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 Dell13, 15 rats for each group was enough for a power of 0.8 (80% chance of finding statistical significance, if the specified effect exists).</li>
	<li>House all rats in classical cages under isolation conditions in a conventional facility equipped with a changing station. Instigate breeding in an IVC (individually ventilated cage) rack, with SPF (specific pathogen free) rats being specifically purchased.</li>
	<li>Use the following housing conditions: temperature range from 19-24 &#176;C; relative humidity: 40-60%; ventilation: frequency of air renewal: 10-15 per h; light/dark cycle: 12 h-12 h.</li>
	<li>Sterilize all caging equipment and use irradiated bedding. Systemically implement cage enrichment by placing sterile cardboard trays and towels inside the cages.</li>
	<li>Allow access to the zone under restricted conditions: new lab coat, mask, gloves, footwear, and hair bonnet at the entrance.</li>
	<li>Carry out colony health monitoring on sentinel animals housed on dirty bedding on an annual basis, according to the Felasa14 recommendations for conventional facilities. Keep four to five animals per cage, with the appropriate irradiated diet and provide acidified water ad libitum. Monitor daily.</li>
	<li>Transfer rats elected for surgical intervention in filtering top cages to the surgical rooms 2 h before surgery.</li>
</ol>
2. Surgical Procedure
<ol>
	<li>Prepare the surgical instruments: fine scissors, a clamp, two hemostatic clamps, and a needle holder. During all the procedures, wear gloves, a mask, a hood, and a lab coat.</li>
	<li>Randomly divide the rats into two groups (PRP and saline solution).</li>
	<li>Take the rat out of its cage and weigh it. Use numbered ear tags to identify the rats. To avoid desiccation of the eyes, place waterdrops on the cornea.</li>
	<li>Anesthetize the rat intraperitoneally with xylazine (10 mg/kg of body weight) and ketamine (80 mg/kg of body weight). To confirm anesthetization, place the animals under cardio-respiratory monitoring and check for any eye reflexes.</li>
	<li>Inject 50 &#181;L of buprenorphine subcutaneously&#160; (0.01-0.05 mg/kg&#160; every 8-12 h) in the neck area as a painkiller.</li>
	<li>Shave the left hind limb with an electric shaver, disinfect it with 3 alternating scrubs of iso-betadine/alcohol (diluted 1:10) solution and place the rat on a warm pad (20 &#176;C) under a dissecting microscope. Place the rat on lateral decubitus with the leg to be operated on in a superior position. Hold the paw with surgical forceps.</li>
	<li>Using the scissors, make a small lateral incision (20-25 mm) in the skin surrounding the left Achilles tendon, and dissect the fascia using fine scissors to expose the Achilles tendon complex (Figure 2a).</li>
	<li>Remove the plantaris tendon with a pair of scissors (Figure 2b).</li>
	<li>Cut the Achilles tendon transversally 5 mm proximal to its calcaneal insertion and remove a 5-mm long portion using scissors. Do not suture the tendon (Figure 2c, 2d).</li>
	<li>Suture the fascia and then the skin with resorbable yarn doing an overjet suture (continuous suture). Monofilament sutures can be used as well to prevent wicking from the skin into the surgical incision.</li>
	<li>Place the rat under a heating lamp until awake and then place the rat back in a cage (58 x 38 cm) (Nota bene: No immobilization is imposed).</li>
</ol>
3. PRP Preparation15
<ol>
	<li>Anesthetize the donor rats with xylazine (10 mg/kg of body weight) and ketamine (80 mg/kg of body weight) by intraperitoneal injection. The PRP injection is carried out 2 h postoperatively without any re-anesthetic.</li>
	<li>Inject 50 &#181;L of buprenorphine subcutaneously as a painkiller.</li>
	<li>Collect whole blood (20 mL per rat, final bleeding) by cardiac puncture into sterile tubes containing 3.2% buffered sodium citrate (0.109 M, anticoagulant). Then euthanize the rat by intracardiac injection.</li>
	<li>To obtain PRP, centrifuge the blood for 10 min at 150 x g and room temperature. This initial low speed centrifugation step yields two distinct phases: a lower phase consisting of red blood cells (RBCs) that accounts for about 80-90% of the total volume, and an upper phase consisting of platelet-rich plasma (PRP) (typically 10-20% of the blood sample).</li>
	<li>Gently collect the PRP (upper phase) in a secondary plastic tube using a plastic transfer pipette. As the interface between PRP and red blood cells is very loose, do not pipette too close to the interface. When handled properly, contaminating RBCs in PRP should be below 0.05 x 103 cells/&#181;L. Discard the remaining lower phase and the primary blood collection tube.</li>
	<li>Determine the volume of collected PRP and measure the platelet count on a hematology analyzer. These values are required for calculations to adjust platelet concentration in the next step. A blood cell count is also useful to evaluate potential contamination with RBCs and WBCs. Platelet concentration in PRP at this stage is typically 1 &#8211; 1.5 x106/&#181;L.</li>
	<li>Perform a second centrifugation of the PRP for 10 min at 1,000 x g and room temperature. This high-speed centrifugation step yields a loose platelet pellet and a supernatant consisting of autologous platelet-poor plasma (PPP). Using the values determined in step 3.6, calculate the volume of supernatant to be discarded to concentrate the PRP and reach a final concentration of 2.5 x106/&#181;L.</li>
	<li>Gently collect the supernatant (PPP) in a secondary plastic tube using a plastic transfer pipette, leaving about two thirds of the final volume calculated in step 3.7. As the pellet is loose, a significant quantity of platelets is lost when PPP is discarded, hence potentially reducing the final desired concentration.</li>
	<li>Carefully resuspend the platelet pellet with the remaining supernatant by gentle repeated pipetting. Measure the platelet count on this concentrated PRP. If required, add the appropriate volume of autologous PPP to reach the final target concentration. 
	NOTE: Use the PRP within 3 h of preparation.</li>
	<li>Add 50 &#181;L of CaCl2 (11 mEq 10 mL) per mL of PRP to activate the platelets.</li>
	<li>Inject 50 &#181;L of fresh PRP or saline solution with a 21G needle directly into the sutured operation site about 1 h after preparation. During this time, keep PRP at room temperature.</li>
	<li>Monitor the functional recovery of the rats closely during the days after surgery and before the removal of the Achilles tendon.</li>
</ol>
4. Removal of Achilles tendon and Biomechanical Testing16
<ol>
	<li>5, 15, or 30 days later5, weigh the rat. Then, anesthetize with xylazine (10 mg/kg of body weight) and ketamine (80 mg/kg of body weight) by intraperitoneal injection.</li>
	<li>Inject 50 &#181;L of buprenorphine subcutaneously as a painkiller. Remove the tendon before sacrificing the animal to keep the physiological conditions for as long as possible.</li>
	<li>Shave the left hind limb and place the rat under a dissecting microscope. Place the rat on lateral decubitus with the leg to be operated on in a superior position. Hold the paw with fingers.</li>
	<li>Make a small incision (10 mm) in the previous operation site and extend until the triceps suralis is exposed.</li>
	<li>To remove the healing Achilles tendon, cut the calcaneal bone transversally 5-10 mm distal to its Achilles tendon attachment. Then dissect a part of the triceps suralis, which is attached to the Achilles tendon (Figure 3b). The muscle part has to be big enough to fit in the cryo-jaw (Figure 3c-d). Place the sample immediately into the cryo-jaw using forceps (Figure 3e).</li>
	<li>After removing the muscle-tendon-bone complex, euthanize the rat by intracardiac injection with Nembutal (200 mg/kg). Confirm death by assessing the absence of heart rhythm and respiration for 15 min.</li>
	<li>Place the muscle unit into the upper jaw (Figure 3e), close it and place it vertically into a universal testing machine (106.2 kN, Figure 3a). Then fix the bone between the lower clamps of the machine (Figure 3f).</li>
	<li>Add liquid nitrogen into both of the upper jaw basins to freeze the muscle, so that it is an order of magnitude stiffer than the tendon and does not deform during the tensile test.</li>
	<li>When the freezing zone reaches the metal clamp border, start the tensile test, so the tendon will preserve its structure. Set the displacement rate of the machine at a constant speed of 1 mm/s until rupture. Record the ultimate tensile strength (UTS) given in Newtons (N) on a computer.</li>
	<li>To calculate the cross-sectional area, place two cameras perpendicular to each other, forming an elliptical shape, and take pictures.</li>
	<li>To account for the difference in the cross-sectional area of the healing tendons, normalize the UTS to a unit area (N per square millimeter), which represents the mechanical stress experienced by the tissue.</li>
</ol>
5. Histological Analysis
NOTE: 15 tendons of each group underwent histological analysis.
<ol>
	<li>After its removal, submerge the Achilles tendon immediately in 4% paraformaldehyde to preserve its structure (1 mm of tissue is fixed per hour, so the time of fixation varies depending on the size of the tendon).</li>
	<li>Replace the paraformaldehyde by 70% ethanol, and leave the sample for at least one night in this solution.</li>
	<li>Place the tendon in a little plastic container with holes and place that container in a fresh 70% ethanol solution for 1 h.</li>
	<li>Place the container in 95% ethanol for 1 h. Repeat.</li>
	<li>Place the container in 100% ethanol for 1 h. Repeat.</li>
	<li>Place the container in 100% xylene for 1 h. Repeat.</li>
	<li>Now place the plastic container, which contains the tendon, in liquid paraffin (56 &#176;C) and leave it overnight.</li>
	<li>Using an embedding station (equipped with molten wax, a hot plate, and a cold plate), choose a metallic mold that best fits the tissue sample, and fill it with a little liquid paraffin so that the base is covered. 
	NOTE: Orientate the sample to obtain vertical sections at the microtome.</li>
	<li>When the sample is correctly positioned, fill up the mold with liquid paraffin and place it onto the refrigerating machine. Let it cool down for at least 15 min.</li>
	<li>Remove from the paraffin block and place it on ice for at least 30 min.</li>
	<li>Prepare several clean glass slides and put some droplets of deionized water on them.</li>
	<li>At the microtome, cut the blocks in sections of 5 &#181;m (only use sections from the middle part of the tendon) and place the section on a slide.</li>
	<li>Place the slides in a heating oven at 65 &#176;C for several min, so that the paraffin just starts to melt to bond the tissue to the glass.</li>
	<li>Place the slides containing paraffin sections in a slice holder.</li>
	<li>Now deparaffinize and rehydrate the tissue by placing it in the following successive baths: 
	8 min in Xylene (Bath 1) 
	4 min in Xylene (Bath 2) 
	2 min in 100% ethanol 
	2 min in 95% ethanol 
	2 min in 70% ethanol 
	2 min in deionized water</li>
	<li>For Hematoxylin-Eosin staining, place the tissue in the following solutions.
	<ol>
		<li>Immerse the tissue for 8 min in hematoxylin solution (1 g hematoxylin monohydrate, 0.2 g KIO3, 50 g AIK (SO4)2.12H2O; 200 mL glycerin; adjust to 1 L with distilled water).</li>
		<li>Immerse the tissue for 8 min under running tap water.</li>
		<li>Immerse the tissue for 30 s in eosin solution (0.25% eosin in 100 mL of distilled water containing 200 &#181;L of undiluted acetic acid).</li>
		<li>Immerse the tissue for 2 min under running tap water.</li>
	</ol>
	</li>
	<li>For the Masson&#39;s Trichrome Staining, follow these steps.
	<ol>
		<li>After rehydration, place the sections in a container with iron alum (0.5 g (NH4)Fe(SO4)2.12H2O in 10 mL H2O) and close the container. Heat it up for 3 min at 280 W in a microwave.</li>
		<li>Let it cool down for a minute and then rinse it with deionized water.</li>
		<li>Place the sections in a closed container with Regaud&#39;s hematoxylin solution (0.5 g hematoxylin dissolved in 50 mL H2O at 50 &#176;C, then add 5 mL 95% ethanol and 5 mL glycerin), heat it up for 90 s at 280 W.</li>
		<li>Rinse it with deionized water.</li>
		<li>Place the sections in a picric acid solution (at saturation in methanol) for 3 min, rinse for 5 min under running tap water and then place it in deionized water for a few seconds.</li>
		<li>Now place the sections in acid fuchsin (0.5 g in 50 mL H2O containing 0.25 mL of undiluted acetic acid) for 5 s and rinse under running tap water until the dye disappears.</li>
		<li>Incubate the sections in phosphomolybdic acid solution (0.5 g in 50 mL H2O) for 5 min, place it for a few seconds in deionized water.</li>
		<li>Place the sections in light green (0.5 g in 50 mL H2O containing 0.25 mL of undiluted acetic acid) solution for another 5 min and place it under running tap water until the dye disappears.</li>
	</ol>
	</li>
	<li>Now dehydrate the tissue again.
	<ol>
		<li>Immerse the section for 1 min in 70% ethanol.</li>
		<li>Immerse the section for 1 min in 95% ethanol.</li>
		<li>Immerse the section for 1 min in 100% ethanol.</li>
		<li>Immerse the section for 1 min in Xylene (Bath 1).</li>
		<li>Immerse the section for 1 min in Xylene (Bath 2).</li>
		<li>Remove the sections without removing too much xylene and put a drop of mounting medium on the sample.</li>
		<li>Put a coverslip on top (without provoking any bubbles) and let it harden for at least 2 h.</li>
		<li>Hydrolyze 5 histological sections (each of a different tendon) of each group in 6 N HCl for 3 h and determine the concentration of collagen by measuring hydroxyproline as an index in 5 &#181;m unstained sections.</li>
	</ol>
	</li>
	<li>Normalize the results by the section area using the manufacturer&#39;s software.</li>
	<li>Stain some of the sections with Light Green Stain to visualize and quantify fibrillar collagen by computer assisted analysis.</li>
	<li>Scan the sections and convert the pictures in nuances of grey using the IrfanView software. Use the software for semi-quantification (e.g., Quantity One).</li>
</ol>
6. Molecular Evaluation
<ol>
	<li>After tendon rupture, which occurs during mechanical testing, take the samples, snap freeze them in liquid nitrogen, and store them at -80 &#176;C (PRP and saline solution)17.</li>
	<li>Use a commercial Total RNA kit to isolate total RNA17.</li>
	<li>Measure the expression of collagen (Col I and Col III), Matrix Metalloproteinases (MMP-2, MMP-3 and MMP-9), and tenomodulin (TNMD) by RT-PCR17,18.</li>
	<li>Normalize the expression levels of mRNA to the levels of 28S19.</li>
</ol>

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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>

No conflicts of interest declared.

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&amp;eacute;tamine 1000 CEVA)</td><td>CEVA Sant&amp;eacute; Animale</td><td>none</td><td>anesthetic</td></tr><tr><td>Buprenorphin (Veterg&amp;eacute;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 &amp;amp; Johnson</td><td></td><td></td></tr><tr><td>Nembutal</td><td>CEVA Sant&amp;eacute; 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></tbody></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 ...

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Research

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Medicine

9.2K Views.  University of Liège. Dit protocol beschrijft het evaluatieproces van genezing van pezen bij ratten die zijn ingespoten met allogene platelet rich plasma (PRP) of zoutoplossing na het verwijderen van deel van de achillespees. De voortgang van de genezing van de pees wordt geëvalueerd op verschillende tijdstippen met behulp van verschillende types van analyses.

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

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Bioengineering

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Preparing Activated Platelet-Rich Plasma for Culturing Human Adipose-Derived Stem Cells

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of cultured cells, implying a possible use in regenerative medicine. Here, a double-spin method was used to prepare PRP from whole blood. PRP was further activated by autologous thrombin. The platelet count was measured in the activated PRP and the proliferation-stimulating effect in human adipose-derived stem cells (hASCs) was examined. The resulting platelet count was 11.5-times higher in PRP than in whole blood plasma. The proliferation of hASCs was markedly enhanced by incubation with 1% PRP. The described method can be used to reproducibly prepare PRP with a high concentration of platelets. PRP prepared by this method markedly promotes proliferation of hASCs.

In this report, we describe a double-spin method for preparing activated platelet-rich plasma (PRP). Using autologous thrombin, human adipose-derived stem cells (hASCs) were cultured. Activated PRP was shown to promote proliferation of hASCs.

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of ...

Developmental Biology

A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found at the end of all long bones in children that is responsible for longitudinal bone growth. Once damaged, cartilage tissue within the growth plate can undergo premature ossification and lead to unwanted bony repair tissue, which forms a "bony bar." In some cases, this bony bar can result in bone growth deformities, such as angular deformities, or it can completely halt longitudinal bone growth. There is currently no clinical treatment that can fully repair an injured growth plate. Using an animal model of growth plate injury to better understand the mechanisms underlying bony bar formation and to identify ways to inhibit it is a great opportunity to develop better treatments for growth plate injuries. This protocol describes how to disrupt the rat proximal tibial growth plate using a drill-hole defect. This small animal model reliably produces a bony bar and can result in growth deformities similar to those seen in children. This model allows for investigation into the molecular mechanisms of bony bar formation and serves as a means to test potential treatment options for growth plate injuries.

The growth plate is a cartilaginous region in children's long bones where longitudinal growth occurs. When injured, bony tissue can form and impair growth. We describe a rat model of growth plate injury that leads to bony repair tissue, allowing the study of repair mechanisms and growth plate regeneration strategies.

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited effect on the repair of cartilage and on bone regeneration. Platelet-rich plasma (PRP) is an autologous bioactive substance that can repair cartilage injury and accelerate bone regeneration effectively. However, reporting of PRP preparation protocols in clinical studies is highly inconsistent, with the majority of studies providing insufficient information to allow the protocol to be reproduced. We describe a repeatable method of preparing PRP visually, the treatment of KOA using PRP intra-articular injection, and methods of evaluating the outcome. PRP was prepared using manual double centrifugation. The PRP layer was extracted from peripheral blood and used for knee joint cavity injection. Evaluations included assessments of blood platelet concentrations and clinical outcomes. Preparation of PRP by manual centrifugation requires less apparatus and is less costly than plasma filtration or centrifugation using equipment. The centrifugation time of our double centrifugation method was 6 and 5 minutes for the respective centrifugations at forces of 800 and 1400 x g, respectively, to allow for the consistent preparation of standardized PRP. However, a manual method is susceptible to operator error, and PRP batch preparation is not available. Intra-articular injection of PRP proved to be an effective treatment for knee osteoarthritis. The entire treatment procedure took less than 30 minutes, the blood platelet concentration of PRP could be standardized, and treatment was proven to be effective when evaluated by follow-up.

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection, including preparation, procedures, and evaluation.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...

Understanding Tendon Impingement &#8211; Murine Explant Model for Studying Mechanobiology

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

Author Spotlight: Unraveling the Mechanobiology of Tendon Impingement &#8211; A Multiaxial Murine Hind Limb Explant Model 

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a ...

Cyclic Mechanical Loading for Inducing Tendinopathy in Rat Achilles Tendon

A Passive Ankle Dorsiflexion Testing System for an In Vivo Model of Overuse-induced Tendinopathy

Tendinopathy is a chronic tendon condition that results in pain and loss of function and is caused by repeated overload of the tendon and limited recovery time. This protocol describes a testing system that cyclically applies mechanical loads via passive dorsiflexion to the rat Achilles tendon. The custom-written code consists of pre- and post-cyclic loading measurements to assess the effects of the loading protocol along with the feedback control-based cyclic fatigue loading regimen. 
We used 25 Sprague-Dawley rats for this study, with 5 rats per group receiving either 500, 1,000, 2,000, 3,600, or 7,200 cycles of fatigue loads. The percentage differences between the pre- and post-cyclic loading measurements of the hysteresis, peak stress, and loading and unloading moduli were calculated. The results demonstrate that the system can induce varying degrees of damage to the Achilles tendon based on the number of loads applied. This system offers an innovative approach to apply quantified and physiological varying degrees of cyclic loads to the Achilles tendon for an in vivo model of fatigue-induced overuse tendon injury.

Author Spotlight: Integrating Mechanical and Biological Analysis in Tendinopathy Research 

This protocol presents a testing system used to induce quantifiable and controlled fatigue injuries in a rat Achilles tendon for an in-vivo model of overuse-induced tendinopathy. The procedure consists of securing the rat's ankle to a joint actuator that performs passive ankle dorsiflexion with a custom-written MATLAB script.

Tendinopathy is a chronic tendon condition that results in pain and loss of function and is caused by repeated overload of the tendon and limited recovery ...

Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited self-repairing capacity and, most importantly, no definitive treatments, tendinopathy still influences patients&#8217; life quality negatively. Tendon-derived stem cells (TDSCs), as primary precursor cells of tendon cells, play an essential role in both the development of tendinopathy, and functional and structural restoration after tendinopathy. Thus, a method that can in vitro mimic the in vivo differentiation of TDSCs into tendon cells would be useful. Here, the present protocol describes a method based on a three-dimensional (3D) uniaxial stretching system to stimulate the TDSCs to differentiate into tendon-like tissues. There are seven stages of the present protocol: isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon stem cell construct, assembly of the uniaxial-stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue. The effectiveness was demonstrated by histology. The entire procedure takes less than 3 weeks. To promote extracellular matrix deposition, 4.4 mg/mL ascorbic acid was used in the stimulation culture medium. A separated chamber with a linear motor provides accurate mechanical loading and is portable and easily adjusted, which is applied for the bioreactor. The loading regime in the present protocol was 6% strain, 0.25 Hz, 8 h, followed by 16 h rest for 6 days. This protocol could mimic cell differentiation in the tendon, which is helpful for the investigation of the pathological process of tendinopathy. Moreover, the tendon-like tissue is potentially used to promote tendon healing in tendon injury as an engineered autologous graft. To sum up, the present protocol is simple, economic, reproducible and valid.

A three-dimensional uniaxial mechanical stimulation bioreactor system is an ideal bioreactor for tenogenic-specific differentiation of tendon-derived stem cells and neo-tendon formation.

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

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