Gli autori riconoscono sostegno finanziario NIH / NIDCR R00DE018212 (SPH), NIH/NIDCR-R01DE022032 (SPH), NIH / NIDCR T32 DE07306 (AJ, JDL), NIH / NCRR S10RR026645, (SPH) e Dipartimenti di Scienze dentali prevenzione e di riparazione e Scienze oro-facciali, UCSF. Inoltre, gli autori riconoscono Xradia Graduate Fellowship (AJ), Xradia Inc., Pleasanton, CA. Gli autori ringraziano il Dott. Kathryn Grandfield, UCSF per la sua assistenza con elaborazione dei dati; Drs. Stephen Weiner e Gili Naveh, Weizmann Institute of Science, Rehovot, Israele, il dottor Ron Shahar, The Hebrew University di Gerusalemme, Israele per le loro discussioni perspicaci specifici del dispositivo situ di caricamento. Gli autori desiderano inoltre ringraziare Biomateriali e Bioingegneria microCT Imaging Strutture presso UCSF per l&#39;uso di micro XCT e il dispositivo situ di caricamento.

Stabulazione degli animali e l&#39;eutanasia: Tutti gli animali utilizzati in questa manifestazione sono stati alloggiati in condizioni esenti da organismi patogeni in accordo con le linee guida della cura degli animali e uso Comitato Istituzionale (IACUC) e l&#39;Istituto Nazionale della Salute (NIH). Fornire gli animali con lo standard hard-pellet ratto chow e ad acqua lib. Euthanize animali attraverso un metodo in due fasi di asfissia anidride carbonica, toracotomia bilaterale secondo il protocollo standard di UCSF approvato dalla IACUC. Eseguire test biomeccanico entro 24 ore di sacrificio di animali per evitare il degrado del tessuto. 1. Preparazione e dissezione di un ratto mandibola o mascella <ol><li> Rimuovere mandibole ratto da recidere delicatamente allegati tessuto e tessuto muscolare membranose preservando tutta la mandibola, compreso il processo coronoideo e il processo condilare (Figura 1) 15. </li><li> Hemimandibles separate in autoefully taglio del tessuto fibroso di sinfisi mandibolare con una lama di bisturi. Nota: I processi coronarici e condilari e ramo della mandibola (figura 1) devono essere rimossi se fisicamente ostacolano il test biomeccanico del molare 2 °. </li><li> Tagliare gli incisivi senza esporre la camera pulpare per non ostacolare il caricamento del molare. </li></ol> 2. Preparazione del campione per in situ compressione in fase di caricamento (figura 2) <ol><li> Immobilizzare il campione su uno stub acciaio utilizzando un materiale che è significativamente più rigido del campione sperimentali prima di caricare in un dispositivo in situ di carico (Figura 2A). Nota: polimetilmetacrilato (PMMA) è stato usato per immobilizzare il campione in questo studio e eventuale eccedenza, è stato rimosso utilizzando un esploratore dentale. </li><li> Allineare la superficie occlusale del molare (s) di interesse parallelo con il disco di preparato AFM metallo con un regolo in entrambipiani (cioè mesiale-distale e vestibolo-linguale). </li><li> Creare una depressione con un corpo contundente che circonda i molari. Nota: Questo spazio dovrebbe servire come un &quot;fossato&quot; per contenere il liquido in eccesso e mantenere l&#39;idratazione dei tessuti durante il caricamento in situ. </li><li> Preparare la superficie del dente per costruire concentrici (Figura 2B) o eccentrico (Fig. 2C) carico con un composito dentale. Incidere la superficie del dente di interesse con il 35% gel di acido fosforico sulla superficie occlusale per 15 sec. </li><li> Sciacquare il mordenzante accuratamente con acqua deionizzata e asciugare la superficie utilizzando una siringa ad aria / acqua o una bomboletta ad aria compressa. Con un esploratore, diffondere una goccia di collante in cuspidi aperte in uno strato sottile. Curare il composito con una luce di polimerizzazione dentale. Nota: Tutti i passi che coinvolgono i compositi devono essere eseguiti senza luce diretta di una lampada. Tali condizioni sarebbe indesiderabile accelerare il processo di polimerizzazione, e could impedire il corretto posizionamento del composito. L&#39;illuminazione è accettabile. </li><li> Rimuovere il collante in eccesso dai denti adiacenti con un bel bisturi o lametta. </li><li> Posizionare fluido composito dentale sulla superficie a seguito della preparazione della superficie e diffondere nelle scanalature del molare (s) di interesse utilizzando un esploratore dentale. </li><li> Esporre il composito alla luce polimerizzazione dentale per 30 sec. </li><li> Modellare un accumulo occlusale di circa 3-4 mm utilizzando un composito di resina dentale, dal piano occlusale del molare (s) di interesse e polimerizzare per 30 sec. </li><li> Ridurre all&#39;inizio della formazione di composito a una superficie piana parallela per consentire un regime loading coerente in tutti i campioni utilizzando un regolo e un manipolo ad alta velocità. Nota: Durante il test biomeccanico, altri campioni devono essere conservati in tris-fosfato soluzione tamponata (TBS) con 50 mg / ml di penicillina e la streptomicina 15. </li></ol> 3. Caricamento Drift dispositivo eRigidità, Proprietà del materiale Differenziare Capability, in situ carico del comune Fibrosa <ol><li> Fissare il campione con l&#39;accumulo composito sull&#39;incudine della fase di carico e di test per il caricamento uniforme come illustrato nella Figura 2B. </li><li> Inserire un carta dell&#39;articolazione sulla superficie del composito seguita caricando il campione ad un carico finito per controllare carico concentrico o eccentrico (Figure 2B e 2C). </li><li> Posizionare Kimwipe TBS-imbevuto intorno al provino per garantire esemplare idratazione. Effettuare una depressione intorno al campione e riempirlo con TBS per mantenere l&#39;organo idratato durante l&#39;imaging. </li><li> Carico massimo di ingresso e velocità di spostamento nel software Deben per comprimere il molare di un carico di picco desiderato ad un tasso di spostamento a seguito immobilizzazione del emimandibola. Nota: letture tipiche dovrebbero includere un carico reazionaria come il materiale è compresso nel tempo (sensibilità dinamometri = 0.1N). Dal tempo di caricamento e di spostamento-tempo, una curva carico-spostamento per il materiale compresso deve essere ottenuta 16-18. Utilizzando i dati raccolti dai cicli di carico, varie proprietà del comune può anche essere determinate. La rigidità del giunto deve essere calcolato dalla pendenza della porzione lineare (ultime 30% dei dati) della fase di caricamento del carico curva della cilindrata 19. </li></ol> 4. La colorazione dei tessuti molli, il Pdl, con acido fosfotungstico (PTA) Nota: Per aumentare il contrasto attenuazione dei raggi X, il Pdl deve essere trattata con soluzione al 5% PTA 20. <ol><li> Soluzione colorante Backfill PTA in un ambiente pulito da 1,8 ml in vetro carpule e mettere carpule caricato nella siringa. </li><li> Iniettare la soluzione lentamente (5 min / carpule) nel PDL-spazio di denti adiacenti per evitare danni strutturali ai tessuti parodontali molare di interesse circostanti. Nota: I passaggi di cui sopra dovrebbe be ripetuto fino a circa 5 carpule completi (9 ml) di soluzione vengono iniettati e lasciato scorrere nei tessuti circostanti. Gli esemplari prepped possono anche essere messi a bagno durante la notte nella soluzione PTA rimanente (8 ore). </li></ol> 5. Le impostazioni di scansione μ-XCT consigliati Eseguire m-XCT con le seguenti impostazioni di scansione: <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> Obiettivo Ingrandimento </td><td> 4X, 10X </td></tr><tr><td></td><td> 1.800 immagini </td></tr><tr><td> X-ray tensione del tubo </td><td> 75 kVp (50 kVp per i campioni PTA macchiato) </td></tr><tr><td></td><td> 8 W </td></tr><tr><td> Tempo di esposizione </td><td> ~ 8-25 sec * </td></tr><tr><td></td><td> ~ 4 micron (obiettivo 4X), ~ 2 micron (obiettivo 10X) ** </td></tr></tbody></table> * Tempo di esposizione può variare in base alla geometria e densità ottica del campione e X-ray tube voensione. ** Pixel di risoluzione effettiva sarà leggermente diverso in base alla configurazione della sorgente, del campione, e il rivelatore.

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Gli autori non hanno nulla da rivelare.

Il primo passo per stabilire questo protocollo coinvolto valutare la rigidità del telaio di carico utilizzando un corpo rigido. Sulla base dei risultati, la rigidità era significativamente superiore che consente l&#39;utilizzo del dispositivo di caricamento per ulteriori test di campioni con valori di rigidezza significativamente più bassi. Il secondo passo evidenziato la capacità dello strumento di distinguere diversi valori di rigidità utilizzando due fasi della curva carico-scarico generati utilizzando un corpo ...

Diversi metodi sperimentali continuano ad essere utilizzati per studiare la biomeccanica delle articolazioni diartrodiali e fibrosi. Metodi specifici per la biomeccanica organo dente includono l&#39;uso di estensimetri 1-3, metodi fotoelasticità 4, 5, Moiré interferometria 6, 7, elettronico modello speckle interferometria 8, e la correlazione di immagini digitali (DIC) 9-14. In questo studio, l&#39;approccio innovativo comprende non invasivo utilizzando raggi X per esporre le strutture interne di un giunto fibroso (tessuti mineralizzati e le loro interfacce costituiti da zone più morbide, e interfacciamento tessuti come legamenti) con carichi equivalenti a condizioni in vivo. Verrà utilizzato un dispositivo situ loading accoppiato ad un microscopio micro-X-ray. Il carico-tempo e le curve carico-spostamento saranno raccolte come il molare di interesse all&#39;interno di un ratto emi-mandibola appena raccolto viene caricato. Il main obiettivo dell&#39;approccio presentato in questo studio è sottolineare l&#39;effetto della morfologia tridimensionale del dente-osso dal confronto a: 1) vuoto e quando caricato, e quando 2) concentricamente ed eccentricamente caricati. Eliminando la necessità di esemplari di taglio, e per eseguire esperimenti sugli interi organi intatti in condizioni di bagnato consentirà la massima conservazione dello stato di stress in 3D. Si apre una nuova area di ricerca nella comprensione dei processi dinamici del complesso sotto diversi scenari di carico. In questo studio, i metodi di biomeccanica test PDL all&#39;interno di un giunto fibroso intatto di un ratto Sprague Dawley, una joint considerato come un ottimo sistema modello bioingegneria saranno dettagliati. Gli esperimenti comprenderanno la simulazione dei carichi della masticazione in condizioni idratati, al fine di sottolineare tre importanti caratteristiche del giunto come si riferiscono alla biomeccanica livello di organi. I tre punti comprendono: 1) forza reazionaria vs spostamento:spostamento dente all&#39;interno della presa alveolare e la sua risposta reazionaria al carico, 2) tridimensionale (3D) configurazione spaziale e morfometria: relazione geometrica del dente con la presa alveolare, e 3) variazioni letture 1 e 2, a causa di un cambiamento nella asse di carico, cioè da concentrica a carichi eccentrici. Le tre letture fondamentali della tecnica proposta può essere applicata per indagare la natura adattiva giunti nei vertebrati a causa di cambiamenti di esigenze funzionali, e / o malattia. Le variazioni nelle previsioni di cui sopra, in particolare la correlazione tra i carichi reazionari con spostamento, e la conseguente carico di tempo e di carico-spostamento curve reazionari a differenti tassi di carico possono essere applicati per evidenziare i cambiamenti globali nel biomeccanica articolare. Efficacia del protocollo proposto sarà valutata mediante accoppiamento letture prove meccaniche di morfometria 3D e biomeccanica complessive del giunto.

<table border="0" cellpadding="0" cellspacing="0" width="652">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Bard Parker Blade</td>
			<td style="width:101px;">BD</td>
			<td style="width:123px;">MEDC-001054</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">AFM metal disk</td>
			<td style="width:101px;">Ted Pella</td>
			<td style="width:123px;">16218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Polymethyl methacrylate&#160;</td>
			<td style="width:101px;">GC America</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Uni-Etch</td>
			<td style="width:101px;">Bisco</td>
			<td style="width:123px;">E5502EBM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Optibond Solo Plus</td>
			<td style="width:101px;">Kerr Corp</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Filtek Flow</td>
			<td style="width:101px;">3M</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Hurculite Ultra</td>
			<td style="width:101px;">Kerr</td>
			<td style="width:123px;">34346</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Tris buffer</td>
			<td style="width:101px;">Mediatech Inc.</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Articulating paper</td>
			<td style="width:101px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:169px;">Phosphotungstic Acid</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:123px;">HT152</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ compressive loading and correlative noninvasive imaging of the bone-periodontal ligament-tooth fibrous joint

Questo studio dimostra un protocollo di test nuovi biomeccanica. Il vantaggio di questo protocollo prevede l&#39;uso di un dispositivo in situ carico accoppiato ad un microscopio a raggi X ad alta risoluzione, permettendo la visualizzazione di elementi strutturali interne sotto carichi fisiologici simulati e bagnata. I campioni sperimentali comprenderanno legamento osso parodontale integro (PDL)-dente articolazioni fibrose. I risultati illustrano tre caratteristiche importanti del protocollo in quanto possono essere applicate alla biomeccanica livello di organi: 1) forza reazionaria vs spostamento: lo spostamento del dente nella presa alveolare e la sua risposta reazionaria a carico, 2) tridimensionale (3D) configurazione spaziale e morfometria: relazione geometrica del dente con la presa alveolare, e 3) variazioni di letture 1 e 2, a causa di un cambiamento di asse, cioè dal concentrico a carichi eccentrici. Efficacia del protocollo proposto sarà valutata mediante accoppiamento meccanico teletture Sting morfometria 3D e biomeccanica complessive del giunto. Inoltre, questa tecnica risalto sulla necessità di equilibrare condizioni sperimentali, carichi specificamente reazionarie precedentemente all&#39;acquisizione tomogrammi di articolazioni fibrose. Va osservato che il protocollo proposto è limitato al test campioni ex vivo sotto condizioni, e che l&#39;uso di agenti di contrasto per visualizzarne la risposta meccanica dei tessuti molli potrebbe portare a conclusioni errate circa tessuto e livello organo biomeccanica.

Stima di carico dispositivo &quot;gioco&quot;, &quot;push back&quot;, rigidità, e la deriva del sistema sotto un carico costante Backlash: Tra carico e scarico porzioni del ciclo, esiste una pausa di 3 secondi durante i quali retromarce nel motore prima vera scarico comincia, cioè come il campione si allontana dalla ganascia superiore (Figura 3). Questo periodo viene indicato come un gioco nel sistema, che rappresenta un intervall...

Watch this Scientific Journal Video about In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint at JoVE.com

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In questo studio, sarà discusso l&#39;uso di un dispositivo in situ loading accoppiato con tomografia micro-computerizzata a raggi X per la biomeccanica articolare fibrosi. Letture sperimentali identificabili con una variazione complessiva delle biomeccanica articolare saranno: 1) forza reazionaria contro lo spostamento, vale a dire lo spostamento del dente nella presa alveolare e la sua risposta reazionaria a carico, 2) tridimensionale (3D) configurazione spaziale e morfometria, cioè geometrico rapporto del dente con la presa alveolare, e 3) variazioni letture 1 e 2 a causa di un cambiamento di asse, cioè carichi concentrici o eccentrici.

In situ carico di compressione e Correlativo imaging non invasiva del Bone-parodontale legamento-dente Fibrosa comune

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

in-situ-compressive-loading-correlative-noninvasive-imaging-bone

Research

JoVE Journal

Bioengineering

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

13.2K Views.  University of California San Francisco.  In questo studio, sarà discusso l'uso di un dispositivo in situ loading accoppiato con tomografia micro-computerizzata a raggi X per la biomeccanica articolare fibrosi. Letture sperimentali identificabili con una variazione complessiva delle biomeccanica articolare saranno: 1) forza reazionaria contro lo spostamento, vale a dire lo spostamento del dente nella presa alveolare e la sua risposta reazionaria a carico, 2) tridimensionale (3D) configuraz...

Video: In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Ad alta risoluzione in fibra ottica per Microendoscopy In situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Ricerca

Bioingegneria

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

Dimostrare l&#39;Usi del romanzo Spectrometer forza gravitazionale su Stretch e misura proteine ​​fibrose

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.

In sintesi in vitro di Native, Spaziatura fibrosa lungo e segmentale collagene spaziatura lungo

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

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

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

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

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

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

FIBS-enabled non invasiva Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Electrospinning Growth Factor Releasing Microsfere in Fibrosi Ponteggi

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

Studiare la Stechiometria di Epidermal Growth Factor Receptor in cellule intatte con correlativa Microscopia

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

Installazione ottimizzata e protocollo per l'Imaging di dominio magnetico con In Situ isteresi misura

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Nanosensori per rilevare attività della proteasi In Vivo per la diagnostica non invasiva

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Microscopia a luce correlativa (CLEM) per il monitoraggio e la proteina virale di Imaging strutture in cellule di Cryo-immobilizzata associate

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...