Integra modello di rigenerazione dermica è stato gentilmente fornito da Integra LifeSciences Corporation. Fonti di fondi a sostegno del lavoro: Questo lavoro è stato parzialmente finanziato dal CIRM-BMBF precoce II Premio traslazionale e il Centro FONDAP per la regolazione del genoma sia per JTE (Nr 15.090.007.).

1 Preparazione di Ponteggi <ol><li> Generare campioni dei ponteggi utilizzando 12 millimetri pugni biopsia. </li><li> Per introdurre molecole bioattive o cellule nel patibolo, drenare i ponteggi da loro stringendo delicatamente con una garza sterile. Poi reidratare i ponteggi aggiungendo 160 ml di una soluzione contenente le molecole bioattive o cellule di interesse. Ricontrollare il successo di bioattivazione con cellule attraverso saggi metaboliche come MTT saggi. </li><li> Se necessario, fissare i composti o le cellule da testare all&#39;interno del ponteggio fornendo loro, ad esempio in una soluzione di fibrina-trombina o un idrogel, ancora attraverso reidratazione come descritto al punto 1.2. NOTA: Questo passaggio può essere utile quando i composti o le cellule non attribuiscono meccanicamente al materiale. Si può anche controllare le dinamiche di rilascio dei composti .. </li><li> Preparare le maglie titanized, che saranno collocati sotto ogni impalcatura in ogni gruppo di sperimentazione e di controllo, da Cutting i pezzi a forma rotonda, di circa 14 mm di diametro. NOTA: La maglia titanized funziona come un confine fisico richiesto per delimitare il tessuto rigenerato dal letto della ferita. </li></ol> 2. Animali <ol><li> Prima attuazione del modello presentato, consultare le leggi di protezione degli animali corrispondenti e ottenere il permesso da parte delle autorità locali. In questo lavoro, tutti gli esperimenti sono stati effettuati con topi, secondo l&#39;attuale atto benessere degli animali tedesca e approvati dal governo distretto dell&#39;Alta Baviera (Regierung von Oberbayern). </li><li> Scegliere con cura l&#39;animale e la tensione. Anche se il modello è stato stabilito per i topi, che può essere tradotto ad altri animali comuni, come ratti, conigli e maiali. </li><li> Nel caso in cui si sta lavorando con gli animali pelo, radere la parte posteriore degli animali prima dell&#39;impianto degli scaffold. NOTA: Questo lavoro utilizza topi di 6 a 8 settimane di età con un peso corporeo compreso tra 20 ge 25 g, ma diverse età e bpesi ody possono anche essere utilizzati. </li></ol> 3 Anestesia <ol><li> Effettuare l&#39;operazione in condizioni sterili. Al fine di mantenere la normale temperatura corporea dell&#39;animale, utilizzare un tappetino riscaldante. </li><li> Prima di inalazione anestesia gli animali ricevono buprenorfina alla dose di 0,05-0,1 mg / kg di peso corporeo, per via sottocutanea ogni 8 ore. </li><li> Posizionare l&#39;animale in anestesia per inalazione (isoflurano) oppure applicare procedure standard equivalenti. Confermare l&#39;anestesia sia premendo delicatamente le dita dei piedi dell&#39;animale o tramite un pizzico di pelle. </li><li> Per evitare essiccosi, iniettare 0,5 ml di soluzione salina fisiologica sottocute all&#39;inizio dell&#39;operazione. </li></ol> 4. escissione della pelle <ol><li> Mettere l&#39;animale in posizione prona e segnare la linea mediana della schiena del mouse con una punta fine penna permanente (Figura 1A). </li><li> Definire l&#39;area di escissione. Mettere i difetti nell&#39;area mostrata in Figuri 1C. Si noti che se i difetti siano troppo caudalmente, gli animali sono più inclini a rimuovere le medicazioni ed i ponteggi. </li><li> Crea rotondo difetti bilaterali utilizzando una biopsia del punzone 10 mm (Figura 1B). Il punzone deve essere attentamente forzato contro la pelle e non deve essere usato per tagliare completamente attraverso la pelle, ma per delineare l&#39;area di escissione (Figure 1C e 1E). </li><li> Sollevare delicatamente la pelle sensibile con una pinza e incidere lungo il cerchio marcato con le forbici chirurgiche sottili (Figura 1F). In caso di sanguinamento, comprimere delicatamente con una garza sterile. Una descrizione passo-passo della escissione è mostrato in Figura 1. NOTA: Il difetto viene creato con un punzone inferiore a quello per la generazione dei ponteggi, per compensare l&#39;allargamento del difetto dovuto alla elasticità della pelle. </li></ol> 5. Scaffold impianto <ol><li> Al fine di rendere lo spazio per il titasciuta maglia, estendere la separazione della pelle e dei tessuti sottostanti alle frontiere ferita per 2-4 mm (Figura 2A). </li><li> Posizionare il titanized maglia nel difetto direttamente sul letto della ferita e sotto i bordi della ferita (Figure 2A e B). </li><li> Luogo Ponteggi direttamente sulla maglia. </li><li> Suturare i ponteggi ai bordi della ferita adiacenti con 4 a 6 singoli nodi, lasciando i bordi leggermente sopra il patibolo (Figura 2C). NOTA: Evitare l&#39;uso di suture biodegradabili. </li><li> Suturare una ferita medicazione trasparente sopra i difetti di proteggere il ponteggio pur consentendo il monitoraggio della ferita (Figura 2D). </li></ol> 6. postoperatoria Cura <ol><li> Mantenere un mouse per gabbia per evitare la manipolazione reciproca della medicazione e ponteggi. </li><li> Valutare lo stato generale dell&#39;animale su una base quotidiana per attività osservando motore, il peso corporeo, segni di dolore, tolleranzaper la medicazione e auto-mutilazione. Monitorare anche la zona della ferita di sanguinamento, locale e segni sistemici di infezione e la posizione della medicazione. </li><li> Per ridurre al minimo il dolore dell&#39;animale, iniettare buprenorfina ogni giorno alla dose di 0,05-0,1 mg / kg di peso corporeo o farmaci analgesici equivalente. </li><li> Se il mouse rimuove o danneggia la medicazione della ferita, sostituire o ricollegare sotto anestesia. </li></ol> 7 Eutanasia e Espianto dell&#39;impalcatura <ol><li> A intervalli di tempo desiderati, sacrificare gli animali da overdose di pentobarbital (150 mg / kg). </li><li> Segnare il sito di escissione pelle con un pennarello indelebile, che dovrebbe includere i ponteggi e quanto del mouse schiena pelle possibile (Figura 3A). </li><li> Incidere la pelle lungo le linee segnate con un paio di forbici o un bisturi. Staccare tutta la pelle, compresi i ponteggi e la rete titanized, dal tessuto sottostante attraverso la preparazione smussato (Fifigura 3B). </li><li> Posizionare il tessuto espiantato sdraiati su una piastra di Petri (Figura 3C e D) </li></ol> 8 Visualizzazione e quantificazione della rete vascolare <ol><li> La transilluminazione: <ol><li> Configurare un dispositivo transilluminazione, montando una piastra di Petri di sopra di una forte sorgente di luce bianca (100 Watt lampadina standard). </li><li> Fissare una fotocamera digitale sopra il transilluminatore per ottenere immagini digitali. </li><li> Posizionare i campioni a testa in giù sulla piastra di Petri sul dispositivo transilluminazione. </li><li> Scattare foto dei ponteggi completi in modalità macro, nonché delle aree di uguali dimensioni di pelle normale. Conservare le immagini in formato TIFF per ulteriori analisi digitale. </li><li> Salvare il tessuto per ulteriori analisi biochimica o istologica. </li></ol></li><li> Segmentazione digitale e quantificazione: <ol><li> Scaricare e installare il software VesSeg-Tool, che può essere ottenuto free di carica a: <a href="http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255">http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255</a> . </li><li> Apri l&#39;immagine con il software VesSeg-Tool. </li><li> Selezionare l&#39;area dell&#39;immagine oggetto del ponteggio per l&#39;analisi digitale (Immagine → Seleziona). </li><li> Per aumentare il contrasto delle navi usare l&#39;opzione &quot;Isteresi soglia&quot; (Immagine → valorizzazione Vaso filtro → Isteresi soglia → Top-Hat trasformazione → Calcolare la valorizzazione nave). </li><li> Procedere con la segmentazione della mappa imbarcazione (confini Immagine → valorizzazione Vaso filtro → Isteresi soglia → Threshold). Selezionare la prima soglia (&quot;copertura Vessel&quot;), così ogni pixel, che è anche lontanamente nave simile, sarà etichettato come nave. Selezionare la seconda soglia (&quot;copertura Background&quot;), in modo che solo i pixel che sono particolarmente nave-come saranno etichettati come i vasi. </li><li> Controllare la proposta automatica di segmentazione manualmente per aggiungere le navi non catturati e per eliminare i falsi positivi strutture comuni, che di solito sono strutture lunghe e sottili vasi simili, come peli o suture. </li><li> Calcolare la lunghezza dei vasi e la superficie totale coperta da navi (statistiche Immagine → Immagine → statistiche immagine binaria). NOTA: Per il calcolo della nave lunghezze, navi in risultante cartina nave sono assottigliate a linee con una larghezza di un pixel 20. </li></ol></li><li> Analizzare l&#39;area pelle nativo sotto gli stessi parametri i ponteggi, assegnando un valore di 100% per il tessuto nativo e riguardano il patibolo a quel valore. Per esempio, se la percentuale di pixel bianchi è del 60% nel tessuto normale e il 30% nel ponteggio, rappresenterebbe una vascolarizzazione 50% del ponteggio. Nota: La copertura vasi bianco viene assegnato l&#39;area di un quadrato attorno al patibolo. Regolare il numero di pixel bianchi consi coperturadering che l&#39;area del cerchio è significativamente più piccolo (π x R 2) del quadrato (4 x R 2). </li></ol>

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Conflitto di interesse: Tutti gli autori: Nessuno Informativa finanziari: Nessuno degli autori ha un interesse finanziario in uno qualsiasi dei prodotti, dispositivi, o farmaci citati in questo manoscritto.

Vi è la necessità di stabilire approcci di successo nel migliorare la perfusione sanguigna in ingegneria tessutale costrutti, che richiede lo sviluppo di nuovi metodi affidabili per studiare i processi di vascolarizzazione all&#39;interno dei biomateriali. Metodi comuni per la preparazione di scaffold vascolarizzazione ex vivo visibile includono l&#39;uso della microscopia, che fornisce uno strumento ad alta risoluzione. Nella maggior parte dei casi, però, questo metodo è limitato all&#39;analisi di piccole...

Negli ultimi decenni, l&#39;ingegneria dei tessuti ha aperto una nuova opzione terapeutica per sostituire difetti del tessuto con le cellule del corpo 1. Al fine di sostenere il processo fisiologico di rigenerazione tissutale, ponteggi sono progettati come una struttura biodegradabile, che fornisce uno scenario in cui le cellule dal letto della ferita possono crescere e ripristinare il difetto 2,3. Vascolarizzazione insufficiente è considerata il principale ostacolo, che trattiene la svolta clinico di scaffold bioartificiali 4. Con la ricrescita delle cellule, la domanda di sostanze nutritive e di ossigeno aumenta e vascolarizzazione del materiale diventa essenziale. Vascolarizzazione insufficiente o ritardato può quindi portare a necrosi centrale dei prodotti dell&#39;ingegneria tissutale 5. Inoltre, i vasi sanguigni forniscono le cellule immunocompetenti e rimuovere i residui metabolici nella zona rigenerante. Gli alti tassi di infezione e la bassa rigenerazione sono soloalcune delle conseguenze di una insufficiente perfusione sanguigna osservato in ingegneria dei tessuti, che mirano ad essere evitato aumentando la vascolarizzazione dei ponteggi 6,7. Diverse strategie che mirano a migliorare la vascolarizzazione attenzione sul ruolo chiave del biomateriale stesso e la microstruttura del patibolo. Ci sono sforzi di ricerca intensivi per sviluppare nuovi approcci nello spostare il processo di guarigione dalla riparazione alla rigenerazione, quindi (ri) generazione di un tessuto con le proprietà fisiologiche più vicino a quello da ristrutturare 8,9. Biomateriali che sono stati studiati e valutati con riferimento al loro potenziale rigenerativo inclusi collagene, fibrina, chitosano e alginato 10,11. Questi biomateriali possono essere utilizzati e combinati come una spina dorsale per la costruzione di nuove impalcature utilizzando diverse strategie come decellularization tessuto, auto-assemblaggio, prototipazione rapida e elettrospinning 12. Per ENHANCE propria capacità rigenerativa del corpo, ponteggi possono essere bioattivato. L&#39;incorporazione di crescita angiogenico ricombinante fattori di 13 o vettori di geni che codificano per fattori 14 ha dimostrato di migliorare la vascolarizzazione del patibolo. L&#39;uso di cellule staminali è stato ampiamente dimostrato di essere una strategia promettente per migliorare la vascolarizzazione, dove le cellule stromali mesenchimali e le cellule progenitrici endoteliali hanno guadagnato più attenzione 15,16. Altri approcci tentano di costruire costrutti che contengono le reti dei vasi prefabbricati prima del trapianto 17. Nonostante intensi sforzi nella progettazione patibolo e la loro bio-attivazione, nessuna strategia è migliorata vascolarizzazione a livello clinicamente significativo e, con l&#39;eccezione di sostituzioni dermici in massicce ustioni, la traduzione di materiali bioengineered nella routine clinica si sta solo posto esitante 18 . Una delle ragioni per cui vascolarizzazionedi costrutti di tessuto artificiale è ancora un problema irrisolto, è la difficoltà di valutare il successo delle nuove tecnologie negli approcci in vivo. Anche se gli esperimenti in vitro possono fornire importanti intuizioni del potenziale vascolarizzazione di ponteggi, modelli animali appropriati sono tenuti a studiare i parametri chiave quali la biocompatibilità del materiale, la sicurezza e l&#39;efficacia del trattamento e, di particolare importanza, la vascolarizzazione del tessuto costruire. Pertanto, strumenti affidabili per visualizzare e quantificare le reti dei vasi sanguigni in vivo sono essenziali. In questo studio presentiamo un metodo semplice e affidabile che permette la visualizzazione e la quantificazione della rete vascolare all&#39;interno ponteggi espiantati. Questo metodo si basa sulla transilluminazione tessuto e segmentazione digitale. Poiché questo metodo è non invasivo, permette ulteriori analisi molecolari e istologiche del materiale bersaglio.

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			<td style="width:431px;">Comments/Description</td>
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			<td height="40" style="height:40px;width:319px;">Ethilon P-3 13 mm 3/8 circle 5-0</td>
			<td style="width:223px;">Ethicon, Norderstedt, Germany</td>
			<td style="width:119px;">698G</td>
			<td>Ethilon polyamid-6 precision point-reverse cutting suture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Biopsy punches (10 mm)</td>
			<td style="width:223px;">Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td>
			<td style="width:119px;">P1050</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Biopsy punches (12 mm)</td>
			<td style="width:223px;">Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td>
			<td style="width:119px;">P1250</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Digital camera&#160;</td>
			<td style="width:223px;">Ricoh, Hannover, Germany</td>
			<td style="width:119px;">Cx1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gazin Mullkompresse&#160;</td>
			<td style="width:223px;">Lohmann und Rauscher, Neuwied, Germany</td>
			<td style="width:119px;">13622</td>
			<td>Sterile gauze (10 cm x 10 cm)</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Double-layer collagen-based scaffold (8 &#39;x 10&#39;)</td>
			<td style="width:223px;">Integra Life Science Corporation, Plainsboro, NJ, USA</td>
			<td style="width:119px;">88101</td>
			<td></td>
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			<td height="40" style="height:40px;">Isoflurane, liquid-gas for inhalative anesthesia&#160;</td>
			<td style="width:223px;">Baxter, Unterschleissheim, Germany</td>
			<td style="width:119px;">100196040</td>
			<td></td>
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			<td height="40" style="height:40px;">Pentobarbital, 16 g / 100 ml</td>
			<td style="width:223px;">Fa. Merial, Hallbergmoos</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
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			<td height="40" style="height:40px;width:319px;">Nuri Nu/Nu Nude mice, CrLNU-Foxn1nu</td>
			<td style="width:223px;">Charles River, Sulzfeld, Germany</td>
			<td style="width:119px;">Strain code 088</td>
			<td>Athymic nude mice, 6 to 8 weeks of age and with a body weight between 20 to 25 g&#160;</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Buprenorphine (0.3 mg/ml)</td>
			<td style="width:223px;">Essex Pharma GmbH, Munich, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Titanized mesh (15 cm x 15 cm), extralight</td>
			<td style="width:223px;">PFM Medical AG, K&#246;ln, Germany</td>
			<td style="width:119px;">6000029</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:319px;">Tissucol Duo S Immuno 2 ml</td>
			<td style="width:223px;">Baxter Germany GmbH, Unterschlei&#223;heim, Germany</td>
			<td style="width:119px;">B1332020110614</td>
			<td>Fibrin-thrombin solution&#160;</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Transparent adhesove drape (30.5 cm x 26 cm)</td>
			<td style="width:223px;">KCI Medical Products, Wimborne Dorset, UK</td>
			<td style="width:119px;">M6275009/10</td>
			<td></td>
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a full skin defect model to evaluate vascularization of biomaterials in vivo

Vascolarizzazione insufficiente è considerata uno dei principali fattori limitanti il ​​successo clinico di costrutti tissutale. Al fine di valutare nuove strategie che mirano a migliorare la vascolarizzazione, metodi affidabili sono tenuti ad effettuare la in-crescita di nuovi vasi sanguigni in impalcature visibili bio-artificiale e quantificare i risultati. Negli ultimi due anni, il nostro gruppo ha introdotto un modello di difetto completo della pelle che consente la visualizzazione diretta dei vasi sanguigni da parte transilluminazione e fornisce la possibilità di quantificazione attraverso la segmentazione digitale. In questo modello, si crea chirurgicamente difetti pieni di pelle nella parte posteriore di topi e li sostituisce con il materiale testato. Molecole o cellule di interesse possono anche essere incorporati in tale materiale, per studiare il loro effetto potenziale. Dopo un periodo di osservazione di uno di propria scelta, i materiali vengono espiantati per la valutazione. Ferite bilaterali offrono la possibilità di fare confronti interni tha minimizzare gli artefatti tra individui nonché di diminuire il numero di animali necessari per lo studio. In confronto ad altri approcci, il nostro metodo offre una semplice, efficace analisi affidabile ed economica. Abbiamo implementato questo modello come strumento di routine per eseguire lo screening ad alta risoluzione durante il test vascolarizzazione di diversi biomateriali e approcci bio-attivazione.

Un difetto pelle piena bilaterale affidabile può essere creato nel topo (Figura 1) in cui la pelle può essere sostituito da un biomateriale in studio (Figura 2). Qui, senza grandi complicazioni si osservano durante o dopo la procedura operativa, né segni macroscopici di infezione o reazione da corpo estraneo. In rari casi, una impalcatura si perde quando un mouse rimuove. Contrazione della ferita non è mai stato osservato (Figura 3). Tissue transilluminazione permes...

Watch this Scientific Journal Video about A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo at JoVE.com

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascolarizzazione è la chiave per approcci in ingegneria dei tessuti successo. Pertanto, tecnologie affidabili sono necessari per valutare lo sviluppo di reti vascolari in tessuto-costrutti. Qui vi presentiamo un metodo semplice e conveniente per visualizzare e quantificare vascolarizzazione in vivo.

Una pelle piena Difetto modello per valutare Vascolarizzazione di Biomateriali<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

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Research

JoVE Journal

Bioengineering

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

12.1K Views.  Technische Universität München.  Vascolarizzazione è la chiave per approcci in ingegneria dei tessuti successo. Pertanto, tecnologie affidabili sono necessari per valutare lo sviluppo di reti vascolari in tessuto-costrutti. Qui vi presentiamo un metodo semplice e conveniente per visualizzare e quantificare vascolarizzazione in vivo. 

Video: A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Tridimensionale pelle umana Ricostruire Modello: uno strumento per studiare la pelle normale e progressione Melanoma

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Ricerca

Bioingegneria

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

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

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

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

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 ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Generazione di una tridimensionale a tutto spessore della pelle Equivalent e Automated Ferimento

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambienti sandwich di sfruttare Cellulari / Materiale Interazioni

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Ingegneria dei tessuti da parte intrinseca Vascolarizzazione in un<em&gt; In Vivo</em&gt; Camera ingegneria dei tessuti

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

Un metodo per la valutazione tempestività e l&#39;accuratezza delle risposte volitivo motore a vibrotattili stimoli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

Un modello di embrione di Zebrafish per nel Vivo visualizzazione e analisi videomicroscopia del biomateriale-collegata dello stafilococco aureo infezione

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

Un modello affidabile e riproducibile critiche medie femorale difetto segmentale in ratti stabilizzato con un Fixator esterno personalizzato

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

In Vivo a due colori immagini fotone di cellule reporter geneticamente taggate nella pelle

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

Modello calvariale di aumento osseo nel coniglio per la valutazione della crescita ossea e della neovascolarizzazione nei materiali di sostituzione ossea

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...