Os autores reconhecem o apoio do financiamento do NIH / NIDCR R00DE018212 (SPH), NIH/NIDCR-R01DE022032 (SPH), NIH / NIDCR T32 DE07306 (AJ, JDL), NIH / NCRR S10RR026645, (SPH) e os Departamentos de prevenção e reparação Odontologia e Ciências orofaciais, UCSF. Além disso, os autores reconhecem Xradia Graduate Fellowship (AJ), Xradia Inc., Pleasanton, CA. Os autores agradecem ao Dr. Kathryn Grandfield, UCSF por sua assistência com pós-processamento de dados; drs. Stephen Weiner e Gili Naveh, Weizmann Institute of Science, Rehovot, Israel, Dr. Ron Shahar, da Universidade Hebraica de Jerusalém, Israel por suas discussões criteriosas específicas para o dispositivo de carga em situ. Os autores também gostariam de agradecer Biomateriais e Bioengenharia microCT Imagem Facilidade na UCSF para o uso de Micro XCT eo dispositivo de carga em situ.

Alojamento dos animais e eutanásia: Todos os animais utilizados neste demonstração foram alojados em condições livres de patógenos, de acordo com as diretrizes do Animal Care e Use Comitê Institucional (IACUC) e do Instituto Nacional de Saúde (NIH). Fornecer animais com ração hard-pellet padrão e água ad lib. Eutanásia animais através de um método de duas etapas de asfixia dióxido de carbono, toracotomia bilateral, de acordo com o protocolo padrão da UCSF aprovado pelo IACUC. Realizar testes biomecânicos nas 24 horas após o sacrifício dos animais, para evitar a degradação do tecido. 1. Preparação e dissecção de um rato mandíbula ou maxila <ol><li> Retirar mandíbulas de ratos por meio do corte do tecido gentilmente anexos e tecido muscular membranosas preservando toda a mandíbula, incluindo o processo e o processo coronoide condilar (Figura 1) 15. </li><li> Hemimandíbula separados de carroefully cortando o tecido fibroso da sínfise mandibular com uma lâmina de bisturi. Nota: Os processos de coronárias e do côndilo e ramo da mandíbula (Figura 1) deve ser removido se obstruir fisicamente testes biomecânicos do 2 º molar. </li><li> Corte os incisivos, sem expor a câmara pulpar, para não dificultar o carregamento do molar. </li></ol> 2. Preparação de amostras para in situ à compressão de carga (Figura 2) <ol><li> Imobilizar a amostra sobre um topo de aço, utilizando um material que é significativamente mais duro do que o espécime experimentais antes de colocá-lo em um dispositivo de carregamento em situ (Figura 2A). Nota: polimetilmetacrilato (PMMA) foi utilizado para imobilizar a amostra em estudo e este excesso, se algum, foi removido usando um explorador dental. </li><li> Alinhe a superfície oclusal do molar (s) de interesse paralelo com o disco de amostra AFM de metal usando uma borda reta em ambosaviões (ou seja, mesial-distal e vestíbulo-lingual). </li><li> Criar uma calha com um instrumento contundente ao redor dos molares. Nota: Este espaço deve servir como um &quot;fosso&quot; para conter o excesso de líquido e manter a hidratação do tecido durante o carregamento em situ. </li><li> Preparar a superfície do dente ao construir-se para concêntrica (Figura 2B) ou excêntrico (Figura 2C) usando uma carga composta dental. Gravar a superfície do dente de interesse com o gel de ácido fosfórico a 35% na superfície oclusal de 15 seg. </li><li> Lavar o produto corrosivo abundantemente com água desionizada e secar a superfície usando uma seringa de ar / água ou um recipiente de ar comprimido. Com um explorador, espalhe uma gota de o agente de ligação em cúspides abertas em uma camada fina. Cure o composto com uma luz de cura dental. Nota: Todas as etapas que envolvem compósitos deve ser realizada sem a luz direta de uma lâmpada. Tais condições seriam indesejavelmente acelerar o processo de polimerização, e could evitar a colocação adequada do composto. Iluminação do quarto é aceitável. </li><li> Retire o excesso de agente de ligação a partir de dentes adjacentes com um bom bisturi ou lâmina de barbear. </li><li> Coloque compósito dentário fluida na superfície após a preparação da superfície e espalhá-lo dentro de ranhuras do molar (s) de interesse, utilizando um explorador dental. </li><li> Exponha o composto à luz de cura dental para 30 seg. </li><li> Moldar um acúmulo oclusal de cerca de 3-4 mm usando uma resina composta dental, a partir do plano oclusal do molar (s) de interesse e cura luz por 30 segundos. </li><li> Reduzir o início da formação de compósito com uma superfície plana paralela ao permitir que um regime de carga consistente em todos os espécimes, usando uma régua e um dispositivo manual de alta velocidade. Nota: Durante o teste biomecânico, outras amostras devem ser armazenadas em tris-fosfato de solução tamponada (TBS) com 50 mg / ml de penicilina, estreptomicina e 15. </li></ol> 3. Carregando Deriva de dispositivos eRigidez, capacidade material de propriedade de diferenciação, in situ Carregando da fibrosa Joint <ol><li> Fixar o espécime com o acúmulo de compósito sobre a bigorna da fase de carregamento, e de teste para uma carga uniforme, como mostrado na Figura 2B. </li><li> Coloque um papel de articulação sobre a superfície do compósito, seguido por carregar as amostras para uma carga finita para verificar a existência de carregamento concêntrica ou excêntrica (Figuras 2B e 2C). </li><li> Coloque Kimwipe TBS-embebido em torno da amostra para garantir a hidratação da amostra. Faça uma calha ao redor da amostra e preenchê-lo com TBS para manter o órgão hidratado durante o exame. </li><li> Carga de pico de entrada e taxa de deslocamento para o software Deben para comprimir o molar para uma carga de pico desejado a uma taxa de deslocamento após a imobilização do hemimandíbula. Nota: as leituras típicas deve incluir uma carga reacionário como o material é comprimido ao longo do tempo (sensibilidade do transdutor de carga = 0,1N). A partir do tempo de carga e tempo de deslocamento, a curva de carga-deslocamento para o material compactado deve ser obtida 16-18. Utilizando os dados recolhidos a partir dos ciclos de carga, várias propriedades do conjunto pode também ser determinada. A rigidez da junta deve ser calculada tomando o declive da porção linear (cerca de 30% do último dos dados) da fase de carregamento da carga versus deslocamento da curva 19. </li></ol> 4. Coloração de Tecidos Moles, o PDL, com ácido fosfotúngstico (PTA) Nota: Para aumentar o contraste de raios-X de atenuação, o PDL devem ser coradas com solução de 5% PTA 20. <ol><li> Solução de coloração Backfill PTA em uma limpa 1,8 ml carpule de vidro e coloque carpule carregado na seringa. </li><li> Injectar solução lentamente (5 min / carpule) para o espaço PDL de dentes adjacentes para evitar danos estruturais nos tecidos periodontais circundantes molar de interesse. Nota: Os passos acima deve be repete-se até cerca de 5 tubetes anestésicos completos (9 ml) de solução são injectados e deixada fluir para os tecidos circundantes. Os espécimes prepped também pode ser embebido durante a noite em solução de PTA restante (8 horas). </li></ol> 5. Recomendados Configurações de digitalização μ-XCT Realize m-XCT com as seguintes configurações de digitalização: <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> Objetivo Ampliação </td><td> 4X, 10X </td></tr><tr><td></td><td> 1.800 imagens </td></tr><tr><td> Tensão do tubo de raios-X </td><td> 75 kVp (50 kVp para amostras de PTA manchado) </td></tr><tr><td></td><td> 8 W </td></tr><tr><td> Tempo de Exposição </td><td> ~ 8-25 seg * </td></tr><tr><td></td><td> ~ 4 mm (objetiva de 4X), ~ 2 m (objetiva de 10X) ** </td></tr></tbody></table> * O tempo de exposição pode variar com base na geometria e a densidade óptica da amostra e do tubo de raios-X voltage. ** Resolução real de pixels será ligeiramente diferente com base na configuração da fonte, espécime, e detector.

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Os autores não têm nada a revelar.

O primeiro passo para estabelecer este protocolo envolveu a avaliação da rigidez da estrutura de carregamento por meio de um corpo rígido. Com base nos resultados, a rigidez foi significativamente mais elevada que permita a utilização do dispositivo de carregamento para mais testes de amostras com valores de rigidez significativamente inferiores. O segundo passo destaque a capacidade de o instrumento para distinguir diferentes valores de rigidez, utilizando duas fases da curva de carga-descarga gerados pela utiliza...

Vários métodos experimentais continuar a ser utilizado para investigar a biomecânica de articulações diartrodiais e fibrosas. Métodos específicos para a biomecânica do órgão de dente incluem o uso de medidores de tensão 1-3, métodos de fotoelasticidade 4, 5, Moiré interferometria 6, 7, eletrônico padrão speckle interferometria 8 e correlação de imagens digitais (DIC) 9-14. Neste estudo, a abordagem inovadora inclui não-invasivo através de raios-X para expor as estruturas internas de um conjunto fibroso (tecidos mineralizados e suas interfaces que consistem em zonas mais suaves, e uma interface de tecidos, tais como ligamentos) com cargas equivalentes para as condições in vivo. Um dispositivo de carregamento situ acoplados a um microscópio de micro-raios-X serão utilizados. O tempo de carga e as curvas de deslocamento de carga será recolhida como o molar de interesse dentro de um rato hemi-mandíbula recém-colhida é carregada. A mobjetivo ain da abordagem apresentada neste estudo é enfatizar o efeito da morfologia tridimensional do dente-osso, comparando as condições em: 1) sem carga e quando carregado, e quando 2) carregado de forma concêntrica e excêntrica. Eliminando a necessidade para os espécimes de corte, e para realizar experiências em órgãos inteiros intactos em condições de humidade para permitir a máxima preservação do estado de stress em 3D. Isso abre uma nova área de investigação para compreender os processos dinâmicos do complexo em vários cenários de carga. Neste estudo, os métodos para a biomecânica testes PDL dentro de um conjunto fibroso intacto de uma ratazana Sprague Dawley, um conjunto considerado como um sistema modelo de bioengenharia óptima será detalhado. Experimentos irá incluir a simulação de cargas da mastigação em condições hidratados, a fim de destacar três características importantes do conjunto como eles se relacionam com a biomecânica do nível do órgão. Os três pontos incluem: 1) força reacionária versus deslocamento:deslocamento do dente dentro do alvéolo e sua resposta reacionária do carregamento, 2) tridimensional (3D) de configuração espacial e morfometria: relação geométrica do dente com o alvéolo, e 3) mudanças nas leituras 1 e 2, devido a uma mudança na eixo de carga, ou seja, a partir de concêntrico para cargas excêntricas. Os três leituras fundamentais da técnica proposta pode ser aplicado para investigar a natureza adaptável de articulações em vertebrados, quer devido à mudança das exigências funcionais, e / ou doença. Mudanças nas leituras acima referidas, nomeadamente a correlação entre as cargas reacionários com deslocamento, e resultando em tempo de carga e carga-deslocamento curvas reacionárias em diferentes taxas de carregamento pode ser aplicada para destacar as mudanças globais em biomecânica. Eficácia do protocolo proposto será avaliado pelo acoplamento de leituras de testes mecânicos para morfometria 3D e biomecânica globais do conjunto.

<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

Este estudo demonstra um protocolo de testes de novas biomecânica. A vantagem deste protocolo inclui a utilização de um dispositivo de carregamento em situ acoplados a um microscópio de alta resolução de raio-X, permitindo assim a visualização dos elementos estruturais internos sob cargas fisiológicas simulados e condições de humidade. Espécimes experimentais incluirá ligamento periodontal osso intacto (PDL) de dentes articulações fibrosas. Os resultados irão ilustrar três características importantes do protocolo, como eles podem ser aplicados a biomecânica nível órgão: 1) força reacionária versus deslocamento: deslocamento do dente dentro do alvéolo e sua resposta reacionária do carregamento, 2) tridimensional (3D) configuração espacial e morfometria geométrica: relação do dente com o alvéolo, e 3) mudanças nas leituras 1 e 2, devido a uma mudança no eixo de carga, ou seja, a partir de concêntrica a cargas excêntricas. A eficácia do protocolo proposto será avaliada por acoplamento te mecânicaleituras picada a morfometria 3D e biomecânica global da articulação. Além disso, esta técnica vai enfatizar a necessidade de equilibrar as condições experimentais, as cargas especificamente reacionários antes de adquirir tomografias de articulações fibrosas. Deve notar-se que a proposta de protocolo é limitada a testes de amostras ex vivo sob condições, e que o uso de agentes de contraste para visualizar a resposta mecânica do tecido mole pode levar a conclusões erradas sobre tecidos e órgãos de nível biomecânica.

Estimativa de carga dispositivo &quot;backlash&quot;, &quot;pushback&quot;, rigidez, e deriva do sistema sob uma carga constante Folga: Entre carga e descarga de porções do ciclo, existe uma pausa de 3 segundos durante o qual engrena inversa dentro do motor antes da verdadeira descarga começa, por exemplo, como o modelo afasta-se da mandíbula superior (Figura 3). Este período é designado por uma folga no sistema, o que represe...

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

Neste estudo, será discutido o uso de um dispositivo de carga acoplada situ com tomografia de raios de micro-X calculado para biomecânica fibrosos. Leituras experimentais identificáveis ​​com uma mudança total na biomecânica incluem: 1) força reacionária versus deslocamento, deslocamento de dentes ou seja, dentro do alvéolo e sua resposta reacionária do carregamento, 2) tridimensional (3D) de configuração espacial e morfometria, ou seja geométrica relação do dente com o alvéolo, e 3) mudanças nas leituras 1 e 2, devido a uma mudança no eixo de carga, ou seja, cargas concêntricas ou excêntricas.

In situ compressão de carga e de imagem não invasivos Correlative do Osso-Ligamento periodontal de dentes fibrosa Joint

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 visualizações.  University of California San Francisco.  Neste estudo, será discutido o uso de um dispositivo de carga acoplada situ com tomografia de raios de micro-X calculado para biomecânica fibrosos. Leituras experimentais identificáveis ​​com uma mudança total na biomecânica incluem: 1) força reacionária versus deslocamento, deslocamento de dentes ou seja, dentro do alvéolo e sua resposta reacionária do carregamento, 2) tridimensional (3D) de configuração espacial e morfometria, ...

Vídeo: 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.

De alta resolução de fibra óptica para Microendoscopy In situ Celular imagem

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

Bioengenharia

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.

Demonstrar os usos do Spectrometer Novel Força Gravitacional para esticar e Medida Proteínas fibrosas

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.

Em síntese in vitro de Nativo, Espaçamento fibroso Long e Colágeno Espaçamento segmentar Longo

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.

Projeto de um biorreator Carregando Mecânica Biaxial para Engenharia de Tecidos

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 habilitado 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 ...

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.

Estudando a estequiometria do Receptor do Factor de Crescimento Epidérmico em células intactas utilizando Microscopia Correlativo

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.

Configuração otimizada e protocolo para a imagem latente magnética domínio em Situ histerese medicão

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.

Nanosensors para detectar Protease atividade In Vivo para diagnóstico não-invasivo

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.

Correlativo luz microscopia eletrônica de varredura (CLEM) para rastreamento e Imaging proteína Viral associado estruturas nas células Cryo-imobilizado

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

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

Isolamento, Expansão e Diferenciação de Células-Tronco Mesenquimais do Coxim de Gordura Infrapatelar da Articulação Sufocante de Cabra

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...