Name: Forfatter Spotlight: Optrævling af mekanobiologien af seneimpingement - En multiaksial murin bagbenseksplanteringsmodel
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

Forfatterne er taknemmelige for støtte og hjælp fra Jeff Fox og Vidya Venkatramani fra University of Rochester Center for Muskuloskeletal Research's Histology, Biochemistry, and Molecular Imaging (HBMI) Core, delvist finansieret af P30AR06965. Derudover vil forfatterne gerne takke Center for Light Microscopy and Nanoscopy (CALMN) ved University of Rochester Medical Center for hjælp med multifotonmikroskopi. Denne undersøgelse blev finansieret af R01 AR070765 og R01 AR070765-04S1 samt 1R35GM147054 og 1R01AR082349.

Alt dyrearbejde blev godkendt af University of Rochester Committee on Animal Resources.
1. Fremstilling af vævskulturmedier
<ol><li>Kultur alle eksplanter i Dulbeccos modificerede ørnemedium (1x DMEM) med 1% v / v penicillin-streptomycin og 200 μM L-ascorbinsyre i en inkubator ved 37 ° C og 5% CO2. Til den indledende 48 timers forbehandling dyrkes hver enkelt i 70 ml dyrkningsmedier suppleret med 100 nM dexamethason74. Efter forbehandli...

Forståelse af seneimpingement - Murine Explant Model til at studere mekanobiologi

Den eksperimentelle murine bagbenseksplantatplatform parret med vævskulturprotokollen beskrevet i denne undersøgelse giver en passende model til undersøgelse af mekanobiologien af impingementdrevet fibrocartilagedannelse ved indsættelsen af akillessenen. Nytten af denne explant-model demonstreres af de repræsentative resultater, som indikerer vedligeholdelse af cellelevedygtighed samtidig med signifikant og rumligt heterogen ændring i toluidinblå farvning efter 7 dages statisk impingement. Disse resultater tyder p...

Et væld af sener, herunder akillessenen og rotatormanchetsenerne, oplever knoglepåvirkning på grund af normal anatomisk positionering1,2,3,4. Senimpingement genererer trykbelastning rettet på tværs til den langsgående fiberakse5,6,7. Regioner af senepåvirkning demonstrerer en unik fibrocartilagefænotype, hvor krympede, runde celler (fibrochondrocytter) er indlejret i et uorganiseret kollagennetværk med markant øget glycosaminoglycan (GAG) indhold2,3,4,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24. Tidligere undersøgelser tyder på, at det forskellige mekaniske miljø produceret af senepåvirkning opretholder denne GAG-rige matrix ved at drive aflejringen af store aggregerede proteoglycaner, især aggrecan, selvom de underliggende mekanismer er uklare1,3,12,13,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39. Mens fibrocartilage er et normalt træk i impinged regioner af sunde sener, afvigende proteoglycan metabolisme forbundet med overdreven fibrocartilage dannelse er et kendetegnende træk ved tendinopati, en almindelig og invaliderende sygdom, der uforholdsmæssigt opstår i kronisk impinged sener1,40,41,42,43,44,45,46,47,48,49. Derfor er senepåvirkning klinisk anerkendt som en vigtig ydre faktor, der driver flere af de mest almindelige sendinopatier, herunder rotatormanchetsygdom og insertionel akillessendinopati (IAT)50,51,52. I øjeblikket er behandling af tendinopati ineffektiv. For eksempel kræver ca. 47% af patienterne med IAT kirurgisk indgreb efter mislykket konservativ behandling med variable postoperative resultater53,54,55,56. På trods af det tilsyneladende forhold mellem impingement og tendinopati er de mekanobiologiske mekanismer, hvormed celler i impinged senesans og reagerer på deres mekaniske miljø, dårligt beskrevet, hvilket slører forståelsen af tendinopatipatogenese og resulterer i utilstrækkelig behandling.
Explant modeller er nyttige værktøjer i undersøgelsen af senemekanobiologi57,58. Som et første skridt i retning af at forstå mekanobiologien af senepåvirkning har flere tidligere undersøgelser undersøgt cellulær respons efter anvendelse af simpel uniaxial kompression på celler eller udskåret seneeksplanter 27,29,30,31,32,33,34,39. Imidlertid mangler celler in vitro ekstracellulære og pericellulære matricer, der letter belastningsoverførsel, sekvestrerer vigtige vækstfaktorer og cytokiner frigivet ved mekanisk deformation og tilvejebringer substrat for fokale adhæsionskomplekser, der spiller en rolle i mekanotransduktion57,59. Derudover kan hverken in vitro- og exciserede eksplantationsundersøgelser rekapitulere det multiaksiale mekaniske belastningsmiljø, der genereres af senepåvirkning in vivo, hvilket afhænger af anatomiske træk i det impingerede område 5,6. I forbindelse med indsættelsen af den impingerede akillessene omfatter dette omgivende væv såsom retrocalcaneal bursa og Kagers fedtpude 60,61,62,63. Omvendt tillader in vivo-modeller af senepåvirkning 25,28,36,37,38,64,65,66 minimal kontrol over størrelsen og hyppigheden af belastning, der påføres direkte på senen, hvilket er en velkendt begrænsning af in vivo-modeller til undersøgelse af senemekanobiologi57,58,67,68,69,70. På grund af udfordringer med at måle senebelastning in vivo er det interne belastningsmiljø, der genereres i disse modeller, ofte dårligt karakteriseret.
I dette manuskript præsenterer vi en brugerdefineret eksperimentel platform, der genskaber impingement af akillesseneindsættelsen på calcaneus inden for hele murin bagbenseksplanter, der, når de parres med denne vævskulturprotokol, opretholder levedygtighed over 7 dage i explantagekultur og giver mulighed for undersøgelse af de biologiske følgevirkninger af senepåvirkning. Platformen er bygget på en 3D-printet polymælkesyrebase (PLA), der danner grundlaget for fastgørelsen af grebene og 3D-printet PLA-volumenreduktionsindsats. Grebene bruges til at klemme overbenet og knæet proksimalt til Achilles myotendinøse kryds med det kaudale aspekt af bagbenet opad, så akillessenen kan afbildes ovenfra ved hjælp af en ultralydssonde eller omvendt mikroskop (figur 1A). Volumenreduktionsindsatsen glider langs et spor på basen og reducerer det krævede volumen vævskulturmedier. En flettet linje viklet rundt om bagpoten føres ud af platformen ved hjælp af basisdesignet og et 3D-trykt PLA-klip. Ved at trække i snoren er bagpoten dorsiflexed, og akillesseneindsættelsen er stødt mod calcaneus, hvilket resulterer i forhøjet tværgående trykbelastning 5,6 (figur 1A). Platformen er indeholdt i et akrylbad, der opretholder bagbenet eksplanter nedsænket i vævskulturmedier. Fastgørelse af den stramme streng til ydersiden af badet med tape opretholder ankeldorsiflexion for at producere statisk impingement af akillesseneindsættelsen. CAD-filer til 3D-printede komponenter leveres i flere formater (supplerende fil 1), hvilket giver mulighed for import til en række kommercielle og gratis open source CAD-software til modifikation, der passer til eksperimentelle behov. Hvis adgang til 3D-printere ikke er tilgængelig til fremstilling, kan CAD-filer leveres til online 3D-udskrivningstjenester, der udskriver og sender delene til lave omkostninger.
Det er vigtigt, at triceps surae-Achilles musculotendinous kompleks spænder over både knæ- og ankelled 71,72,73. Derfor påvirkes trækbelastning i akillessenen af knæbøjning. Knæforlængelse placerer akillessenen under spænding, mens knæbøjning reducerer spændinger. Ved først at strække knæet ud og derefter passivt dorsiflexere anklen, kan trykbelastninger ved den impingerede indsættelse overlejres på trækstammer. Omvendt, ved passivt dorsiflexing anklen med knæet bøjet, reduceres trækbelastningen, og trykbelastningen forbliver. Den nuværende protokol undersøger tre sådanne betingelser. 1) Ved statisk påvirkning er foden dorsiflexet til < 110° i forhold til skinnebenet for at ramme indsættelsen, med knæet bøjet for at reducere spændingen. 2) For baseline-spændingsgruppen forlænges anklen over 145 ° dorsiflexion med knæet strakt ud, hvilket genererer overvejende trækbelastning ved indsættelsen. 3) For den ubelastede gruppe dyrkes eksplanter i en petriskål med knæ og ankel i neutrale positioner i fravær af eksternt påført belastning. De ovenfor nævnte vinkler måles fotografisk i forhold til et koordinatsystem, hvor foden og skinnebenet er parallelle i en vinkel på 180° og vinkelret i en vinkel på 90°.
De vigtigste trin i protokollen omfatter 1) dissektion af bagbenseksplanter og omhyggelig fjernelse af hud og planttaris sener; 2) eksplantekultur efter en 48 timers forbehandling af dexamethason; 3) vævssektionering og histologisk farvning; og 4) farvebilledanalyse for at vurdere fibrobruskdannelse. Efter dissektion forbehandles hver eksplantation af bagekstremiteter i 48 timer i dyrkningsmedier suppleret med dexamethason74. Kontralaterale lemmer fra hver mus tildeles separate eksperimentelle grupper til parvis sammenligning, hvilket hjælper med at kontrollere biologisk variabilitet. Efter forbehandling placeres eksplanter i platforme som beskrevet ovenfor og dyrkes i yderligere 7 dage (figur 1B). Yderligere sammenligninger foretages med en forbehandlet (dag 0) gruppe, hvor eksplantater fjernes umiddelbart efter 48 timers forbehandling.
Efter explant-kultur trimmes bagbenene ned, formalin fikseres, afkalkes og indlejres i paraffin. Seriel sektionering i sagittal orientering giver visualisering af akillessenen fra det myotendinøse kryds til calcaneal indsættelse, samtidig med at sektionsdybden kan spores gennem hele senen. Terminal deoxynukleotidyltransferase (TdT)-medieret dUTP X-nick-mærkning (TUNEL) bruges til at visualisere DNA-skade sekundært til apoptose og vurdere levedygtighed. Toluidinblå histologi og brugerdefineret farvebilledanalyse udføres for at kvantificere ændringer i GAG-farvning. Toluidinblå farvede vævssektioner bruges derefter til SHG-billeddannelse til at karakterisere ændringer i collagefiberorganisation (figur 1B).
De fremlagte repræsentative resultater tyder på ændret histologisk farvning af den GAG-rige matrix og disorganisering af det ekstracellulære kollagennetværk genereret af 7 dages statisk impingement i modellen. Denne model kan bruges til at udforske molekylære mekanismer, der ligger til grund for impingement-drevet fibrocartilaginøs ændring.

<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

Senens påvirkning af knoglen genererer et multiaksialt mekanisk belastningsmiljø med markant forhøjet tværgående trykstamme, som fremkalder en lokaliseret fibrocartilagefænotype karakteriseret ved akkumulering af glycosaminoglycan (GAG) -rig matrix og ombygning af kollagennetværket. Mens fibrocartilage er et normalt træk i impinged regioner af sunde sener, overskydende GAG aflejring og disorganisering af kollagen netværk er kendetegnende træk ved tendinopati. Følgelig er impingement klinisk anerkendt som en vigtig ydre faktor i initiering og progression af tendinopati. Ikke desto mindre er mekanobiologien, der ligger til grund for senepåvirkning, stadig underundersøgt. Tidligere bestræbelser på at belyse det cellulære respons på senepåvirkning har anvendt uniaxial kompression på celler og udskåret seneeksplanter in vitro. Imidlertid mangler isolerede celler et tredimensionelt ekstracellulært miljø, der er afgørende for mekanorespons, og både in vitro og udskårne eksplantatundersøgelser undlader at rekapitulere det multiaksiale belastningsmiljø, der genereres af senepåvirkning in vivo, hvilket afhænger af anatomiske træk i det impingerede område. Desuden mangler in vivo-modeller af senepåvirkning kontrol over det mekaniske belastningsmiljø. For at overvinde disse begrænsninger præsenterer vi en ny murine bagbenseksplanteringsmodel, der er egnet til at studere mekanobiologien af akillessenepåvirkning. Denne model opretholder akillessenen in situ for at bevare lokal anatomi og reproducerer det multiaksiale belastningsmiljø, der genereres ved påvirkning af akillesseneindsættelsen på calcaneus under passivt påført ankeldorsiflexion, mens cellerne bevares i deres oprindelige miljø. Vi beskriver en vævskulturprotokol, der er integreret i denne model, og præsenterer data, der etablerer vedvarende eksplanteringslevedygtighed over 7 dage. De repræsentative resultater viser forbedret histologisk GAG-farvning og nedsat kollagenfiberjustering sekundært til impingement, hvilket tyder på forhøjet fibrocartilagedannelse. Denne model kan let tilpasses til at undersøge forskellige mekaniske belastningsregimer og giver mulighed for manipulation af molekylære veje af interesse for at identificere mekanismer, der medierer fænotypisk ændring i akillessenen som reaktion på impingement.

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.

Forfatter Spotlight: Optrævling af mekanobiologien af seneimpingement - En multiaksial murin bagbenseksplanteringsmodel 

Murine bagben explant model til undersøgelse af mekanobiologi af akillesseneimpingement

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.

Repræsentative billeder af TUNEL farvede vævssektioner viser minimale apoptotiske kerner i kroppen af akillessenen efter 7 dages eksplantatkultur på tværs af eksperimentelle grupper (figur 2A). Kvantificering af disse billeder giver bevis for, at vævskulturprotokollen opretholder op til 78% levedygtighed i gennemsnit inden for akillessenen efter 7 dages eksplantatkultur under belastningsforhold (figur 2B).
Kvalitativt værdsætte...

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

Vi præsenterer en brugerdefineret eksperimentel platform og vævskulturprotokol, der genskaber fibrocartilaginøs forandring drevet af impingement af akillesseneindsættelsen i murine bagekstremiteter med vedvarende cellelevedygtighed, hvilket giver en model, der er egnet til at udforske mekanobiologien af senepåvirkning.

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

Vi studerer mekanobiologien, der ligger til grund for senepåvirkning, og den proces, hvormed denne unikke mekaniske efterspørgsel driver lokaliseret fiberbruskdannelse i sundhed og sygdom. Her søger vi at karakterisere matrix til modellering i forhold til rumligt heterogene mønstre af multiaksial mekanisk stamme, genereret af impingement og at identificere molekylære mekanismer, der formidler dette respons. In vitro modeller til undersøgelse af impingement mekanobiologi har anvendt simpel kompression til isolerede seneceller eller kunstig uniaxial kompression til delvis og hel seneeksplantering.Etablerede dyremodeller for senepåvirkning, manipulering af ekstern kilde til senepåvirkning in vivo oftest kirurgisk og udforsker biologi efter genoptagelse af fysisk aktivitet. In vitro-modeller har betydelige begrænsninger, da isolerede celler mangler deres tredimensionelle ekstracellulære miljø, hvilket er afgørende for mekanorespons. Mens udskårne eksplanteringsmodeller omgår denne begrænsning, undlader begge at genskabe multiaksiale belastningsmønstre genereret af impingement in vivo.Omvendt giver dyremodeller begrænset evne til at måle eller kontrollere indre vævsstammer. Vores Murine Hind Limb Explant Model til undersøgelse af impingement mekanobiologi opretholder celler inden for deres ekstracellulære miljø og bevarer lokal anatomi af den impinged akillesseneindsættelse in situ, hvilket muliggør kontrolleret recept på impingement gennem passivt anvendt ledbevægelse for at genskabe multiaksiale mønstre af vævsstamme, der er målbare og velkarakteriserede.

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

Research

JoVE Journal

Bioengineering

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.

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.

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 dissektion for at generere murin bagben Explant model til undersøgelse af sener impingement

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.