Gli autori ringraziano Texas A &amp; M University Ingegneria e Experiment Station (TEE) per il sostegno finanziario di questa ricerca. Lindsay Nail ringrazia il sostegno della Texas A &amp; M University Louis Stokes Alliance for Minority partecipazione (LSAMP) e la National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP). Dawei Zhang grazie alla Texas A &amp; M University Tesi Fellowship.

1. Synthesizing PCL-DA macromero <ol><li> Eseguire la reazione acrylation. <ol><li> Pesare 20 g di PCL-diol (M n = ~ 10.000 g / mol) in un 250 ml pallone da fondo dotati di ancoretta magnetica Teflon coperto. </li><li> Sciogliere il PCL-diolo in DCM. <ol><li> Aggiungere 120 mLlof DCM al pallone (concentrazione = 0,17 g / ml). </li><li> Collocare un setto di gomma liberamente nel collo del pallone in modo da evitare l&#39;accumulo di pressione evitando anche l&#39;evaporazione di DCM. </li><li> Mescolare la soluzione per ~ 30 min a ~ 250 rpm per sciogliere completamente il polimero. </li></ol></li><li> Aggiungere ~ 6,6 mg di 4-dimetilamminopiridina (DMAP) alla soluzione e sciogliere sotto agitazione. </li><li> Collocare un setto di gomma saldamente nel collo del pallone. Lasciare che la soluzione per continuare a mescolare. </li><li> Il setto di gomma, eliminare delicatamente il pallone con N 2 per ~ 3 min utilizzando un positivo N 2 ago pressione in ingresso e un ago apertocome valvola di sfogo. </li><li> Rimuovere l&#39;ingresso N 2 e uscita. </li><li> Aggiungere 0,56 ml (4,0 mmoli) di trietilammina (Et 3 N) goccia a goccia con una siringa di vetro dotato di un ago inserito attraverso il setto di gomma. </li><li> Aggiungere 0,65 ml (8,0 mmoli) di cloruro di acriloile gocciolata mediante una siringa di vetro dotato di un ago inserito attraverso il setto di gomma. </li><li> Restituisce l&#39;ingresso N 2 al pallone e permettere ai contenuti di mescolare sotto pressione positiva di N 2 per ~ 30 min. </li><li> Preriscaldare bagno d&#39;olio a 55 ° C. </li><li> Dopo l&#39;assegnato ~ 30 min, rimuovere l&#39;ingresso N 2 e sostituire il setto con un condensatore. </li><li> Immergere il pallone nel bagno d&#39;olio preriscaldato. </li><li> Lasciare il contenuto del pallone per mescolare per 20 ore. </li><li> Dopo il assegnato 20 ore, togliere il pallone dal bagno d&#39;olio e lasciare raffreddare il contenuto alla RT. </li><li> Utilizzando un evaporatore rotante, rimuovere DCM solvente dal pallone. </li></ol></li><li> Purify il greggio prodotto PCL-DA. <ol><li> Per il matraccio, aggiungere ~ 135 ml di acetato di etile e sciogliere il greggio PCL-DA. </li><li> Filtro a gravità la soluzione attraverso carta da filtro in un pallone a fondo tondo pulite 250 ml. (Nota: La soluzione può addensare sulla carta da filtro, non facilmente passando caso affermativo, applicare accuratamente leggero calore con una pistola di riscaldamento..) </li><li> Utilizzando un evaporatore rotante, rimuovere acetato di etile solvente dal pallone. </li><li> Per il matraccio, aggiungere ~ 140 ml di DCM e sciogliere il greggio PCL-DA. </li><li> Trasferire il contenuto di un 500 ml imbuto separatore. </li><li> Per l&#39;imbuto, aggiungere 13,5 ml di 2 M di carbonato di potassio (K 2 CO 3). </li><li> Tappare l&#39;imbuto. Mescolare delicatamente i due strati invertendo l&#39;imbuto e ruotare delicatamente una o due volte, avendo cura di scaricare la pressione tramite il rubinetto. Ripetere 3 volte. </li><li> Sostituire il tappo con uno strato di Parafilm e lasciare che la miscela da separare O / N (~ 12 ore). </li><li> Raccogliere il fondo, ostrato di Ganic in un pallone da 250 ml Erlenmeyer. </li><li> Aggiungere ~ 5 g di solfato di magnesio anidro (MgSO4) al pallone e miscelare delicatamente. </li><li> Gravity filtrare il composto attraverso carta da filtro qualitativo e in un ambiente pulito da 250 ml pallone fondo. </li><li> Utilizzando un evaporatore rotante, rimuovere DCM solvente dal pallone. </li><li> Secco sotto alto vuoto per rimuovere residui di DCM. (Nota: PCL-DA deve essere conservato al riparo dalla luce.) </li><li> Confermare acrylation con 1 H NMR 22,23. </li></ol></li></ol> 2. Preparare il SMP Scaffold (Figura 1) <ol><li> Preparare il modello di sale fuso. <ol><li> Utilizzare un setaccio 425 micron per ottenere cloruro di sodio (NaCl) particelle ~ 460 ± 70 micron di diametro. (Nota: dimensione media delle particelle può essere confermata da microscopia elettronica a scansione [SEM] le immagini con il software ImageJ.) 14 </li><li> Per una fiala di vetro 3 ml (ID = 12.9 mm), aggiungere 1,8 g di NaCl precedentemente setacciata. </li><li> Aggiungere lentamente, in quattro porzioni, 7,5% in peso (in base al peso del sale) acqua DI (0,146 g) al flaconcino. Mescolare con una spatola metallica dopo l&#39;aggiunta di ogni porzione di acqua. </li><li> Tappare la fiala, avvolgere nel tessuto e posizionare verticalmente in una provetta da centrifuga. Centrifugare per 15 minuti a 3220 x g. </li><li> Rimuovere il tappo e lasciare asciugare O / N (~ 12 ore). </li></ol></li><li> In una nuova fiala di vetro, preparare una &quot;soluzione macromero&quot; combinando 0,15 g di PCL-DA per ml di DCM. (Nota:. Per un patibolo, ~ 1 ml di soluzione devono essere preparati) Cap e mescolare la soluzione ad alta velocità su un vortex per ~ 1 min. </li><li> In una nuova fiala di vetro 3 ml, preparare una &quot;soluzione di fotoiniziatore&quot; basata su 10% in peso 2,2-dimetossi-2-fenil acetofenone (DMP) in 1-vinil-2-pirrolidinone (NVP). Combinare 0.115 g di DMP in 1 ml di NVP. (Nota: Per un ponteggio, ~ è richiesto di 180 ml.) Cap, avvolgerlo in un foglio di alluminio (per bloccare la luce) e mescolare la soluzione ad alta velocità su un vortexper ~ 1 min. (Nota: se preparata in anticipo, la soluzione deve essere conservata in frigorifero e al riparo dalla luce.) </li><li> Avvolgere la fiala contenente la soluzione macromero (escluso il tappo) con un foglio di alluminio (per bloccare la luce) e inserirlo tramite pipetta 15 vol% (in base al volume totale della soluzione macromero) della soluzione fotoiniziatore. Cap e mescolare la soluzione ad alta velocità su un vortex per ~ 1 min. </li><li> Avvolgere il flaconcino contenente il modello di sali fusi (escluso il tappo), con un foglio di alluminio (per bloccare la luce) e aggiungere tramite pipetta la soluzione macromero / fotoiniziatore precedentemente preparato (~ 0,6 ml o fino a quando il modello è completamente coperto). </li><li> Tappare la fiala, avvolgere nel tessuto e posizionare verticalmente in una provetta da centrifuga. Centrifugare per 10 minuti a 1.260 xg per distribuire la soluzione macromero tutto il modello. </li><li> Togliere il foglio di alluminio, stappando la fiala ed esporre alla luce UV (365 nm, 25 W) per 3 min. O asciugare / N. </li><li> Rimuovere la &quot;scaffol sale contenentid &quot;dalla fiala con una pinzetta dopo aver realizzato e fratturazione della parte superiore della fiala di vetro. </li><li> In un becher da 400 ml, ~ preparare 200 ml di acqua / etanolo solvente (1: 1 vol: vol). </li><li> Mantenere il ponteggio sommerso in acqua / etanolo solvente per 4 giorni con variazioni giornaliere solvente. </li><li> Rimuovere il patibolo dal solvente e asciugare O / N. </li></ol> 3. L&#39;applicazione di Polydopamine Coating per SMP Scaffold (Figura 1) <ol><li> In 400 ml becher munito di ancoretta Teflon coperto, preparare ~ 200 ml di una soluzione di dopamina cloridrato (2 mg / ml in tampone Tris 10 mM, pH = 8,5, 25 ° C). Mescolare a ~ 150 rpm. </li><li> Inserire un ago monouso (lunghezza = 40 mm; scartamento = 20) nel scaffold, ~ metà della distanza attraverso il ponteggio. Avvolgere un filo attorno al mozzo ago. </li><li> Immergere il ponteggio (con il mozzo dell&#39;ago sopra la superficie della soluzione) nella soluzione dopamina agitazione ancorando il filo al bordo del becher. </li><li> Degassare il ponteggio inserendo una siringa nel mozzo dell&#39;ago e usarlo per estrarre aria dal ponteggio. (Nota: degassamento è completa quando non più aria può essere rimosso e la soluzione è completamente infiltrato il ponteggio.) </li><li> Mantenere il ponteggio immerso nella soluzione dopamina agitazione per 16 ore. </li><li> Rimuovere il ponteggio dalla soluzione e rimuovere l&#39;ago. Risciacquare con acqua deionizzata e asciugare in un forno sotto vuoto a temperatura ambiente per 24 ore. </li><li> Posizionare il patibolo in un forno 85 ° C per 1 ora. </li><li> Lasciare che il patibolo raffreddare a temperatura ambiente. L&#39;impalcatura cilindrico finale sarà ~ 6 mm di diametro x ~ 5 mm di altezza. </li></ol> 4. La valutazione del comportamento &quot;Self-fitting&quot; <ol><li> Preparare un &quot;irregolare modello di difetto CMF&quot; utilizzando un foglio di plastica rigida con spessore ~ ​​5 mm. Utilizzare un trapano per creare un vuoto all&#39;interno del foglio di plastica con un diametro medio di poco inferiore ~ 6 mm, come mostrato nella Figura 2A. </li><li> In abEaker, acqua deionizzata calore (che rappresenta l&#39;uso clinico di soluzione fisiologica) ad una temperatura di ~ 60 ° C. </li><li> Posizionare il patibolo nel bicchiere di ~ 60 ° C acqua. Utilizzare pinzette per spingere l&#39;impalcatura di sotto della superficie dell&#39;acqua, esponendo tutti i settori per l&#39;acqua. Continuare per ~ 2 minuti o fino a quando il ponteggio viene notevolmente ammorbidito (Figura 2B). </li><li> Rimuovere il patibolo dal bicchiere e premere immediatamente (a mano) nel modello di difetto. </li><li> Lasciare raffreddare a RT (~ 5-10 min) (Figura 2C). </li><li> Rimuovere dal difetto di osservare la nuova forma provvisoria fissa e il ritorno dello stato relativamente più rigido (Figura 2D). </li></ol> 5. Test Memoria plasmare il comportamento <ol><li> Utilizzando un analizzatore dinamico meccanico (DMA, ad esempio un TA Instruments Q800 qui usato), eseguire una prova di compressione deformazione controllata ciclica termica meccanica su un ponteggio in due cicli (N) per determinare la forma fissità (R f) eforma di recupero (R r) (Figura 3). <ol><li> Equilibrare a 60 ° C (T alto) per 5 min. </li><li> Comprimere una massima deformazione (ε m = 50%) a 50% / min. </li><li> Tenere a ε m (5 min). </li><li> Raffreddare a 25 ° C (T basso) e mantenere per 10 minuti per fissare la forma temporanea. </li><li> Rimuovere il carico. </li><li> Misurare la deformazione ultima nello stato privo di stress (ε u). </li><li> Riscaldare a 60 ° C (T alto) e mantenere per 10 minuti per recuperare la forma permanente. </li><li> Misurare il ceppo recuperato (ε p). </li><li> Mentre ancora a 60 ° C (T alto), avviare il ciclo ° 2 (N = 2) comprimendo la scaffold al 50% dell&#39;altezza recuperato dopo il 1 ° ciclo (N = 1). </li><li> Ripetere 5.1.3-5.1.8 Per N = 2. </li><li> Calcolare R f e R r per N = 1 e 2 con le seguenti equazioni: R f (N) = [ε u (N) / ε m] R r (N) = [ε m - ε p (N)] / [ε m - ε p (N -1)] </li></ol></li></ol> 6. Visualizing dimensione dei pori e Pore interconnettività <ol><li> Usando la microscopia elettronica a scansione (SEM, ad esempio, una FEI Quanta SEM usati nel presente documento), osservare la dimensione dei pori e pori interconnettività. <ol><li> Con una pinzetta per tenere il patibolo SMP, immergere in N liquido 2 per 1 min. </li><li> Rimuovere da N 2 liquido e frattura lungo il centro del ponteggio con una lama di rasoio pulito. </li><li> Utilizzando nastro di carbonio, apporre una delle due metà SMP scaffold sul campionein scena con la superficie fratturata rivolta verso l&#39;alto. </li><li> Sputter cappotto con Au-Pt (~ 4 nm). </li><li> Acquisire l&#39;immagine SEM ad una tensione di accelerazione consigliata di 10-15 kV (Figura 4A). </li></ol></li></ol> 7. Prova di Bioattività in vitro <ol><li> In una provetta da centrifuga da 50 ml, aggiungere ~ 30 ml di 1X SBF 24. </li><li> Ottenere un ponteggio nella sua originale forma permanente cilindrico moulding. Tagliare il ponteggio a metà (lungo il bordo circolare) usando una lama pulita. </li><li> Inserire un individuo a metà patibolo nel tubo da centrifuga preparato e berretto. </li><li> Mantenere il tubo a 37 ° C in un bagno d&#39;acqua in condizioni statiche senza modifiche SBF. </li><li> Dopo 14 giorni, rimuovere l&#39;impalcatura dalla SBF e asciugare all&#39;aria per 24 ore. </li><li> Con del nastro carbonio, apporre il patibolo sul palco del campione con la superficie fratturata rivolta verso l&#39;alto. </li><li> Sputter cappotto con Au-Pt (~ 4 nm). </li><li> Acquisire l&#39;immagine SEM ad un relodato tensione di accelerazione di 10-15 kV (Figura 4B). </li></ol>

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A., Kratz, K., Klein, F., L&#252;zow, K., Lendlein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermomechanical+behaviour+of+biodegradable+shape-memory+polymer+foams.">Thermomechanical behaviour of biodegradable shape-memory polymer foams.</a> 1190, (2009).</li><li>Luo, X., Mather, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+shape+memory+elastomeric+composites.">Preparation and characterization of shape memory elastomeric composites.</a> Macromolecules. 42, 7251-7253 (2009).</li><li>Lendlein, A., Schmidt, A. M., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AB-polymer+networks+based+on+oligo(&#603;-caprolactone)+segments+showing+shape-memory+properties.">AB-polymer networks based on oligo(&#603;-caprolactone) segments showing shape-memory properties.</a> Proc Natl Acad Sci. 98, 842-847 (2001).</li></ol>

The authors have nothing to disclose.

Questo protocollo descrive la preparazione di una impalcatura basato PCL polydopamine rivestite cui sé-montaggio comportamento, nonché osteoinduttività e bioattività, rende di interesse per il trattamento di difetti ossei irregolari CMF. Aspetti del protocollo può essere modificato per cambiare varie caratteristiche patibolo. Il protocollo inizia con acrylation di un PCL-diolo per consentire cura UV. Nell&#39;esempio riportato, il PCL-diolo M n è ~ 10.000 g / mol. Tuttavia, ...

Attualmente considerato il gold standard di cranio-maxillo-facciale (CMF) Trattamenti difetto osseo, trapianto di innesti autologhi raccolti è ostacolato da procedure di innesto complesse, sito donatore la morbilità e la disponibilità limitata 1. Una particolare difficoltà sta plasmando e fissa l&#39;autotrapianto rigida saldamente nel difetto al fine di ottenere osteointegrazione e per prevenire il riassorbimento dell&#39;innesto. L&#39;ingegneria dei tessuti è stata studiata come una strategia alternativa per autotrapianto e sostituti ossei sintetici (ad esempio cemento osseo) 2,3. Fondamentale per il successo di un approccio di ingegneria tissutale è un ponteggio con un set specifico di proprietà. In primo luogo, al fine di raggiungere l&#39;osteointegrazione, il ponteggio deve formare stretto contatto con il tessuto osseo adiacente 4. Il ponteggio deve essere anche osteoconduttivo, permettendo la migrazione delle cellule, la diffusione dei nutrienti e neotissue deposizione 4,5. Questo comportamento viene generalmente raggiunta con sca biodegradabileffolds esibendo un poro morfologia fortemente interconnesso. Infine, il ponteggio deve essere bioattivo in modo da promuovere l&#39;integrazione ed il legame con circostante tessuto osseo 5. Qui, vi presentiamo un protocollo per preparare una impalcatura ingegneria dei tessuti con queste proprietà. È importante sottolineare che questa impalcatura mostra la capacità di &quot;auto-fit&quot; in difetti irregolari CMF grazie al suo comportamento memoria di forma 6. Polimeri a memoria di forma Thermoresponsive (SMPS) sono noti per subire un cambiamento di forma in seguito all&#39;esposizione al calore 7,8. SMP è formata da &quot;NETPoint&quot; (cioè legami crociati chimici o fisici), che determinano la forma permanente e &quot;segmenti di commutazione&quot;, che mantengono la forma temporanea e recuperano la forma permanente. I segmenti di commutazione presentano una temperatura di transizione termica (T trans) corrispondente sia alla transizione vetrosa (Tg) o fondere transizione (T m) del polimero. ComeDi conseguenza, SMP può essere deformata in sequenza in una forma temporanea a T&gt; T trans, fisso a forma temporaneo T &lt;T trans, e recuperato alla forma permanente a T&gt; T trans. Così, un ponteggio SMP potrebbe realizzare &quot;self-fitting&quot; all&#39;interno di un difetto CMF come segue 6. Dopo l&#39;esposizione a scaldare salina (T&gt; T trans), un ponteggio SMP sarebbe diventato malleabile, permettendo un ponteggio cilindrica genericamente preparato per essere in un difetto irregolare pressato a mano, con recupero della forma promuovere l&#39;espansione del ponteggio al confine difetto. Al raffreddamento (T &lt;T trans), l&#39;impalcatura sarebbe tornato al suo stato relativamente più rigida, con forma fissità mantenere la sua nuova forma temporanea all&#39;interno del difetto. In questo protocollo, una impalcatura SMP è preparato da policaprolattone (PCL), un polimero biodegradabile studiato ampiamente per la rigenerazione dei tessuti e altre applicazioni biomediche 9-11. Per la memoria di forma, the T m di PCL funge T trans e varia tra 43 e 60 ° C, a seconda del peso molecolare del PCL 12. In questo protocollo, il T trans (vale a dire T m) del patibolo è 56,6 ± 0,3 ° C 6. Per raggiungere osteoconduttività, un protocollo è stato sviluppato per rendere scaffold SMP basati PCL con pori altamente interconnessi basati su un metodo 6,13,14-colata solvente particolato lisciviazione (SCPL). Polycaprolactone diacrilato (PCL-DA) (M n = ~ 10.000 g / mol) è stata utilizzata per permettere una rapida, reticolazione fotochimica ed è stato sciolto in diclorometano (DCM) per consentire solventi colata sul modello sale. Dopo polimerizzazione fotochimica ed evaporazione del solvente, il modello di sale è stato rimosso per lisciviazione in acqua. La dimensione media del sale regola dimensione dei pori patibolo. È importante sottolineare che il modello sale era fusa con acqua prima di solvente di fusione per ottenere pori interconnectivity. Bioattività è stata impartita al ponteggio SMP dalla formazione in situ di un rivestimento polydopamine sulle pareti dei pori 6. Bioattività è spesso introdotto ponteggi dall&#39;inclusione di vetro o vetroceramica riempitivi 15. Tuttavia, questi possono dare origine a indesiderati fragili proprietà meccaniche. La dopamina ha dimostrato di formare un aderente, polydopamine strato sottile su una varietà di substrati 16-19. In questo protocollo, l&#39;impalcatura SMP è stato sottoposto ad una soluzione leggermente basica (pH = 8,5) di dopamina per formare un rivestimento di nanothick polydopamine su tutte le superfici murali pori 6. Oltre a migliorare idrofilicità superficiale per migliorare l&#39;adesione cellulare e la diffusione, polydopamine ha dimostrato di essere bioattivo in termini di formazione di idrossiapatite (HAP) in seguito all&#39;esposizione al liquido corporeo simulato (SBF) 18,20,21. In un ultimo passaggio, l&#39;impalcatura rivestito viene esposto a trattamento termico a 85 ° C (T&gt; T trans) which porta a ponteggi densificazione. Il trattamento termico è stato precedentemente notato per essere essenziale per il comportamento a memoria di forma patibolo, forse a causa di domini cristallini PCL riorganizzazione di maggiore vicinanza 14. Abbiamo inoltre descritti i metodi per caratterizzare il comportamento auto-montaggio all&#39;interno di un modello di difetto irregolare, plasmare il comportamento in termini di memoria prove di compressione meccanica ciclico-termici deformazione controllata (cioè recupero della forma e modellare la fissità), poro morfologia, e in bioattività vitro. Strategie per adattare le proprietà impalcature sono anche presentati.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polycaprolactone-diol (Mn ~ 10,000 g/mol)</td>
			<td>Sigma-Aldrich</td>
			<td>440752</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma-Aldrich</td>
			<td>D65100</td>
			<td>Dried over 4A molecular sieves</td>
		</tr>
		<tr>
			<td>4-dimethylaminopyridine (DMAP)</td>
			<td>Sigma-Aldrich</td>
			<td>D5640</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine (Et3N)</td>
			<td>Sigma-Aldrich</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Acryloyl chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A24109</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>319902</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Carbonate (K2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>209619</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous magnesium sulfate (MgSO4)</td>
			<td>Fisher</td>
			<td>M65</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2-dimethoxy-2-phenyl acetophenone (DMP)</td>
			<td>Sigma-Aldrich</td>
			<td>196118</td>
			<td></td>
		</tr>
		<tr>
			<td>1-vinyl-2-pyrrolidinone (NVP)</td>
			<td>Sigma-Aldrich</td>
			<td>V3409</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>459844</td>
			<td></td>
		</tr>
		<tr>
			<td>Dopamine Hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>H8502</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffer (2mol/L)</td>
			<td>Fisher</td>
			<td>BP1759</td>
			<td>Used at 10 mM concentration, pH = 8.5</td>
		</tr>
		<tr>
			<td>Sieve</td>
			<td>VWR</td>
			<td>47729-972</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Transilluminator (365 nm, 25 W)</td>
			<td>UVP</td>
			<td>95-0426-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynamic Mechanical Analyzer (DMA)</td>
			<td>TA Instruments</td>
			<td>Q800</td>
			<td></td>
		</tr>
		<tr>
			<td>High Resolution Sputter Coater</td>
			<td>Cressington</td>
			<td>208HR</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope (SEM)</td>
			<td>FEI</td>
			<td>Quanta 600</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of a bioactive, pcl-based "self-fitting" shape memory polymer scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

La risultante SMP scaffold basato PCL è in grado di auto-montaggio in un modello CMF difetto (Figura 2). Dopo una breve esposizione a caldo salina (~ 60 ° C), il patibolo cilindrica ammorbidisce permettendo al patibolo da premere manualmente in ed espandere all&#39;interno del modello difetto. Dopo raffreddamento a RT, l&#39;impalcatura è fissato nella sua nuova forma temporanea che è trattenuta dopo la rimozione dal difetto. Il comportamento a memoria di forma di un pon...

Watch this Scientific Journal Video about Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold at JoVE.com

Fabrication of a Bioactive, PCL-based &quot;Self-fitting&quot; Shape Memory Polymer Scaffold

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Realizzazione di una sede a PCL Bioactive, &quot;Self-fitting&quot; Shape Memory Polymer Ponteggio

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

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Research

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Bioengineering

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

12.5K Views.  Texas A&M University. Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Video: Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Shape Memory Polymers per la Cultura cella attiva

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

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Bioingegneria

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1 Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2 Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3 Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5 Without this 'activation' process, SurgiLux films are readily removed.6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, &#039;SurgiLux&#039;: Preparation and Demonstration

The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.

Un Based Chitosano, Laser attivato sottile pellicola adesiva chirurgici, &#39;SURGILUX&#39;: Preparazione e dimostrazione

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Test microtrazione Environmentally-controllato di meccanica-adattivi nanocompositi polimerici per<em&gt; Ex vivo</em&gt; Caratterizzazione

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

Fabbricazione microfluidica di fibre polimeriche e bioibride con dimensioni e forma predesignate

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

Produzione robusta fibra naturale preforme Utilizzando batterica cellulosa come Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Fabbricazione di meccanicamente Tunable e Bioactive metallo Ponteggi per applicazioni biomediche

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Fabbricazione di invertito colloidale cristallo poli (glicole etilenico) Scaffold: una piattaforma Cell Culture tridimensionale per il fegato Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Fabbricazione e caratterizzazione dei fantasmi di tessuto ottico contenente macrostruttura

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Microdissection di segmenti di tessuto renale primario e incorporazione con tecnologia romanzo costrutto senza impalcatura

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

Fabbricazione scalabile di Stretchable, Dual Channel, organo di Microfluidic chip

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...