B., F&#228;ssler, R., Brandau, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenomodulin+is+necessary+for+tenocyte+proliferation+and+tendon+maturation.">Tenomodulin is necessary for tenocyte proliferation and tendon maturation.</a> Mol. Cell. Biol. 25, 699-705 (2005).</li><li>Lambert, C. A., Colige, A. C., Munaut, C., Lapi&#232;re, C. M., Nusgens, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+pathways+in+the+over-expression+of+matrix+metalloproteinases+in+human+fibroblasts+by+relaxation+of+mechanical+tension.">Distinct pathways in the over-expression of matrix metalloproteinases in human fibroblasts by relaxation of mechanical tension.</a> Matrix Biol. 20, 397-408 (2001).</li><li>Nurden, A. T., Nurden, P., Sanchez, M., Andia, I., Anitua, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+and+wound+healing.">Platelets and wound healing.</a> Front. Biosci. 13, 3532-3548 (2008).</li><li>Woodall, J., Tucci, M., Mishra, A., Benghuzzi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+effects+of+platelet+rich+plasma:+a+study+on+HL-60+macrophage-like+cells.">Cellular effects of platelet rich plasma: a study on HL-60 macrophage-like cells.</a> Biomed. Sci. Instrum. 43, 266-271 (2007).</li><li>Taylor, D. W., Petrera, M., Hendry, M., Theodoropoulos, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+the+use+of+platelet-rich+plasma+in+sports+medicine+as+a+new+treatment+for+tendon+and+ligament+injuries.">A systematic review of the use of platelet-rich plasma in sports medicine as a new treatment for tendon and ligament injuries.</a> Clin. J. Sport Med. 21, 344-352 (2011).</li><li>Mazzocca, A. D., 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+positive+effects+of+different+platelet-rich+plasma+methods+on+human+muscle,+bone,+and+tendon+cells.">The positive effects of different platelet-rich plasma methods on human muscle, bone, and tendon cells.</a> Am. J. Sports Med. 40, 1742-1749 (2012).</li><li>McCarrel, T. M., Minas, T., Fortier, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+leukocyte+concentration+in+platelet-rich+plasma+for+the+treatment+of+tendinopathy.">Optimization of leukocyte concentration in platelet-rich plasma for the treatment of tendinopathy.</a> J. Bone Joint Surg. Am. 94 (1-8), e143 (2012).</li><li>Boswell, S. 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=Increasing+platelet+concentrations+in+leukocyte-reduced+platelet-rich+plasma+decrease+collagen+gene+synthesis+in+tendons.">Increasing platelet concentrations in leukocyte-reduced platelet-rich plasma decrease collagen gene synthesis in tendons.</a> Am. J. Sports Med. 42, 42-49 (2014).</li><li>Virchenko, O., Aspenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+can+one+platelet+injection+after+tendon+injury+lead+to+a+stronger+tendon+after+4+weeks?:+Interplay+between+early+regeneration+and+mechanical+stimulation.">How can one platelet injection after tendon injury lead to a stronger tendon after 4 weeks?: Interplay between early regeneration and mechanical stimulation.</a> Acta Orthop. 77, 806-812 (2006).</li></ol>

Platelets are essential for the early inflammatory phase of the tendon healing process. When these platelets are exposed to binding tissue or factors that induce coagulation, they will release growth factors that are stocked in &#945; granules. Due to this interaction, extracellular matrix macromolecules are synthetized and mesenchymal cells proliferate. Platelets also have a chemotactic activity on progenitor cells in the blood circulation, enhancing angiogenesis and stimulating cellular differentiation<sup class="xref"...

Coagulation, inflammatory processes, and the immunity modulation roles of platelets are well known1. More recently, it has been demonstrated that they also have restorative properties2,3. Indeed, various cytokines and growth factors (VEGF, PDGF, TGF-B, IGF-I, and HGF) are released by platelets during degranulation. These growth factors promote angiogenesis, tissue remodeling, and wound healing (bone, skin, muscle, tendon)2. Centrifuging autologous blood produces platelet rich plasma (PRP) which contains high platelet concentrations depending on the isolation method (between 3 and 10 times blood baseline concentrations). Indeed, various PRP preparation techniques cannot provide an identical final product. Up to now, there has been no international general agreement on this issue. Overall, PRP could be an attractive therapeutic option for treating chronic musculoskeletal conditions, such as tendinopathy, plantar fasciitis, osteoarthritis, and nonunion4. It was used for the first time in oral surgery and implantology4 to improve and accelerate bone healing after placing a dental implant. In this study, we describe a reproducible method that allows the acquisition of PRP for animal experimentation4.
Since lesions of tendons are frequently observed in sportsmen and physical workers, enhancing the healing process and thus reducing the time for recovery are of great interest5. New treatment methods that are developing often involve the use of growth factors, and the administration of PRP is a simple and minimally invasive way to deliver a blend of endogenous growth factors4.
Several in vitro or animal studies have demonstrated that the administration of plasma containing a high level of platelets, by releasing biological mediators, can stimulate tendon and ligament repair by releasing biological mediators6,7,8,9. Furthermore, other studies have shown that PRP can stimulate type I and III collagen synthesis in tendon cells9,10,11. It has also been suggested that PRP can decrease the activation of matrix metalloproteinases (MMPs) and therefore reduce the degradation of the matrix. Cells that are involved in the inflammation process can produce MMP-9, which plays a role in tissue remodeling (physiologic and pathologic) induced by inflammation12.
Based on this information, we hypothesized that a single PRP injection into the sectioned Achilles tendons of rats could improve the recovery process and the mechanical strength of the repaired tissue. This is tested by measuring the biomechanical properties of the healing tendons during the recovery process and by performing histological and molecular analyses to evaluate collagen remodeling in the newly formed tissue. The aim of the study was to observe if a single injection of allogeneic PRP could influence the healing of sectioned Achilles tendons.

<table>
	<tbody>
		<tr>
			<td>Xylazine (Xyl-M)</td>
			<td>VMD</td>
			<td>none</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Ketamin (J&#233;tamine 1000 CEVA)</td>
			<td>CEVA Sant&#233; Animale</td>
			<td>none</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Buprenorphin (Veterg&#233;sic Multidosis)</td>
			<td>ALSTOE</td>
			<td>none</td>
			<td>Painkiller</td>
		</tr>
		<tr>
			<td>iso-Betadine</td>
			<td>MEDA-Pharma</td>
			<td>none</td>
			<td>Desinfectant</td>
		</tr>
		<tr>
			<td>resorbable yarn Vicryl 6/0</td>
			<td>Johnson &#38; Johnson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nembutal</td>
			<td>CEVA Sant&#233; Animale</td>
			<td>none</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Preserves structure of the tissue</td>
		</tr>
		<tr>
			<td>Isopropanol 100%</td>
			<td>VWR</td>
			<td>20,922,364</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 95%</td>
			<td>VWR</td>
			<td>20,823,362</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR</td>
			<td>28973.363</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>VWR</td>
			<td>LEIC3950.1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Millipore</td>
			<td>1.15938.0025</td>
			<td>Colorant</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Millipore</td>
			<td>1.15935.0100</td>
			<td>Colorant</td>
		</tr>
		<tr>
			<td>Eukitt</td>
			<td>Sigma-Aldrich</td>
			<td>3989</td>
			<td>Mounting Medium</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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</table>

effects of allogeneic platelet-rich plasma (prp) on the healing process of sectioned achilles tendons of rats: a methodological description

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily.
Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.

The results are expressed as the mean &#177; standard deviation of the mean and were compared with analysis of variance (ANOVA). A two-way ANOVA and post hoc test de Scheff&#233;, which is a parametric test, were used.
The ultimate tensile strength (UTS) required to provoke a rupture of the non-injured Achilles tendons of rats was 42.0 &#177; 5.7 N (n = 10). The tensile strength increased significantly (p&#60;0.0001) in...

Watch this Scientific Journal Video about Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description at JoVE.com

Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This protocol describes the evaluation process of healing tendons in rats that have been injected with allogeneic platelet rich plasma (PRP) or saline solution after removing part of the Achilles tendon. The progress of tendon healing is evaluated at several time points using different types of analyses.

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a ...

The overall goal of this procedure is to observe the effects of a single injection of Platelet-Rich Plasma, or PRP, on the resistance and healing process of surgically-sectioned rat Achilles tendons at various time points. This method can help answer quick questions on musculoskeletal field about tendon lesions. The main advantage of this technique is that the surgical, biomechanical, and PRP progression procedures are well-standardized.The implications of these techniques extend toward the use of PRP in humans, because these types of lesion and the tendon healing process are similar between rats and human. Though this method can provide insight into PRP preparation for musculoskeletal injuries, this injection technique can also be applied to other systems, such as heart or skin disease. Visual demonstration of the method is very important, because surgical and biomechanical steps require consistent reproducibility in order to obtain optimal results.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 one-to-10 dilution of iso-Betadine solution and place the animal on a 20-degree Celsius heating pad under a dissecting microscope in the lateral decubitus 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 transversely, five millimeters proximal to its calcaneal insertion, and remove a five-millimeter long portion.Then use resorbable yarn and continue with 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 times 10 to the sixth platelets per microliter.Next, gently re-suspend 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 one hour at room temperature.Then load the platelets into a one-milliliter syringe equipped with a 21-gauge needle and inject 50 microliters of the platelets directly into the suture site of each animal. At the appropriate experimental endpoint, shave the left hind limb of the anesthetized experimental animal, and position the rat under a dissecting microscope as just demonstrated. Holding the paw with fingers, make a 10-millimeter incision in the surgical site to expose the triceps suralis.To remove the Achilles tendon, cut the calcaneal bone transversally, five to 10 millimeters distal to its attachment and dissect a piece of the triceps suralis large enough to fit into the cryo-jaw. Use forceps to place the sample immediately into the upper jaw of the cryo-jaw and close the instrument. Place the cryo-jaw vertically into a universal testing machine, and fix the calcaneal bone between the lower clamps.To calculate the cross-sectional area, place cameras perpendicular to each other. Add liquid nitrogen into both of the upper jaw basins to freeze the muscle to an order of magnitude stiffer than the tendon, so that it does not deform during the tensile test. When the freezing zone reaches the metal clamp border, set the displacement rate of the machine at a constant speed of one millimeter per second until rupture, and begin the tensile test, using a computer to record the ultimate tensile strength in Newtons.To account for differences in the cross-sectional areas of the healing tendons, normalize the ultimate tensile strength to a unit area that represents the mechanical stress experienced by the tissue. Then when the tendon ruptures, snap freeze the ruptured tendon samples in liquid nitrogen and store them at negative 80 degrees Celsius until RNA extraction and RTPCR analysis. In this representative experiment, a greater force was required to induce tendon rupture in the PRP-injected group than in the control saline-treated group at every time point tested, suggesting a more robust healing process or better mechanical properties within the PRP-treated animals.Although the PRP group demonstrated a higher intra-group scatter than did the saline group, the cross-sectional area of the tendons was determined to be larger in the PRP-injected group at days 15 and 30 after lesion induction. Further, Masson's trichrome staining revealed a higher presence fibrillar collagen, a key component in tendon morphology and strength, in PRP-treated animals on days five and 15, with a similar intensity of staining observed in both groups by day 30 after injection. After watching this video, you should have a good understanding of how to induce a rat standard lesions, and to sample the tendon to evaluate their healing process after a PRP injection.

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Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface ...

Platelet-Rich Plasma Lysate for Treatment of Eye Surface Diseases

Various ocular surface diseases are treated with blood-derived eye drops. Their use has been introduced in clinical practice because of their metabolite ...

Ultrasound Evaluation of Achilles Tendon Morphology

Measurement of Healthy and Injured Triceps Surae Morphology

Author Spotlight: Exploring the Complexities of Achilles Tendon Injuries &#8212; Research and Future Directions

Achilles tendon injuries occur throughout the lifespan and can negatively affect quality of life and overall health. Achilles tendinopathy is generally ...