Gli autori sono grati a Shad Schipke e Daniel Chesser per la loro assistenza con la progettazione e la realizzazione del sistema e Myles Valtresa per l&#39;assistenza con la preparazione del manoscritto.

1. Raccolta e preparazione dei tessuti <ol><li> Le valvole aortica devono essere raccolti da suini adulti peso non superiore a £ 120 subito dopo la morte. </li><li> Lavare le valvole due volte con soluzione salina tampone fosfato sterile (PBS) e il trasporto al laboratorio in ghiaccio. </li><li> Tutte le fasi successive devono essere eseguite in condizioni sterili. </li><li> Assicurarsi che volantini non mostrano alcun segno di degenerazione, lacrimazione o calcificazione. Rimuovere volantini dalla radice aortica, tagliando 1 / 3 della distanza dal l&#39;anello. </li><li> Volantini in luogo singoli pozzetti di una piastra ben sei Incubare per una notte con medie da 3 ml di coltura Dulbecco modificato Aquila integrato con 1% di soluzione anti-biotic/anti-mycotic e il 10% di siero fetale bovino a 37 ° C e 5% di CO 2. </li><li> In alternativa, le cellule isolate possono essere seminati in sei pozzetti piastre di coltura e utilizzato il dispositivo di pressione. Isolamento di cellule endoteliali e valvola di cellule interstiziali può essere eseguita come descritto in precedenza 13, 14. </li></ol> 2. Studi di pressione <ol><li> Una misura della pressione del sistema è stato progettato per studiare gli effetti mechanobiological di pressione ciclica nel tessuto della valvola aortica 15. </li><li> Accedere al computer e aprire il programma LabVIEW (Figura 1). </li><li> Calibrazione: <ol><li> Prima della sperimentazione, il sistema dovrebbe essere opportunamente calibrato. </li><li> Collegare l&#39;alimentatore al circuito. Questo alimenta le elettrovalvole che controllano il flusso di aria all&#39;interno e all&#39;esterno della camera. </li><li> Assicurarsi che l&#39;aria compressa è collegata al sistema e aprire l&#39;alimentazione di aria alla velocità completa. </li><li> Accendere l&#39;amplificatore di segnale. Assicurarsi che la lettura della tensione è 0,00. Regolare se necessario </li><li> L&#39;interfaccia LabVIEW dispone di un interruttore la scritta &quot;TEST / RECORD&quot;. Assicurarsi che l&#39;interruttore è impostato su &quot;TEST&quot;. Fare clic sul pulsante &quot;Air Supply&quot; per aprire l&#39;elettrovalvola di ingresso. </li><li> Utilizzando il regolatore di pressione del gas, pressurizzare la camera con aria compressa a 1 PSI. La pressione nella camera può essere letto usando l&#39;indicatore di pressione digitale situato sul retro finale piatto della camera. Una volta che la pressione è equilibrata, registrare la lettura della tensione da amplificatore di segnale. Ripetere l&#39;operazione per PSI 2, 3, 4 e 5. </li><li> Costruire una curva di calibrazione di pressione contro di tensione. La pressione deve essere convertito dal PSI al mmHg. L&#39;equazione dal grafico può essere inserito nel codice del programma LabVIEW </li></ol></li><li> Rimuovere la placca frontale in alluminio dalla camera di pressione e spruzzare la camera con il 70% di etanolo. Lasciare agire per un minimo di 10 minuti per consentire l&#39;etanolo residuo dei fumi di dissipare. </li><li> Posto a sei pozzetti contenenti i campioni volantino nella camera e sostituire la piastra front-end. Garantire la tenuta è tenuta stringendo i dadi situati sui quattro barre filettate (che si trova ad ogni angolo della piastra finale) a mano. Posizionare la camera di pressione nel 37 ° C incubatore. Un diagramma schematico della camera di pressione è mostrata in figura 2. </li><li> L&#39;interfaccia sarà richiesto all&#39;utente di fornire la quantità di tempo i cicli di sistema tra ingresso e uscita aria compressa. Questi dovrebbero essere regolate per le condizioni diastolica e sistolica e 0.6s 0.4s per imitare, rispettivamente, ad una frequenza di 1Hz. L&#39;utente deve inserire anche un file di dati percorso. </li><li> In LabVIEW, fare clic su Esegui e passare il &quot;TEST / RECORD&quot; passare a &quot;RECORD&quot;. </li><li> Assicurarsi che la pressione è al livello desiderato utilizzando il grafico sull&#39;interfaccia LabVIEW. La pressione può essere regolata utilizzando il regolatore di pressione del gas. </li><li> Eseguire il programma per la lunghezza di tempo desiderato. </li><li> Una volta che l&#39;esperimento è completa, fare clic sul pulsante di arresto su LabVIEW, spegnere l&#39;alimentazione dell&#39;aria e aprire la valvola di scarico in camera di pressione. </li><li> Rimuovere la piastra di front-end dalla camera e recuperare così sei campioni piastra contenente. I campioni possono essere analizzati per l&#39;espressione genica, espressione proteica, istologia, proprietà meccaniche etc </li></ol> 3. Rappresentante dei risultati: Il sistema di pressione è in grado di simulare la pressione massima transvalvolare osservato sotto normotesi, fase I e fase II condizioni ipertesi. Tuttavia, la pressione non era in grado di mimare il gradiente di pressione sistolica, che è essenzialmente zero nel vivo. La frequenza è mantenuta a 1Hz, con un tempo di 0.6s dell&#39;aria in ingresso e un tempo di scarico di 0.4s. Rappresentante pressione forme d&#39;onda di condizioni di pressione normali che elevate ottenuti dal sistema può essere visto in figura 3. <img src="/files/ftp_upload/3316/3316fig1.jpg" alt="Figura 1"/> Figura 1: Schermata dell&#39;interfaccia LabVIEW. <img src="/files/ftp_upload/3316/3316fig2.jpg" alt="Figura 2"/> Figura 2: Schema della camera di pressione A. vista isometrica della camera di pressione; B. Vista laterale della camera di pressione;C. Vista dall&#39;alto della camera di pressione. <img src="/files/ftp_upload/3316/3316fig3.jpg" alt="Figura 3"/> Figura 3: Grafico di simulazione della pressione all&#39;interno della camera di pressione (A) normotesi, (B) Fase I ipertesi, e (C) Fase II condizioni ipertesi.

<ol>
	<li>Freeman, R. V., Otto, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrum+of+calcific+aortic+valve+disease:+pathogenesis,+disease+progression,+and+treatment+strategies.">Spectrum of calcific aortic valve disease: pathogenesis, disease progression, and treatment strategies.</a> Circulation. 111, 3316-3326 (2005).</li><li>Robicsek, F., Thubrikar, M. J., Fokin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cause+of+degenerative+disease+of+the+trileaflet+aortic+valve:+review+of+subject+and+presentation+of+a+new+theory.">Cause of degenerative disease of the trileaflet aortic valve: review of subject and presentation of a new theory.</a> Ann Thorac Surg. 73, 1346-1354 (2002).</li><li>Thubrikar, M. J., Aouad, J., Nolan, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+calcific+deposits+in+operatively+excised+stenotic+or+purely+regurgitant+aortic+valves+and+their+relation+to+mechanical+stress.">Patterns of calcific deposits in operatively excised stenotic or purely regurgitant aortic valves and their relation to mechanical stress.</a> Am J Cardiol. 58, 304-308 (1986).</li><li>Agno, F. S., Chinali, M., Bella, J. N., Liu, J. E., Arnett, D. K., Kitzman, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+valve+sclerosis+is+associated+with+preclinical+cardiovascular+disease+in+hypertensive+adults:+the+Hypertension+Genetic+Epidemiology+Network+study.">Aortic valve sclerosis is associated with preclinical cardiovascular disease in hypertensive adults: the Hypertension Genetic Epidemiology Network study.</a> J Hypertens. 23, 867-8673 (2005).</li><li>Cawley, P. J., Otto, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+calcific+aortic+valve+stenosis+-+fact+or+fiction.">Prevention of calcific aortic valve stenosis - fact or fiction.</a> Annals of Medicine. 41, 100-108 (2009).</li><li>Durst, C. A., Grande-Allen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+physical+characterization+of+a+synchronous+multivalve+aortic+valve+culture+system.">Design and physical characterization of a synchronous multivalve aortic valve culture system.</a> Ann Biomed Eng. 38, 319-3125 (2010).</li><li>Engelmayr, G. C., Soletti, L., Vigmostad, S. C., Budilarto, S. G., Federspiel, W. J., Chandran, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+flex-stretch-flow+bioreactor+for+the+study+of+engineered+heart+valve+tissue+mechanobiology.">A novel flex-stretch-flow bioreactor for the study of engineered heart valve tissue mechanobiology.</a> Ann Biomed Eng. 36, 700-712 (2008).</li><li>Sucosky, P., Padala, M., Elhammali, A., Balachandran, K., Jo, H., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+an+ex+vivo+culture+system+to+investigate+the+effects+of+shear+stress+on+cardiovascular+tissue.">Design of an ex vivo culture system to investigate the effects of shear stress on cardiovascular tissue.</a> J Biomech Eng. 130, 035001-03 (2008).</li><li>Syedain, Z. H., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+cyclic+stretch+bioreactor+for+tissue-engineered+heart+valves.">Controlled cyclic stretch bioreactor for tissue-engineered heart valves.</a> Biomaterials. 30, 4078-4084 (2009).</li><li>Lagana, K., Moretti, M., Dubini, G., Raimondi, M. T. <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+bioreactor+for+the+controlled+application+of+complex+mechanical+stimuli+for+cartilage+tissue+engineering.">A new bioreactor for the controlled application of complex mechanical stimuli for cartilage tissue engineering.</a> Proc Inst Mech Eng H. 222, 705-715 (2008).</li><li>Wartella, K. A., Wayne, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioreactor+for+biaxial+mechanical+stimulation+to+tissue+engineered+constructs.">Bioreactor for biaxial mechanical stimulation to tissue engineered constructs.</a> J Biomech Eng. 131, 044501-044501 (2009).</li><li>Wallis, M. C., Yeger, H., Cartwright, L., Shou, Z., Radisic, M., Haig, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+study+of+a+novel+urinary+bladder+bioreactor.">Feasibility study of a novel urinary bladder bioreactor.</a> Tissue Eng Part A. 14, 339-348 (2008).</li><li>Butcher, J. T., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valvular+endothelial+cells+regulate+the+phenotype+of+interstitial+cells+in+co-culture:+effects+of+steady+shear+stress.">Valvular endothelial cells regulate the phenotype of interstitial cells in co-culture: effects of steady shear stress.</a> Tissue Eng. 12, 905-915 (2006).</li><li>Metzler, S. A., Pregonero, C. A., Butcher, J. T., Burgess, S. C., Warnock, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+Strain+Regulates+Pro-Inflammatory+Protein+Expression+in+Porcine+Aortic+Valve+Endothelial+Cells.">Cyclic Strain Regulates Pro-Inflammatory Protein Expression in Porcine Aortic Valve Endothelial Cells.</a> J Heart Valve Dis. 17, 571-578 (2008).</li><li>Schipke, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Design of a cyclic pressure bioreactor for the ex vivo study of aortic heart valve mechanobiology. , (2008).</li><li>Smith, K. E., Metzler, S. A., Warnock, J. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+immediate-early+gene+responses+to+elevated+pressure+in+porcine+aortic+valve+interstitial+cells.">Differential immediate-early gene responses to elevated pressure in porcine aortic valve interstitial cells.</a> J Heart Valve Dis. 15, 34-41 (2006).</li><li>Brathwaite, D., Weissman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+onset+of+atrial+arrhythmias+following+major+noncardiothoracic+surgery+is+associated+with+increased+mortality.">The new onset of atrial arrhythmias following major noncardiothoracic surgery is associated with increased mortality.</a> Chest. 114, 462-468 (1998).</li><li>Walsh, S. R., Oates, J. E., Anderson, J. A., Blair, S. D., Makin, C. A., Walsh, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postoperative+arrhythmias+in+colorectal+surgical+patients:+incidence+and+clinical+correlates.">Postoperative arrhythmias in colorectal surgical patients: incidence and clinical correlates.</a> Colorectal Dis. 8, 212-216 (2006).</li><li>Walsh, S. R., Tang, T., Gaunt, M. E., Schneider, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+arrhythmias+after+non-cardiothoracic+surgery.">New arrhythmias after non-cardiothoracic surgery.</a> BMJ. 7, 333-333 (2006).</li><li>Walsh, S. R., Tang, T., Wijewardena, C., Yarham, S. I., Boyle, J. R., Gaunt, M. 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Nessun conflitto di interessi dichiarati.

Il sistema di pressione con successo esposti lembi della valvola aortica a pressioni cicliche che erano rappresentativi della pressione diastolica transvalvolare. Tuttavia, non era in grado di simulare la pressione sistolica transvalvolare, come l&#39;unica pressione è sceso a 40 mmHg. Pressione transvalvolare è la differenza tra la pressione in aorta ascendente e del ventricolo sinistro. Durante la diastole, quando la valvola è chiusa, la differenza di pressione è 80mmHg in condizioni normotesi e 90 mmHg e 100 mmHg...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> DMEM </td><td> Sigma </td><td> D5671 </td><td></td></tr><tr><td> Dulbecco PBS </td><td> Sigma </td><td> D5652 </td><td></td></tr><tr><td> Soluzione Anti-mycotic/antibiotic </td><td> Sigma </td><td> A5955 </td><td></td></tr><tr><td> Siero bovino fetale </td><td> ThermoScientific </td><td> SH30070 </td><td></td></tr><tr><td> Viton diaframma elettrovalvole </td><td> McMaster Carr </td><td> 4868K11 </td><td></td></tr><tr><td> Trasduttore di pressione </td><td> Omega Engineering, Inc. </td><td> PX302-200GV </td><td></td></tr><tr><td> Celle di carico condizionatore </td><td> Encore Electronics, Inc. </td><td> 4025-101 </td><td></td></tr><tr><td> Acquisizione dati (DAQ) Modulo </td><td> Misurazione Computing </td><td> PMD1608 </td><td></td></tr></tbody></table>

design of a cyclic pressure bioreactor for the ex vivo study of aortic heart valves

La valvola aortica, che si trova tra il ventricolo sinistro e l&#39;aorta, consente un flusso unidirezionale, impedendo il riflusso nel ventricolo. Lembi valvolari aortici sono composti da cellule interstiziali sospesi all&#39;interno di una matrice extracellulare (ECM) e sono rivestiti con un monostrato di cellule endoteliali. La valvola resiste un ambiente duro, dinamico ed è costantemente esposto a taglio, flessione, trazione e compressione. La ricerca ha dimostrato lesioni calcifica delle valvole malate si verificano nelle zone di elevato stress meccanico a causa della interruzione o danno endoteliale matrice interstiziale 1-3. Quindi, non sorprende che gli studi epidemiologici hanno dimostrato l&#39;alta pressione sanguigna per essere un fattore di rischio importante nell&#39;insorgenza della malattia della valvola aortica 4. L&#39;unica opzione di trattamento attualmente disponibile per la malattia della valvola è la sostituzione chirurgica della valvola malata con una valvola di bioprotesi o meccanico 5. Una migliore comprensione della biologia della valvola in risposta a stress fisici aiuterebbe chiarire i meccanismi della patogenesi della valvola. A sua volta, questo potrebbe aiutare nello sviluppo di terapie non invasive come l&#39;intervento farmaceutico o di prevenzione. Bioreattori Diversi sono stati precedentemente sviluppato per studiare la mechanobiology di valvole cardiache native o attrezzata 6-9. Bioreattori pulsatile sono stati sviluppati per studiare una vasta gamma di tessuti tra cui la cartilagine 10, 11 e osso della vescica 12. Lo scopo di questo lavoro è stato quello di sviluppare un sistema ciclico di pressione che potrebbero essere utilizzati per spiegare la risposta biologica dei lembi valvolari aortici ai carichi di aumento della pressione. Il sistema consisteva in una camera acrilico in cui collocare i campioni e produrre pressione ciclica, viton elettrovalvole a membrana di controllare i tempi del ciclo di pressione, e un computer per controllare dispositivi elettrici. La pressione è stata monitorata mediante un trasduttore di pressione, e il segnale è stato condizionato usando un condizionatore cella di carico. Un programma LabVIEW regolata la pressione utilizzando un dispositivo analogico per pompare aria compressa nel sistema al tasso appropriato. Il sistema ha imitato i livelli di pressione dinamica transvalvolare associati con la valvola aortica; un&#39;onda dente di sega ha prodotto un aumento graduale della pressione, tipico del gradiente di pressione transvalvolare che è presente attraverso la valvola durante la diastole, seguito da un brusco calo di pressione che rappresenta l&#39;apertura della valvola a sistole. Il programma LabVIEW ha permesso agli utenti di controllare l&#39;entità e la frequenza della pressione ciclica. Il sistema è stato in grado di sottoporre campioni di tessuto a condizioni di pressione fisiologiche e patologiche. Questo dispositivo può essere utilizzato per aumentare la nostra comprensione di come valvole cardiache rispondere ai cambiamenti nell&#39;ambiente locale meccanico.

Watch this Scientific Journal Video about Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves at JoVE.com

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Un bioreattore pressione ciclica in grado di sottoporre valvola tessuto cardiaco alle condizioni fisiologiche e patologiche della pressione è stato progettato. Un programma di LabVIEW consente agli utenti di controllare la pressione portata, ampiezza e frequenza. Questo dispositivo può essere utilizzato per studiare la mechanobiology del tessuto cardiaco valvola o cellule isolate.

Progettazione di un bioreattore per la pressione ciclica Ex Vivo Studio delle valvole cardiache aortica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

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Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

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15.6K Views.  Mississippi State University. Un bioreattore pressione ciclica in grado di sottoporre valvola tessuto cardiaco alle condizioni fisiologiche e patologiche della pressione è stato progettato. Un programma di LabVIEW consente agli utenti di controllare la pressione portata, ampiezza e frequenza. Questo dispositivo può essere utilizzato per studiare la mechanobiology del tessuto cardiaco valvola o cellule isolate.

Video: Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

Patient-specific simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered by the absence of in vivo imaging technology for clinically acquiring myocardial fiber orientations. The objective of this project was to develop a methodology to estimate cardiac fiber orientations from in vivo images of patient heart geometries. An accurate representation of ventricular geometry and fiber orientations was reconstructed, respectively, from high-resolution ex vivo structural magnetic resonance (MR) and diffusion tensor (DT) MR images of a normal human heart, referred to as the atlas. Ventricular geometry of a patient heart was extracted, via semiautomatic segmentation, from an in vivo computed tomography (CT) image. Using image transformation algorithms, the atlas ventricular geometry was deformed to match that of the patient. Finally, the deformation field was applied to the atlas fiber orientations to obtain an estimate of patient fiber orientations. The accuracy of the fiber estimates was assessed using six normal and three failing canine hearts. The mean absolute difference between inclination angles of acquired and estimated fiber orientations was 15.4 &deg;. Computational simulations of ventricular activation maps and pseudo-ECGs in sinus rhythm and ventricular tachycardia indicated that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.The new insights obtained from the project will pave the way for the development of patient-specific models of the heart that can aid physicians in personalized diagnosis and decisions regarding electrophysiological interventions.

A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.

Paziente-specifica modellazione del Cuore: Stima di orientamento delle fibre ventricolari

Patient-specific  simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered  by the absence of in vivo imaging technology for ...

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Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Progettazione di un biassiale Carico meccanico bioreattore per l&#39;ingegneria tissutale

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

Il funzionamento di un bioreattore da banco

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Caratterizzazione dei sistemi complessi Utilizzando il Design of Experiments Approccio: Transient Protein Expression in tabacco come un caso di studio

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Anatomically Realistic Neonatal Heart Model for Use in Neonatal Patient Simulators

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff practice clinical interventions such as chest compressions to ensure patient survival in the case of bradycardia or cardiac arrest. The simulators used currently are of low physical fidelity and therefore cannot provide qualitative insight into the procedure of chest compressions. The embedding of an anatomically realistic heart model in future simulators enables the detection of cardiac output generated during chest compressions; this can provide clinicians with an output parameter, which can deepen the understanding of the effect of the compressions in relation to the amount of blood flow generated. Before this monitoring can be achieved, an anatomically realistic heart model must be created containing: two atria, two ventricles, four heart valves, pulmonary veins and arteries, and systemic veins and arteries. This protocol describes the procedure for creating such a functional artificial neonatal heart model by utilizing a combination of magnetic resonance imaging (MRI), 3D printing, and casting in the form of cold injection molding. Using this method with flexible 3D printed inner molds in the injection molding process, an anatomically realistic heart model can be obtained.

This protocol describes a procedure for creating functional artificial neonatal heart models by utilizing a combination of magnetic resonance imaging, 3D printing, and injection molding. The purpose of these models is for integration into the next generation of neonatal patient simulators and as a tool for physiological and anatomical studies.

Modello di cuore neonatale anatomicamente realistici per l'uso in simulatori paziente neonatale

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Microscopia di fluorescenza di luce-foglio per lo studio del cuore murino

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

Un nuovo modello Ex Vivo per la valutazione delle prestazioni del materiale di iniezione sottomucosa endoscopica

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Caratterizzazioni biassiale meccaniche delle valvole atrioventricolari cuore

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...

A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication functions, fostering a deleterious physical environment. The frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device capable of replicating the motion of the joint is described. The friction testing device controls the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces. This study adopts a synovium-on-cartilage configuration, and friction coefficient measurements are presented for tests performed in a phosphate-buffered saline (PBS) or synovial fluid (SF) bath. The testing was performed for a range of contact stresses, highlighting the lubricating properties of SF under high loads. This friction testing device can be used as a biomimetic bioreactor for studying the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.

The present protocol describes a friction testing device that applies simultaneous reciprocal sliding and normal load to two contacting biological counterfaces.

Un dispositivo per test di attrito-bioreattore per lo studio della biomeccanica delle articolazioni sinoviali, della meccanobiologia e della regolazione fisica

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication ...

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

Approvvigionamento e decellularizzazione degli arti posteriori di ratto utilizzando un bioreattore ex vivo basato sulla perfusione per allotrapianto composito vascolarizzato

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...