Name: Author Spotlight: Unraveling the Mechanobiology of Tendon Impingement &amp;#8211; A Multiaxial Murine Hind Limb Explant Model
Uploaded: 2023-12-08T13:30:00
Description: Watch this Scientific Journal Video about Unraveling the Mechanobiology of Tendon Impingement – A Multiaxial Murine Hind Limb Explant Model at JoVE.com

The authors are grateful for support and assistance provided by Jeff Fox and Vidya Venkatramani of the University of Rochester Center for Musculoskeletal Research&#39;s Histology, Biochemistry, and Molecular Imaging (HBMI) Core, funded in part by P30AR06965. Additionally, the authors would like to thank the Center for Light Microscopy and Nanoscopy (CALMN) at the University of Rochester Medical Center for assistance with multiphoton microscopy. This study was funded by R01 AR070765 and R01 AR070765-04S1, as well as 1R35GM147054 and 1R01AR082349.

All animal work was approved by the University of Rochester Committee on Animal Resources.
1. Preparation of tissue culture media
<ol>
	<li>Culture all explants in Dulbecco&#39;s Modified Eagle Medium (1x DMEM) with 1% v/v penicillin-streptomycin and 200 &#181;M L-ascorbic acid in an incubator at 37 &#176;C and 5% CO2. For the initial 48 h pretreatment, culture each explant in 70 mL of culture media&#160;supplemented with 100 nM dexamethasone74</...

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

The experimental murine hind limb explant platform paired with the tissue culture protocol described in this study provide a suitable model for studying the mechanobiology of impingement-driven fibrocartilage formation at the Achilles tendon insertion. The utility of this explant model is demonstrated by the representative results, which indicate maintenance of cell viability concomitant with significant and spatially heterogeneous change in Toluidine blue staining after 7 days of static impingement. These findings sugge...

A multitude of tendons, including the Achilles tendon and rotator cuff tendons, experience bony impingement due to normal anatomical positioning1,2,3,4. Tendon impingement generates compressive strain directed transversely to the longitudinal fiber axis5,6,7. Regions of tendon impingement demonstrate a unique fibrocartilage phenotype in which shrunken, round cells (fibrochondrocytes) are embedded within a disorganized collagen network with markedly increased glycosaminoglycan (GAG) content2,3,4,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24. Prior studies suggest the disparate mechanical environment produced by tendon impingement sustains this GAG-rich matrix by driving the deposition of large aggregating proteoglycans, most notably aggrecan, although the underlying mechanisms are unclear1,3,12,13,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39. While fibrocartilage is a normal feature in impinged regions of healthy tendons, aberrant proteoglycan metabolism associated with excessive fibrocartilage formation is a hallmark feature of tendinopathy, a common and debilitating disease that disproportionately emerges in chronically impinged tendons1,40,41,42,43,44,45,46,47,48,49. Accordingly, tendon impingement is clinically recognized as an important extrinsic factor driving several of the most common tendinopathies, including rotator cuff disease and insertional Achilles tendinopathy (IAT)50,51,52. Currently, treatment of tendinopathy is inefficient. For example, approximately 47% of patients with IAT require surgical intervention after failed conservative management, with variable postoperative outcomes53,54,55,56. Despite the apparent relationship between impingement and tendinopathy, the mechanobiological mechanisms by which cells in impinged tendon sense and respond to their mechanical environment are poorly described, which obscures understanding of tendinopathy pathogenesis and results in inadequate treatment.
Explant models are useful tools in the study of tendon mechanobiology57,58. As a first step towards understanding the mechanobiology of tendon impingement, several prior studies have explored cellular response following the application of simple uniaxial compression to cells or excised tendon explants27,29,30,31,32,33,34,39. However, cells in vitro lack extracellular and pericellular matrices that facilitate strain transfer, sequester important growth factors and cytokines released by mechanical deformation, and provide substrate for focal adhesion complexes that play a role in mechanotransduction57,59. Additionally, both in vitro and excised explant studies fail to recapitulate the multiaxial mechanical strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region5,6. In the context of the impinged Achilles tendon insertion, this includes surrounding tissues such as the retrocalcaneal bursa and Kager&#39;s fat pad60,61,62,63. Conversely, in vivo models of tendon impingement25,28,36,37,38,64,65,66 allow minimal control over the magnitude and frequency of load applied directly to the tendon, which is a well-recognized limitation of in vivo models for studying tendon mechanobiology57,58,67,68,69,70. Given challenges in measuring tendon strain in vivo, the internal strain environment generated within these models is often poorly characterized.
In this manuscript, we present a custom experimental platform that recreates impingement of the Achilles tendon insertion upon the calcaneus within whole murine hind limb explants that, when paired with this tissue culture protocol, maintains viability over 7 days in explant culture and allows for study of the biologic sequelae of tendon impingement. The platform is built upon a 3D printed polylactic acid (PLA) base that provides the foundation for the attachment of the grips and 3D printed PLA volume reduction insert. The grips are used to clamp the upper leg and knee proximal to the Achilles myotendinous junction with the caudal aspect of the hind limb facing upward, allowing the Achilles tendon to be imaged from above using an ultrasound probe or inverted microscope (Figure 1A). The volume reduction insert&#160;slides along a track on the base and reduces the required volume of tissue culture media. A braided line wrapped around the hind paw is routed out of the platform utilizing the base design and a 3D printed PLA clip. By pulling on the string, the hind paw is dorsiflexed, and the Achilles tendon insertion is impinged against the calcaneus, resulting in elevated transverse compressive strain5,6 (Figure 1A). The platform is contained within an acrylic bath that maintains the hind limb explants submerged in tissue culture media. Securing the taut string to the outside of the bath with adhesive tape maintains ankle dorsiflexion to produce static impingement of the Achilles tendon insertion. CAD files for 3D printed components are provided in multiple formats (Supplementary File 1), allowing import into a range of commercial and free, open-source CAD software for modification to suit experimental needs. If access to 3D printers is not available for fabrication, CAD files can be provided to online 3D printing services that will print and ship the parts at low cost.
Importantly, the triceps surae-Achilles musculotendinous complex spans both the knee and ankle joints71,72,73. Consequently, tensile strain in the Achilles tendon is influenced by knee flexion. Knee extension places the Achilles tendon under tension, whereas knee flexion reduces tension. By first extending the knee and then passively dorsiflexing the ankle, compressive strains at the impinged insertion can be superimposed upon tensile strains. Conversely, by passively dorsiflexing the ankle with the knee flexed, tensile strain is reduced, and compressive strain remains. The current protocol explores three such conditions. 1) For static impingement, the foot is dorsiflexed to &#60; 110&#176; with respect to the tibia to impinge the insertion, with the knee flexed to reduce tension. 2) For the baseline tension group, the ankle is extended above 145&#176; of dorsiflexion with the knee extended, generating predominately tensile strain at the insertion. 3) For the unloaded group, explants are cultured in a Petri dish with the knee and ankle in neutral positions in the absence of externally applied load. The angles referred to above are photographically measured relative to a coordinate system where the foot and tibia are parallel at an angle of 180&#176; and perpendicular at an angle of 90&#176;.
Key steps of the protocol include 1) dissection of hind limb explants and careful removal of the skin and plantaris tendon; 2) explant culture following a 48 h dexamethasone pretreatment; 3) tissue sectioning and histological staining; and 4) color image analysis to assess fibrocartilage formation. Following dissection, each hind limb explant is pretreated for 48 h in culture media&#160;supplemented with dexamethasone74. Contralateral limbs from each mouse are assigned to separate experimental groups for pairwise comparison, which helps control biological variability. After pretreatment, explants are positioned into platforms as described above and cultured for 7 more days (Figure 1B). Additional comparisons are made to a pretreated (day 0) group in which explants are removed immediately following the 48 h pretreatment.
After explant culture, hind limbs are trimmed down, formalin fixed, decalcified and embedded in paraffin. Serial sectioning in sagittal orientation provides visualization of the Achilles tendon from the myotendinous junction to the calcaneal insertion while allowing section depth to be tracked through the entire tendon. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP X-nick labeling (TUNEL) is used to visualize DNA damage secondary to apoptosis and assess viability. Toluidine blue histology and custom color image analysis are performed to quantify changes in GAG staining. Toluidine blue stained tissue sections are then used for SHG imaging to characterize alterations in collage fiber organization (Figure 1B).
The provided representative results suggest altered histological staining of the GAG-rich matrix and disorganization of the extracellular collagen network generated by 7 days of static impingement within the model. This model can be utilized to explore molecular mechanisms underlying impingement-driven fibrocartilaginous change.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absorbent underpads</td>
			<td>VWR</td>
			<td>82020-845</td>
			<td>For benchtop dissection</td>
		</tr>
		<tr>
			<td>Acrylic bath</td>
			<td>Source One</td>
			<td>X001G46CB1</td>
			<td>Contains the explant platform submerged in culture media</td>
		</tr>
		<tr>
			<td>Autoclave bin</td>
			<td>Thermo Scientific</td>
			<td>13-361-20</td>
			<td>Used as secondary containment, holds two platforms</td>
		</tr>
		<tr>
			<td>Base</td>
			<td>-</td>
			<td>-</td>
			<td>3D printed from CAD files provided as Supplementary Files</td>
		</tr>
		<tr>
			<td>Braided line</td>
			<td>KastKing</td>
			<td>30lb test</td>
			<td>Used to wrap around paw and apply ankle dorsiflexion</td>
		</tr>
		<tr>
			<td>Clip</td>
			<td>-</td>
			<td>-</td>
			<td>3D printed from CAD files provided as Supplementary Files</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Fisherbrand</td>
			<td>12-541-034</td>
			<td>Rectangular, No. 2, 50 mm x 24 mm</td>
		</tr>
		<tr>
			<td>Cytoseal XYL</td>
			<td>VWR</td>
			<td>8312-4</td>
			<td>Xylene-based mounting media for coverslipping Toluidine blue stained tissue sections</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>MP Biomedical LLC</td>
			<td>194561</td>
			<td>CAS#50-02-2</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO), anhydrous</td>
			<td>Invitrogen by ThermoFisher</td>
			<td>D12345</td>
			<td>CAS#67-68-5, use to solubilize dexamethasone into concentrated stock solutions</td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch Brand</td>
			<td>34-8724-5195-9</td>
			<td>To attach sandpaper to Grip platens</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (1X DMEM)</td>
			<td>Gibco by ThermoFisher</td>
			<td>11965092</td>
			<td>high glucose, (-) pyruvate, (+) glutamine</td>
		</tr>
		<tr>
			<td>EDTA tetrasodium salt dihydrate</td>
			<td>Thermo Scientific Chemicals</td>
			<td>J15700.A1</td>
			<td>CAS#10378-23-1, used to make 14% EDTA solution for sample decalcifcation</td>
		</tr>
		<tr>
			<td>Ethanol, 200 proof</td>
			<td>Thermo Scientific</td>
			<td>T038181000</td>
			<td>CAS#64-17-5, 1 L supply</td>
		</tr>
		<tr>
			<td>Foam biopsy pads</td>
			<td>Leica</td>
			<td>3801000</td>
			<td>Used with processing cassettes, help hold ankle joints in desired position during fixation and decalcification</td>
		</tr>
		<tr>
			<td>Forceps, #SS Standard Inox</td>
			<td>Dumont</td>
			<td>11203-23</td>
			<td>Straight, smooth, fine tips</td>
		</tr>
		<tr>
			<td>Forceps, Micro-Adson 4.75&#34;</td>
			<td>Fisherbrand</td>
			<td>13-820-073</td>
			<td>Straight, fine tips with serrated teeth</td>
		</tr>
		<tr>
			<td>Garnet Sandpaper, 50-D Grit</td>
			<td>Norton</td>
			<td>M600060 01518</td>
			<td>Or other coarse grit sandpaper</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher Chemical</td>
			<td>A38S-500</td>
			<td>CAS#64-19-7, for adjusting pH of sodium acetate buffer used for Toluidine blue histology, as well as 14% EDTA decalcification solution</td>
		</tr>
		<tr>
			<td>Grips</td>
			<td>ADMET</td>
			<td>GV-100NT-A4</td>
			<td>Stainless steel vice grips, screws and springs described in the protocol are included</td>
		</tr>
		<tr>
			<td>Histobond Adhesive Microscope Slides</td>
			<td>VWR</td>
			<td>16005-108</td>
			<td>Sagittal sections of hind limbs explants reliably adhere to these slides through all staining protocols</td>
		</tr>
		<tr>
			<td>In situ Cell Death Detection Kit, TMR Red</td>
			<td>Roche</td>
			<td>12156792910</td>
			<td>TUNEL assay</td>
		</tr>
		<tr>
			<td>Labeling tape</td>
			<td>Fisherbrand</td>
			<td>15-959</td>
			<td>Or any other labeling tape of preference</td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544-100G</td>
			<td>CAS#50-81-7, for culture media formulation</td>
		</tr>
		<tr>
			<td>Neutral buffered formalin, 10%</td>
			<td>Leica</td>
			<td>3800600</td>
			<td>For sample fixation, 5 gallon supply</td>
		</tr>
		<tr>
			<td>Nunc petri dishes</td>
			<td>Sigma-Aldrich</td>
			<td>P7741-1CS</td>
			<td>100 mm diameter x 25 mm height, maintain explants submerged in 70 mL of culture media as described in protocol</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (100X)</td>
			<td>Gibco by ThermoFisher</td>
			<td>15140122</td>
			<td>Add 5 mL to 500 mL 1X DMEM for 1% v/v (1X) working concentration</td>
		</tr>
		<tr>
			<td>Polylactic acid (PLA) 1.75 mm filament</td>
			<td>Hatchbox</td>
			<td>-</td>
			<td>Choose filament diameter compatible with your 3D printer extruder, in color of choice.</td>
		</tr>
		<tr>
			<td>Processing cassettes</td>
			<td>Leica</td>
			<td>3802631</td>
			<td>For fixation, decalcification and paraffin embedding</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent with DAPI</td>
			<td>Invitrogen by ThermoFisher</td>
			<td>P36931</td>
			<td>Mounting media for coverslipping tissue sections after TUNEL</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Fisher BioReagents</td>
			<td>BP1700-50</td>
			<td>CAS#39450-01-6, used for antigen retrieval in TUNEL protocol</td>
		</tr>
		<tr>
			<td>Scissors, Fine</td>
			<td>FST</td>
			<td>14094-11</td>
			<td>Straight, sharp</td>
		</tr>
		<tr>
			<td>Slide Staining Set, 12-place</td>
			<td>Mercedes Scientific&#160;</td>
			<td>MER 1011</td>
			<td>Rack with 12 stain dishes and slide dippers for Toluidine blue histology</td>
		</tr>
		<tr>
			<td>Sodium acetate, anhydrous</td>
			<td>Thermo Scientific Chemicals</td>
			<td>A1318430</td>
			<td>CAS#127-09-3, used to make buffer for Toluidine blue histology</td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Low Profile Microtome Blades</td>
			<td>VWR</td>
			<td>25608-964</td>
			<td>For paraffin sectioning</td>
		</tr>
		<tr>
			<td>Toluidine Blue O</td>
			<td>Thermo Scientific Chemicals</td>
			<td>348601000</td>
			<td>CAS#92-31-9</td>
		</tr>
		<tr>
			<td>Volume Reduction Insert</td>
			<td>-</td>
			<td>-</td>
			<td>3D printed from CAD files provided as Supplementary Files</td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Leica</td>
			<td>3803665</td>
			<td>4 gallon supply for histological staining</td>
		</tr>
	</tbody>
</table>

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 &amp;#8211; A Multiaxial Murine Hind Limb Explant Model 

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

We study the mechanobiology underlying tendon impingement and the process by which this unique mechanical demand drives localized fiber cartilage formation in health and disease. Here we seek to characterize matrix for modeling relative to spatially heterogeneous patterns of multiaxial mechanical strain, generated by impingement and to identify molecular mechanisms mediating this response. In vitro models for studying impingement mechanobiology have applied simple compression to isolated tendon cells or artificial uniaxial compression to partial and whole tendon explants.Established animal models of tendon impingement, manipulating external source of tendon impingement in vivo most often surgically and explore biology following resuming physical activity. In vitro models present significant limitations as isolated cells lack their three dimensional extracellular environment, crucial to mechano-response. While excised explant models circumvent this limitation, both fail to recreate multiaxial strain patterns generated by impingement in vivo.Conversely, animal models offer limited ability to measure or control internal tissue strains. Our Murine Hind Limb Explant Model for studying impingement mechanobiology maintains cells within their extracellular environment and preserves local anatomy of the impinged achilles tendon insertion in situ, allowing controlled prescription of impingement through passively applied joint motion to recreate multiaxial patterns of tissue strain that are measurable and well-characterized.

Representative images of TUNEL stained tissue sections demonstrate minimal apoptotic nuclei within the body of the Achilles tendon after 7 days of explant culture across experimental groups (Figure 2A). Quantification of these images provides evidence that the tissue culture protocol maintains up to 78% viability on average within the Achilles tendon after 7 days of explant culture across loading conditions (Figure 2B).
Qualitatively,...

Watch this Scientific Journal Video about Unraveling the Mechanobiology of Tendon Impingement – A Multiaxial Murine Hind Limb Explant Model at JoVE.com

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 ...

murine-hind-limb-explant-model-for-studying-mechanobiology-achilles

Research

JoVE Journal

Bioengineering

Explant Dissection to Generate Murine Hind Limb Explant Model to Study Tendon Impingement

To begin, transfer 70 milliliters of pre-warmed Dulbecco's Modified Eagle Medium into a Petri dish in a sterile biosafety cabinet and add dexamethasone at a concentration of 100 nano molar. Assemble the surgical tools required for the dissection, which includes smooth, straight, fine tip forceps. Straight fine tip forceps with serrated teeth and straight sharp fine scissors.To dissect hind limb explants. Locate the hip joint of a euthanized mouse positioned supine on the bench top lined with absorbent under pads. Use fine scissors to create a small incision on the skin covering the proximal and anterior aspects of the upper leg.Then use fingers to pull the incision apart to expand and pinch the exposed upper leg. Carefully pull the skin distally to de-glove the hind limb to the level of the ankle. Insert one scissor blade gently under the skin along the dorsal aspect of the foot.Then make an incision that extends towards the toes and continue pulling the skin distally until it is completely removed. Position the mouse to have a clear view of the Achilles tendon insertion onto the posterior aspect of the calcaneus near the ankle. To remove the plant terrace tendon, insert one tip of the smooth fine tip forceps between the two tendons.Pass the tip medially under the plantaris tendon. Then pull it proximally to tear through the plantaris muscle. Then using fine tip serrated forceps, grasp the detached proximal end of the plantaris tendon and pull it distally to completely remove it.At the hip joint, employ fine scissors to precisely sever through the pelvis, isolating the hind limb. Use the scissors to gently remove the remaining pelvis, revealing the femoral head. In the biosafety cabinet transfer the hind limb explant into the dish containing cell culture media and dexamethasone.Relocate the dish to the incubator and subject it to pre-treatment for 48 hours.

Explant Culture and Platforms Loading to Study the Biologic Sequelae of Tendon Impingement

To begin, preheat an adequate amount of sterile culture media while the 48-hour dexamethasone pretreatment of murine hindlimb explant is about to complete. Once the pretreatment ends, move the pretreated explants to the biosafety cabinet. For the unloaded group, aspirate the pretreatment media and transfer the explants to fresh Petri dishes.Add 70 milliliters of culture media to each dish before returning them to the incubator. To prepare the explant platforms for the baseline tension and static impingement group, cut pieces of sandpaper to match the size of the grip platens. For the static impingement group, cut pieces of braided line and pret tie a loose overhand knot halfway along the length of the line.Tear pieces of aluminum foil to cover each acrylic bath and spray them with 70%ethanol. Transfer the prepared components to the biosafety cabinet. Arrange the components such as acrylic bath, base, volume reduction inserts, clip, and grips for platform assembly.Place platform components inside secondary containers of sufficient volume to contain any potential culture media leaks. Submerge the containers in a solution of at least 10%bleach for a minimum of one hour. Rinse the bleach solutions with tap water.After transferring the rinsed components to the biosafety cabinet, use M3 screws and compression springs to affix the platens to the grips. Securely attach the grips to the base employing an M5 screw and insert the M6 screws until they contact the platens. Enhance the platens'functionality by affixing sandpaper using double-sided tape.Close the grips to facilitate the adhesion of the sandpaper to the platens. To load a platform, fully open the grips and use forceps to carefully position the upper leg and knee between the platens. Close the grips gently to hold the limb in place.Use forceps to grasp the exposed femoral head or foot and adjust the knee flexion angle, while gradually closing the forceps to secure it in place. For the baseline tension group, extend the knee joint while closing the grips. In the case of the static impingement group, flex the knee joint while closing the grips.For the static impingement group, place the overhand knot of the string around the distal paw and tighten it securely. Route the string through a slot in the base underneath the explant and through the clip hole. Position the base into the acrylic bath and fasten the clip to the top edge of the bath.To dorsiflex the foot, pull the string until it reaches at least 110 degrees relative to the tibia. Use a permanent marker to mark the string as it exits the clip. Capture a photograph of the explant in this dorsiflexed position for future quantification of the dorsiflexion angle.Remove the base from the setup and attach the volume reduction insert by sliding it along a track on the base. Place the base back into the acrylic bath and reposition the clip on the top edge. Use the marked string as a guide.Pull it to restore the foot to its original dorsiflexion angle and secure the string to the exterior of the bath using tape to maintain a static dorsiflexed position. For the baseline tension group, once the explant is positioned between the grips, take a photograph to quantify the dorsiflexion angle. Then slide the volume reduction insert onto the base and place it into the acrylic bath.Add 125 milliliters of prewarmed culture media to each platform to submerge the explants. After covering the top of the bath with aluminum foil, place the entire setup into a secondary container. Relocate the secondary container to the incubator and culture for seven days.