Name: biomechanical testing of murine tendons
Uploaded: 2019-10-15T14:30:00
Description: Watch this Scientific Journal Video about Biomechanical Testing of Murine Tendons at JoVE.com

De studie werd ondersteund door de NIH/NIAMS (R01 AR055580, R01 AR057836).

Dierstudies werden goedgekeurd door het institutioneel Dierenzorg-en gebruiks Comité van Columbia University. Muizen die in deze studie werden gebruikt, waren van een C57BL/6J-achtergrond en werden gekocht bij het Jackson Laboratory (Bar Harbor, ME, USA). Ze werden ondergebracht in pathogeen-vrije barrière omstandigheden en werden voorzien van voedsel en water ad libitum.
1. ontwikkeling van Custom-Fit 3D gedrukte armaturen voor aangrijpend bot
<ol><li>Bone Image Acquisition en 3D Bone mod...

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S., Connizzo, B. K., Soslowsky, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fiber+re-alignment+in+a+neonatal+developmental+mouse+supraspinatus+tendon+model.">Collagen fiber re-alignment in a neonatal developmental mouse supraspinatus tendon model.</a> Annals of Biomedical Engineering. 40, 1102-1110 (2012).</li><li>Cong, G. T., 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=Evaluating+the+role+of+subacromial+impingement+in+rotator+cuff+tendinopathy:+Development+and+analysis+of+a+novel+murine+model.">Evaluating the role of subacromial impingement in rotator cuff tendinopathy: Development and analysis of a novel murine model.</a> Journal of Orthopaedic Research. 36, 2780-2788 (2018).</li><li>Thomopoulos, S., Birman, V., Genin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Interfaces+and+Attachments+in+Biology.">Structural Interfaces and Attachments in Biology.</a> Infection and Immunity. 35, (2013).</li><li>Boivin, G. P., 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=Biomechanical+properties+and+histology+of+db/db+diabetic+mouse+Achilles+tendon.">Biomechanical properties and histology of db/db diabetic mouse Achilles tendon.</a> Muscles, Ligaments and Tendons Journal. 4, 280-284 (2014).</li><li>Ansorge, H. L., Adams, S., Birk, D. E., Soslowsky, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical,+Compositional,+and+Structural+Properties+of+the+Post-natal+Mouse+Achilles+Tendon.">Mechanical, Compositional, and Structural Properties of the Post-natal Mouse Achilles Tendon.</a> Annals of Biomedical Engineering. 39, 1904-1913 (2011).</li><li>Shu, C. C., Smith, M. M., Appleyard, R. C., Little, C. B., Melrose, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achilles+and+tail+tendons+of+perlecan+exon+3+null+heparan+sulphate+deficient+mice+display+surprising+improvement+in+tendon+tensile+properties+and+altered+collagen+fibril+organisation+compared+to+C57BL/6+wild+type+mice.">Achilles and tail tendons of perlecan exon 3 null heparan sulphate deficient mice display surprising improvement in tendon tensile properties and altered collagen fibril organisation compared to C57BL/6 wild type mice.</a> PeerJ. 6, 5120 (2018).</li><li>Probst, A., 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=A+new+clamping+technique+for+biomechanical+testing+of+tendons+in+small+animals.">A new clamping technique for biomechanical testing of tendons in small animals.</a> Journal of Investigative Surgery. 13, 313-318 (2000).</li><li>Talan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+temperature+of+C57BL/6J+mice+with+age.">Body temperature of C57BL/6J mice with age.</a> Experimental Gerontology. 19, 25-29 (1984).</li><li>Newton, M. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+durations+of+immobilization+in+the+rat+result+in+enhanced+mechanical+properties+of+the+healing+supraspinatus+tendon.">Long durations of immobilization in the rat result in enhanced mechanical properties of the healing supraspinatus tendon.</a> Journal of Biomechanical Engineering. 129, 400-404 (2006).</li><li>Freedman, B. R., Sarver, J. J., Buckley, M. R., Voleti, P. B., Soslowsky, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+and+structural+response+of+healing+Achilles+tendon+to+fatigue+loading+following+acute+injury.">Biomechanical and structural response of healing Achilles tendon to fatigue loading following acute injury.</a> Journal of Biomechanics. 47, 2028-2034 (2014).</li><li>Deymier, A. 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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=Functionally+Distinct+Tendons+From+Elastin+Haploinsufficient+Mice+Exhibit+Mild+Stiffening+and+Tendon-Specific+Structural+Alteration.">Functionally Distinct Tendons From Elastin Haploinsufficient Mice Exhibit Mild Stiffening and Tendon-Specific Structural Alteration.</a> Journal of Biomechanical Engineering. 139, 111003 (2017).</li><li>Mikic, B., Bierwert, L., Tsou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achilles+tendon+characterization+in+GDF-7+deficient+mice.">Achilles tendon characterization in GDF-7 deficient mice.</a> Journal of Orthopaedic Research. 24, 831-841 (2006).</li><li>Sikes, K. 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Murine diermodellen worden vaak gebruikt voor het bestuderen van pees aandoeningen, maar karakterisering van hun mechanische eigenschappen is uitdagend en ongewoon in de literatuur. Het doel van dit protocol is het beschrijven van een tijd efficiënte en reproduceerbare methode voor het testen van de trek van muriene pezen. De nieuwe methoden verkort de tijd die nodig is om een monster te testen van uren tot minuten en elimineerde een belangrijk aangrijpend artefact dat een veelvoorkomend probleem was in eerdere methoden...

Pees aandoeningen zijn gebruikelijk en zeer overwegend onder de veroudering, atletische, en actieve populaties1,2,3. In de Verenigde Staten, 16.400.000 bindweefsel letsels worden gerapporteerd elk jaar4 en rekening voor 30% van alle letsel-gerelateerde Arts Office bezoeken3,5,6,7, 8. De meest getroffen sites zijn de rotator cuff, Achilles pees en Patellaire pees9. Hoewel er een verscheidenheid aan niet-operatieve en operatieve behandelingen zijn onderzocht, met inbegrip van anti-inflammatoire geneesmiddelen, revalidatie, en chirurgische reparatie, uitkomsten blijven slecht, met beperkte terugkeer naar functie en hoge percentages van falen5, 6. Deze slechte klinische uitkomsten hebben fundamentele en translationele studies gemotiveerd om de tendinopathie te begrijpen en nieuwe behandelings benaderingen te ontwikkelen.
Trek-biomechanische eigenschappen zijn de primaire kwantitatieve uitkomsten die de functie pees definiëren. Daarom, laboratorium karakterisatie van tendinopathie en werkzaamheid van de behandeling moet een rigoureuze testen van pees treksterkte eigenschappen omvatten. Talrijke studies hebben methoden beschreven om de biomechanische eigenschappen van pezen te bepalen van diermodellen zoals ratten, schapen, honden en konijnen10,11,12. Echter, weinig studies hebben getest de biomechanische eigenschappen van Murine pezen, voornamelijk te wijten aan de moeilijkheden in het aangrijpend van de kleine weefsels voor trekproeven. Zoals Murine modellen hebben tal van voordelen voor mechanistisch bestuderen tendinopathie, met inbegrip van genetische manipulatie, uitgebreide reagens opties, en lage kosten, ontwikkeling van nauwkeurige en efficiënte methoden voor biomechanisch testen van muriene weefsels is nodig.
Om de mechanische eigenschappen van pezen goed te testen, moet het weefsel effectief worden vastgedraaid, zonder uitglijden of artifeitelijke scheuren in de grip-interface of breken van de groei plaat. In veel gevallen, met name voor korte pezen, wordt het bot aan de ene kant vastgedraaid en wordt de pees aan het andere uiteinde vastgedraaid. Botten worden meestal vastgezet door ze in te sluiten in materialen zoals epoxy hars13 en polymethylmethacrylaat14,15. Pezen worden vaak geplaatst tussen twee lagen schuurpapier, gelijmd met cyanoacrylaat, en beveiligd met behulp van compressie klemmen (als de dwarsdoorsnede plat is) of in een bevroren medium (als de dwarsdoorsnede groot is)15,16,17 . Deze methoden zijn toegepast op biomechanisch testen van muriene pezen, maar uitdagingen ontstaan door de geringe omvang van de specimens en de conformiteit van de groei plaat, die nooit18verbeert. Bijvoorbeeld, de diameter van de Murine Opperarm hoofd is slechts een paar millimeter, waardoor het aangrijpend maken van het bot moeilijk. Specifiek, trekproeven van Murine supraspinatus pees-to-Bone monsters vaak resulteert in falen op de groei plaat in plaats van in de pees of op de pees enthesis. Op dezelfde manier is biomechanisch testen van de Achilles pees een uitdaging. Hoewel de Achilles pees groter is dan andere muriene pezen, is de calcaneus klein, waardoor het moeilijk is om dit bot aan te grijpen. Het bot kan worden verwijderd, gevolgd door het aangrijpend van de twee pees uiteinden; Dit verzet zich echter tegen het testen van de Tendon-naar-bot-bevestiging. Andere groepen melden aangrijpend de calcaneus Bone met behulp van op maat gemaakte armaturen19,20, verankering door klemmen21, fixeren in zelfuithardende kunststof cement22 of met behulp van een conische vorm sleuf22, maar deze eerdere methoden blijven beperkt door de lage reproduceerbaarheid, hoge grijp insufficiëntie en vervelende voorbereidings vereisten.
Het doel van de huidige studie was het ontwikkelen van een nauwkeurige en efficiënte methode voor het testen van Trek-biomechanisch onderzoek van muriene pezen, gericht op de supraspinatus en Achilles pezen als voorbeelden. Met behulp van een combinatie van 3D-reconstructies van native Bone anatomie, solid modeling, en additieve productie, werd een nieuwe methode ontwikkeld om grip te hebben op de botten. Deze armaturen effectief beveiligd de botten, voorkomen groei plaat falen, verminderde preparaat voorbereidingstijd, en verhoogde testen reproduceerbaarheid. De nieuwe methode is gemakkelijk aanpasbaar om andere muriene pezen te testen, evenals pezen bij ratten en andere dieren.

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	<tbody>
		<tr >
			<td >Agarose</td>
			<td >Fisher Scientific</td>
			<td >BP160-100</td>
			<td >Dissovle 1g in 100 ml ultrapure water to make 1% agarose&#160;</td>
		</tr>
		<tr >
			<td >Bruker microCT&#160;</td>
			<td >Bruker BioSpin Corp</td>
			<td >Skyscan 1272&#160;</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >ElectroForce&#160;</td>
			<td >TA Instruments</td>
			<td >3200</td>
			<td>Testing platform</td>
		</tr>
		<tr >
			<td >Ethanol 200 Proof</td>
			<td >Fisher Scientific</td>
			<td >A4094</td>
			<td>Dilute to 70% and use as suggested in protocol</td>
		</tr>
		<tr >
			<td >Fixture to attach grips</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Kimwipes</td>
			<td >Kimberly-Clark</td>
			<td >&#160;S-8115</td>
			<td>As suggested in protocol</td>
		</tr>
		<tr >
			<td >MicroCT CT-Analyser (Ctan)</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >MilliQ water (Ultrapure water)</td>
			<td>Millipore Sigma</td>
			<td>QGARD00R1 (or related purifier)</td>
			<td>100 ml&#160;</td>
		</tr>
		<tr >
			<td >Meshmixer</td>
			<td >Autodesk</td>
			<td >http://www.meshmixer.com/</td>
			<td >Free engineering software used by authors to refine mesh</td>
		</tr>
		<tr >
			<td >Objet EDEN 260VS&#160;</td>
			<td >Stratasys LTD</td>
			<td ></td>
			<td>Precision Prototyping</td>
		</tr>
		<tr >
			<td >Objet Studio</td>
			<td >Stratasys LTD</td>
			<td ></td>
			<td>Used by authors with 3D printer</td>
		</tr>
		<tr >
			<td >PBS - Phosphate-Buffered Saline</td>
			<td >ThermoFisher Scientific</td>
			<td >10010031</td>
			<td >2.5 L of 10% PBS&#160;</td>
		</tr>
		<tr >
			<td >S&#38;T Forceps</td>
			<td >Fine Science Tools</td>
			<td >00108-11</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Scalpel Blade - #11</td>
			<td >Fine Science Tools</td>
			<td >10011-00</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Scalpel Handle - #3</td>
			<td >Fine Science Tools</td>
			<td >10003-12</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >SkyScan 1272</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >Skyscan CT-Vox</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >SkyScan NRecon</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >SolidWorks CAD</td>
			<td >Dassault Syst&#232;mes</td>
			<td >SolidWorks Research Subsription</td>
			<td>Solid modeling computer-aided design used by authors</td>
		</tr>
		<tr >
			<td >SuperGlue</td>
			<td >Loctite</td>
			<td >234790</td>
			<td>As suggested in protocol</td>
		</tr>
		<tr >
			<td >Testing bath</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Thin film grips&#160;</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >VeroWhitePlus</td>
			<td >Stratasys LTD</td>
			<td >NA</td>
			<td>3D printing material used by authors</td>
		</tr>
		<tr >
			<td >WinTest&#160;</td>
			<td >WinTest Software</td>
			<td ></td>
			<td>Used by authors to collect data</td>
		</tr>
	</tbody>
</table>

biomechanical testing of murine tendons

Pees aandoeningen zijn gebruikelijk, beïnvloeden mensen van alle leeftijden, en zijn vaak debilitating. Standaard behandelingen, zoals ontstekingsremmende geneesmiddelen, revalidatie en chirurgische herstelling, falen vaak. Om de functie pees te definiëren en de werkzaamheid van nieuwe behandelingen aan te tonen, moeten de mechanische eigenschappen van pezen uit diermodellen nauwkeurig worden bepaald. Murine diermodellen worden nu veel gebruikt voor het bestuderen van pees aandoeningen en het evalueren van nieuwe behandelingen voor Tendinopathieën; echter, het bepalen van de mechanische eigenschappen van muis pezen is uitdagend. In deze studie, een nieuw systeem werd ontwikkeld voor pees mechanische testen met 3D-gedrukte armaturen die exact overeenkomen met de anatomieën van de opperarmbeen en calcaneus mechanisch testen supraspinatus pezen en Achilles pezen, respectievelijk. Deze armaturen werden ontwikkeld met behulp van 3D reconstructies van inheemse bot anatomie, solid modeling, en additieve productie. De nieuwe aanpak elimineerde artifeitelijke aangrijpende mislukkingen (bijv. falen bij het falen van de groei plaat in plaats van in de pees), verminderde totale testtijd en verhoogde reproduceerbaarheid. Bovendien is deze nieuwe methode gemakkelijk aanpasbaar voor het testen van andere muriene pezen en pezen van andere dieren.

3D-gedrukte armaturen werden gebruikt voor het testen van 8 weken oude Murine supraspinatus en Achilles pezen. Alle mechanisch geteste monsters mislukten bij de enthesis, zoals gekarakteriseerd door microCT-scans, visuele inspectie en video analyse na trekproeven. Een een-op-een vergelijking van de vorige en huidige methoden voor supraspinatus pees testen in ons laboratorium is weergegeven in Figuur 3. In de vorige methode28,29,...

Watch this Scientific Journal Video about Biomechanical Testing of Murine Tendons at JoVE.com

Biomechanical Testing of Murine Tendons

Het protocol beschrijft efficiënte en reproduceerbare Trek-biomechanische testmethoden voor muriene pezen door het gebruik van Custom-Fit 3D-gedrukte armaturen.

Biomechanische beproeving van Muriene pezen

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, ...

Het protocol beschrijft een efficiënte en reproduceerbare methode voor biomechanische testen van muispezen door middel van herbruikbare custom-fit 3D-geprinte armaturen. De nieuwe methoden verminderen de tijd die nodig is om het monster te testen van uren tot minuten en elimineren een belangrijk aangrijpend artefact dat een veelvoorkomend probleem was in eerdere methoden. Deze methode is niet beperkt tot de supraspinatus en achillespezen.Het kan gemakkelijk worden aangepast aan het testen van andere muispezen en pezen van grotere dieren. Om de reproduceerbaarheid en efficiëntie van de aanpak te garanderen, kunnen meerdere cycli van ontwerpprototyping en validatie nodig zijn voor armaturen die hier niet worden beschreven. Om te beginnen voert u een microgecomputte tomografiescan uit van het hele bot in de agarosegel.Nadat u bent reconstructie in dwarsdoorsnedeafbeeldingen, selecteert u de opdracht Bestand openen om de bestandsgegevensset te openen. Open de voorkeuren van het dialoogvenster en selecteer het tabblad Geavanceerd. Gebruik het adaptieve rendering-algoritme om de 3D-modellen te construeren die vloeiender oppervlaktedetails bieden.Gebruik 10 als de plaatsparameter die de afstand in pixels tot het aangrenzende punt definieert dat wordt gebruikt voor het zoeken naar de objectrand. Minimaliseer de tolerantie tot 0.1 om de bestandsgrootte te verkleinen. Als u het interessevolume wilt opgeven, selecteert u handmatig twee afbeeldingen die u wilt instellen als de boven- en onderkant van het geselecteerde VOI-bereik en gaat u vervolgens naar de tweede pagina, Regio van belang.Selecteer handmatig het interessegebied op één doorsnedeafbeelding. Herhaal het selecteren van de ROI voor elke 10 tot 15 dwarsdoorsnedeafbeeldingen. Ga vervolgens naar de derde pagina, Binaire selectie.Klik in het menu Histogram op Van gegevensset om de histogramverdeling van de helderheid van alle afbeeldingen van de gegevensset weer te geven. Klik ook in het menu Histogram op het menu Een 3D-modelbestand maken. Sla een 3D-model van het bot op in STL-bestandsindeling.Importeer in de Meshmixer-software mesh en selecteer Alles om te bewerken. Kies Produceren uit de bewerken toolset en selecteer vervolgens Driehoeksbudget in de gereedschapsset Doel verminderen. Verminder het aantal tricount en accepteer wijzigingen.Resave de nieuw gereduceerde bestand in STL formaat door te kiezen export As.To het ontwerp van een supraspinatus pees humeral bot, gebruik maken van een solide modellering computer-aided ontwerpprogramma om een custom-fit model van humerus-aangrijpende armatuur te creëren. Open het STL-formaatbestand van het opperarmbeen in een solide modelleringsprogramma en sla op als onderdeelbestand in SLDPRT-indeling. Open vervolgens het bestand Deel om handmatig drie anatomisch relevante vlakken te maken, bijvoorbeeld sagittale, coronale en dwarse.Klik op Bestand Nieuw om het onderdeel vaste blokcomponent te maken. Klik op Bestand Nieuw om een assemblagemodel te maken met twee componenten, het vaste blok en het rechter- of linkerhumerusbot. Definieer de oriëntatie van het bot in het blok om ervoor te zorgen dat de hoek tussen de pees en het bot 180 graden is.Zorg ervoor dat het volledige botvolume in het blok past. Selecteer in het venster Montage het blok en klik op Component bewerken op de werkbalk Assemblage. Klik op Invoegen, Onderdelen, Holte.Selecteer Uniform Schalen en voer 0% in als de waarde die u in alle richtingen moet schalen. Onderdruk het botdeel en sla de montage als onderdeel op. Open de cilinder met spouwgedeelte.Maak een schets, zorg ervoor dat u slechts 0,5 millimeter boven de humerale kop laat. Selecteer Geëxtrudeerd knippen in Functies. Snijd de assemblage langs het sagittale vlak om twee symmetrische componenten te creëren die het bot voor- en lateraal passen.Snijd het montagedeel langs het sagittale vlak om twee symmetrische componenten te creëren die het bot voor- en posteriorly passen. Het is van cruciaal belang om ervoor te zorgen dat de humerale kop om te voorkomen dat de groeiplaat falen en om een strakke klaring die het uitschakelen van het opperarmbeen van het model tijdens het testen voorkomt te definiëren. Ga verder zoals beschreven in het manuscript om de achterste component en voorste component af te maken.Sla beide componenten op als afzonderlijke onderdeelbestanden. Druk nu op Patroon invoegen, spiegelen en selecteer Spiegel om 3D-spiegelmodellen te maken voor elk onderdeel van de armatuur voor de tegenovergestelde ledemaat. Selecteer het voorvlak als een spiegel vlak vlakte.Selecteer het onderdeel als het lichaam om te spiegelen. Selecteer het spiegelvlak en maak een schets die alle materialen bevat. Selecteer Uit Functies extruded cut om het originele onderdeel te verwijderen en alleen gespiegeld onderdeel te behouden.Klik op de onderkant van het onderdeel en gebruik als een vliegtuig om een schets te maken. Klik op Sketch Tekst en voeg L of R.This voegt een ets op de onderkant van de armaturen om onderscheid te maken tussen de linker-en rechterkant. Sla alle armatuuronderdelen op in het standaardbestandsformaat van STL ter voorbereiding op 3D-printen.Om de supraspinatus spierpees, opperarmbeenmonster te ontleden, positioneer de geëuthanaseerde muis eerst in een gevoelige positie en maak een incisie in de huid van boven de elleboog van de voorpoot naar de schouder. Gebruik tangen om de huid voorzichtig te verwijderen met stompe dissectie, zodat het spierstelsel van de schouder zichtbaar is. Verwijder het weefsel rond het opperarmbeen totdat het bot is blootgesteld.Houd het opperarmbeen met tangen vast en verwijder voorzichtig de deltoïde- en trapeziusspieren om de coacromiale boog bloot te leggen. Identificeer het wisselstroomgewricht en scheid het sleutelbeen voorzichtig van de acromion met een scalpelmes. Zorg ervoor dat de supraspinatuspees en de benige hechting niet beschadigd worden, verwijder de spier uit zijn scapulaire bevestiging met behulp van een scalpelblad.Maak de humerale kop los van de glenoid en scheurt de gewrichtscapsule en de infraspinatus subscapularis en teres kleine pezen. Disarticulate het ellebooggewricht om het opperarmbeen te scheiden van de ellepijp en straal. Isoleer het opperarmbeen-supraspinatus peesspiermonster en maak overtollige zachte weefsels op het opperarmbeen en opperarmhoofd schoon.Om de achillespees, het calcaneusbot, te ontleden, positioneer je een geëuthanaseerde muis in een gevoelige positie. Zorg ervoor dat de achillespees en de benige bevestiging niet worden beschadigd, verwijder de huid met stompe dissectie, zodat het spierstelsel rond de enkel- en kniegewrichten wordt blootgesteld. Met behulp van een scalpelblad, beginnend bij de achillespees-calcaneus bevestiging, voorzichtig los de gastrocnemius spier van de proximale bijlagen.Verwijder de calcaneus voorzichtig uit de verschillende aangrenzende botten en isoleer het achillespees-calcaneus-monster en maak overtollige zachte weefsels schoon. Om het dwarsdoorsnedegebied van de pees te bepalen, voegt u het bot ondersteboven in om het monster op te schorten in de cryotube gevuld met agarosegel met het bot in de agarosegel en de pees en spier buiten. Na de scan met behulp van microgecomputeerde tomografie, verwijder voorzichtig de spier uit de pees met behulp van een scalpel mes.Plaats het bot in de 3D-geprinte armatuur en bevestig ze aan de testroosters. Steek en lijm de pees tussen een gevouwen dun tissuepapier en klem deze met dunne filmgrepen. Steek het monster en de grepen in een testbad van PBS bij 37 graden Celsius en voer een mechanische test uit.In deze studie, met behulp van herbruikbare 3D-geprinte armaturen grip bot, monster voorbereidingstijd werd teruggebracht van uren tot minuten. Representatieve belastingsvervormingscurven voor trekproeven van supraspinatuspees met behulp van de huidige methoden elimineerden belangrijke grijperartefacten zoals falen van de groeiplaat. De mechanische eigenschappen van supraspinatus en achillespezen tonen een significant effect van seks op basis van ongepaarde T-tests met een P-waarde van minder dan 0,05.Micro CT met succes gemeten de dwarsdoorsnede gebied langs de lengte van supraspinatus pees langs de lengte van de achillespees. Het is belangrijk om te onthouden dat elke anatomische site specifieke ontwerpcriteria heeft die nodig zijn voor effectieve grip. Daarom moet het ontwerp voor elke armatuur dienovereenkomstig worden aangepast.Naast trekproeven tot falen, kunnen deze armaturen worden gebruikt voor cyclische belastingtests, die informatie geven over peesmoeheid en viscoeleastische eigenschappen. Het karakteriseren van de mechanische eigenschappen van murinepezen is ongewoon in de literatuur vanwege een aantal redenen, waaronder de moeilijkheid bij het grijpen van deze kleine weefsels, vervelende en tijdrovende methoden voor het bereiden van specimens en groeiplaatfracturen. Dit protocol presenteert een tijd-efficiënte en reproduceerbare methode voor het testen van muispezen die artefactuele grip mislukkingen heeft geëlimineerd en heeft verdrievoudigd het aantal exemplaren die kunnen worden getest in een dag.

Research

JoVE Journal

Bioengineering

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Gericht Cellular Self-Assembly to Cell-Derived weefsel Rings fabriceren voor Biomechanische Analyse en Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Bepaling van de Transport Rate van xenobiotica en nanomaterialen via de placenta met de<em&gt; Ex vivo</em&gt; Human placentaperfusie Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Biomechanische karakterisering van menselijke zachte weefsels met behulp van inspringen en Trekproeven

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Een experimentele en Finite Element Protocol bij het vervoer van neutrale en geladen opgeloste stoffen over gewrichtskraakbeen Onderzoek

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

A Test Bed to Examine Helmet Fit and Retention and Biomechanical Measures of Head and Neck Injury in Simulated Impact

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during an impact are important factors in protecting the helmet wearer from impact-induced injury. This manuscript aims to investigate impact-induced injury mechanisms in different helmet fit scenarios through analysis of simulated helmeted impacts with an anthropometric test device (ATD), an array of headform acceleration transducers and neck force/moment transducers, a dual high speed camera system, and helmet-fit force sensors developed in our research group based on Bragg gratings in optical fiber. To simulate impacts, an instrumented headform and flexible neck fall along a linear guide rail onto an anvil. The test bed allows simulation of head impact at speeds up to 8.3 m/s, onto impact surfaces that are both flat and angled. The headform is fit with a crash helmet and several fit scenarios can be simulated by making context specific adjustments to the helmet position index and/or helmet size. To quantify helmet retention, the movement of the helmet on the head is quantified using post-hoc image analysis. To quantify head and neck injury potential, biomechanical measures based on headform acceleration and neck force/moment are measured. These biomechanical measures, through comparison with established human tolerance curves, can estimate the risk of severe life threatening and/or mild diffuse brain injury and osteoligamentous neck injury. To our knowledge, the presented test-bed is the first developed specifically to assess biomechanical effects on head and neck injury relative to helmet fit and retention.

Using an anthropometric head and neck, optical fiber-based fit force transducers, an array of head acceleration and neck force/moment transducers, and a dual high speed camera system, we present a test bed to study helmet retention and effects on biomechanical measures of head and neck injury secondary to head impact.

Een Test-Bed helm Fit en retentie en biomechanische maatregelen van hoofd- en nekletsel in gesimuleerde gevolgen te onderzoeken

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during ...

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder regions, collectively referred to as the brachial plexus (BP). Despite recent advances in obstetrical care, the problem of NBPP continues to be a global health burden with an incidence of 1.5 cases per 1,000 live births. More severe types of this injury can cause permanent paralysis of the arm from the shoulder down. Prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of newborn BP nerves when subjected to stretch. Current knowledge of the newborn BP is extrapolated from adult animal or cadaveric BP tissue instead of in vivo neonatal BP tissue. This study describes an in vivo mechanical testing device and procedure to conduct in vivo biomechanical testing in neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads until failure. The camera system also allows monitoring of the failure location during rupture. Overall, the presented method allows for a detailed biomechanical characterization of neonatal BP when subjected to stretch.

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet model.

Methoden voor in vivo biomechanische tests op brachiale plexus in neonatale biggen

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder ...

Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). Smooth muscle cells exhibit a contractile function within the vaginal and cervical walls. Depending on the biochemical environment and the mechanical distension of the organ walls, the smooth muscle cells alter the contractile conditions. The contribution of the smooth muscle cells under baseline physiological conditions is classified as a basal tone. More specifically, a basal tone is the baseline partial constriction of smooth muscle cells in the absence of hormonal and neural stimulation. Furthermore, the ECM provides structural support for the organ walls and functions as a reservoir for biochemical cues. These biochemical cues are vital to various organ functions, such as inciting growth and maintaining homeostasis. The ECM of each organ is composed primarily of collagen fibers (mostly collagen types I, III, and V), elastic fibers, and glycosaminoglycans/proteoglycans. The composition and organization of the ECM dictate the mechanical properties of each organ. A change in ECM composition may lead to the development of reproductive pathologies, such as pelvic organ prolapse or premature cervical remodeling. Furthermore, changes in ECM microstructure and stiffness may alter smooth muscle cell activity and phenotype, thus resulting in the loss of the contractile force.
In this work, the reported protocols are used to assess the basal tone and passive mechanical properties of the nonpregnant murine vagina and cervix at 4-6 months of age in estrus. The organs were mounted in a commercially available pressure myograph and both pressure-diameter and force-length tests were performed. Sample data and data analysis techniques for the mechanical characterization of the reproductive organs are included. Such information may be useful for constructing mathematical models and rationally designing therapeutic interventions for women&#8217;s health pathologies.

This protocol utilized a commercially available pressure myograph system to perform pressure myograph testing on the murine vagina and cervix. Utilizing media with and without calcium, the contributions of the smooth muscle cells (SMC) basal tone and passive extracellular matrix (ECM) were isolated for the organs under estimated physiological conditions.

Biaxiaal basgeluid en passieve testen van het Muriene voortplantingssysteem met behulp van een Myograph-druk

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). ...

The Quantification of Injectability by Mechanical Testing

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more viscous than traditional aqueous injections and may be semi-solid, therefore, their injectability cannot be assumed. This protocol describes a method to objectively assess the injectability of these materials using a standard mechanical tester. The syringe plunger is compressed by the crosshead at a set rate, and the force is measured. The maximum or plateau force value can then be used for comparison between samples, or to an absolute force limit. This protocol can be used with any material, and any syringe and needle size or geometry. The results obtained may be used to make decisions about formulations, syringe and needle sizes early in the translational process. Further, the effects of altering formulations on injectability may be quantified, and the optimum time to inject temporally changing materials determined. This method is also suitable as a reproducible way to examine the effects of injection on a material, to study phenomena such as self-healing and filter pressing or study the effects of injection on cells. This protocol is faster and more directly applicable to injectability than rotational rheology, and requires minimal post processing to obtain key values for direct comparisons.

Presented here is a protocol for quantitatively evaluating the injectability of a material through a syringe-needle system using a standard mechanical testing rig.

De kwantificering van injecteerbaarheid door mechanische testen

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more ...

Optical Coherence Tomography Based Biomechanical Fluid-Structure Interaction Analysis of Coronary Atherosclerosis Progression

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis as one of the leading causes of global death, morbidity and economic burden, novel ways of analyzing and predicting its progression are needed. One such computational method is the use of fluid-structure interaction (FSI) to analyze the interaction between the blood flow and artery/plaque domains. Coupled with in vivo imaging, this approach could be tailored to each patient, assisting in differentiating between stable and unstable plaques. We outline the three-dimensional reconstruction process, making use of intravascular Optical Coherence Tomography (OCT) and invasive coronary angiography (ICA). The extraction of boundary conditions for the simulation, including replicating the three-dimensional motion of the artery, is discussed before the setup and analysis is conducted in a commercial finite element solver. The procedure for describing the highly nonlinear hyperelastic properties of the artery wall and the pulsatile blood velocity/pressure is outlined along with setting up the system coupling between the two domains. We demonstrate the procedure by analyzing a non-culprit, mildly stenotic, lipid-rich plaque in a patient following myocardial infarction. Established and emerging markers related to atherosclerotic plaque progression, such as wall shear stress and local normalized helicity, respectively, are discussed and related to the structural response in the artery wall and plaque. Finally, we translate the results to potential clinical relevance, discuss limitations, and outline areas for further development. The method described in this paper shows promise for aiding in the determination of sites at risk of atherosclerotic progression and, hence, could assist in managing the significant death, morbidity, and economic burden of atherosclerosis.

There is a need to determine which atherosclerotic lesions will progress in the coronary vasculature to guide intervention before myocardial infarction occurs. This article outlines the biomechanical modeling of arteries from Optical Coherence Tomography using fluid-structure interaction techniques in a commercial finite element solver to help predict this progression.

Optische coherentie Tomografie gebaseerde biomechanische vloeistofstructuur interactie analyse van coronaire atherosclerose progressie

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis ...

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

Constructie van een humane Aorta gladde spiercel orgaan-op-A-chip model voor het recapuleren van biomechanische stam in de aortawand

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...