Este trabalho foi financiado pelo departamento de Civil e engenharia ambiental e da faculdade de engenharia da Universidade de Illinois em Urbana-Champaign. Reconhecemos a Ravindra Kinra e Kavita Kinra Fellowship para apoio a pós-graduação de Kavya Mendu. Investigação de microscopia eletrônica de varredura foi realizada nas instalações do laboratório de pesquisa de Material Frederick Seitz e Beckman Institute na Universidade de Illinois em Urbana-Champaign.

Nota: O protocolo descrito aqui, segue as diretrizes de cuidados com animais do Comité de uso e cuidados de Animal institucionais de Illinois.
1. o modelo contratos
<ol><li>Coletar recém-colhidas fêmures bovina de um departamento de Estados Unidos de agricultura USDA-certificada matadouro e transportá-los em sacos de plástico de ar apertado em um refrigerador. Nota: Para o estudo realizado aqui, fêmures foram coletadas de animais que foram 24 a 30 meses, alimentado com milho e pesava cerca de 1.000 - 1.100 libras.</li>
	<li>Congele os fêmures no –20 ° C até o início do processo de preparação de espécime. Esta temperatura mantém os fêmures fresco12,13,14.</li>
</ol>2. corte, limpeza e incorporando os espécimes
<ol><li>Descongele os fêmures congelados em um recipiente com água por cerca de 2 h à temperatura ambiente.</li>
	<li>Corte vários discos de cerca de 10-15 mm de espessura da região média da diáfise usando uma serra de fita mesa top diamante para produzir espécimes com área de seção transversal uniforme do osso cortical.</li>
	<li>Use um kit de dissecação para remover qualquer tecido mole ou carne anexado ao osso cortical.</li>
	<li>As seções transversais dos fêmures obtidas na etapa 2.2 usando uma lâmina de diamante-wafering em uma velocidade baixa serra sob condições de chuva ao longo do eixo longitudinal do osso para obter várias seções aproximadamente cuboidal. Nota: Aqui, apenas a preparação das amostras e zero testes realizados sobre o short – amostras longitudinais são discutidas. No entanto, exceto para a direção do corte, o procedimento de preparação permanece o mesmo para a orientação transversal.</li>
	<li>Limpe os espécimes em uma solução preparada usando 1,5% aniônico limpa e água sanitária de 5% para uma duração de 20 min em um líquido de limpeza ultra-sônico.</li>
	<li>
Incorporar os espécimes de osso cortical em resina acrílica (neste documento methacrylate de polymethyl (PMMA)) para facilitar a manipulação e a estabilidade.
	<ol>
<li>Para incorporar os espécimes, primeiro revesti as paredes do molde com um agente de liberação. Em seguida, misture a resina acrílica e o endurecedor num copo, de acordo com as instruções dadas pelo fabricante do PMMA.</li>
		<li>Coloque um dos espécimes cortar osso cortical em cada molde com a superfície a ser riscado virado para baixo. Deite a mistura de resina acrílica para estes preparados suportes de amostra. Deixe os espécimes cura para uma duração de até 4-5 h.</li>
	</ol>
</li>
	<li>Cortar os espécimes incorporados em discos de espesso de 5 mm, expondo a superfície a ser riscado, usando a baixa velocidade vi e montar os espécimes para discos de metal (alumínio) de diâmetro 34 mm e altura 5 mm usando adesivo de cianoacrilato.</li>
	<li>Embrulhe os espécimes em um indicador embebido em novelos balanceada salina solução (HBSS) e refrigerar a 4 ° C até o uso mais de15,16.</li>
</ol>3. esmerilhamento e polimento de protocolos
Nota: Um pré-requisito para testes de alta precisão em escalas de comprimento pequeno é uma superfície lisa e nivelada de espécimes. Anterior de polimento protocolos13,17 resultar em uma grande rugosidade da superfície, levando a considerável imprecisão na medição. O desafio reside em conseguir baixa média rugosidade da superfície, menos de 100 nm, sobre uma superfície de2 de 3 x 8 mm de grande área.
<ol><li>Moa as amostras de osso cortical bovino à temperatura ambiente utilizando grão 400 e 600 trabalhos de carboneto de silício de grão por 1 min e 5 min, respectivamente. Manter o moedor-polidor a base velocidades de 100 rpm e 150 rpm, respectivamente.</li>
	<li>Máquina de moer os espécimes de osso cortical bovino à temperatura ambiente sobre os documentos de grão 800 e 1.200 para uma duração de 15 min para cada etapa. Manter o moedor-polidor em uma velocidade base de 150 rpm, velocidade da cabeça de 60 rpm e operando com carga de 1 lb.</li>
	<li>Polonês os espécimes usando 3 µm e 1 µm 0,25 µm soluções de suspensão de diamante na mesma ordem em um duro, perfurado, pano não tecido para uma duração de 90 min à temperatura ambiente. Manter a carga de funcionamento de cada etapa em 1 lb com as velocidades de base e cabeça do polidor a 300 rpm e 60 rpm, respectivamente.</li>
	<li>Polir a amostra usando solução de suspensão 0,05 µm da alumina em um pano macio, sintética rayon para uma duração de 90 min no 1 lb com base e cabeça a velocidade de 100 rpm e 60 rpm, respectivamente, também à temperatura ambiente.</li>
	<li>Colocar as amostras em um copo com água desionizada e colocar o béquer em um banho ultra-sônico por 2 min entre cada etapa consecutiva de esmerilhamento e polimento para limpar os resíduos e evitar a contaminação cruzada.</li>
	<li>Ver os as características de superfície usando microscopia ótica e SEM imagem. Nota: Como mostrado na Figura 1, osteomas, canais de Havers, linhas de cimento, regiões intersticiais e lacunas foram observadas em espécimes do osso cortical bovino. Esses métodos de imagem revelam a natureza porosa, heterogênea e anisotrópica de espécimes de osso cortical. Além disso, avançado exame de superfície das amostras foi realizada para avaliar a qualidade da superfície polida. Uma superfície polida representativa é mostrada na Figura 2.</li>
</ol>4. micro teste de alergia
Nota: Micro arranhão de testes é realizado sobre as amostras de osso cortical bovino polido usando um micro testador de zero (Figura 3). Um diamante do indenter de Rockwell com um raio de 200 µm e apex ângulo de 120° é usado para o estudo. O instrumento permite a aplicação de uma carga linear progressiva até 30 s. Além disso, o instrumento é equipado com sensores de alta precisão para medir a carga horizontal, profundidade de penetração e emissões acústicas geradas devido a coçar. O instrumento pode capturar os panoramas de sulcos zero.
<ol><li>Antes de ensaios de amostras de osso cortical, calibre a ponta do indenter de Rockwell usando policarbonato como material de referência3.</li>
	<li>Colocar a amostra de osso cortical no palco e escolha o site de teste de alergia, usando o microscópio ótico configurar integrado ao módulo de micro testador de zero.</li>
	<li>Aplicar uma carga linear progressiva com uma carga inicial de 30 mN e final de carga de 30 s. A taxa de carregamento deve ser definida como 60 N/min e o comprimento de zero a 3 mm.</li>
	<li>Executar a série de testes de zero sobre o curta longitudinal (Figura 3b) amostras de osso cortical bovino, conforme ilustrado na Figura 3.</li>
	<li>Molhe a superfície do espécime com HBSS após um conjunto de todos os três a quatro testes de zero para mantê-los hidratados.</li>
	<li>Analise os dados de teste cutâneo com base na mecânica da fratura não-linear modelagem2.</li>
</ol>

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+technique+for+characterizing+the+fracture+toughness+via+scratch+test+experiments.">An improved technique for characterizing the fracture toughness via scratch test experiments.</a> Wear. 313 (1-2), (2014).</li><li>Akono, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energetic+size+effect+law+at+the+microscopic+scale:+Application+to+progressive-load+scratch+testing.">Energetic size effect law at the microscopic scale: Application to progressive-load scratch testing.</a> J of Nanomech and Micromech. 6 (2), (2016).</li><li>Kataruka, A., Mendu, K., Okeoghene, O., Puthuvelil, J., Akono, A. -. 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Os autores não têm nada para divulgar.

Testes de zero micro induzir uma fratura de modo misto3. Além disso, nas amostras curtas longitudinais osso cortical bovino, processos de fratura são ativados como a sonda cava mais fundo. Para um arranhão de longo de 3 mm, o volume prismático sondado é cerca de 3.600 µm de comprimento, 600 µm de largura e 480 µm profundo. Este grande volume ajudou em prever uma resposta homogeneizada. Um modelo de mecânica de fratura não-linear permitiu-nos extrair a resistência da fratura baseada o <e...

Neste estudo, podemos medir a tenacidade à fratura de osso compacto bovino na mesoescala (osteomas) para a microescala (nível lamelar) usando uma nova técnica de zero micro1,2,3,4, 5. Processos de fratura incluindo propagação iniciação e crack no osso sejam diretamente influenciados pelas escalas de comprimento devido a diferentes componentes estruturais e organização em diferentes níveis de hierarquia. Portanto, avaliar a fratura óssea em menores escalas de comprimento é essencial rendendo um entendimento fundamental de fragilidade óssea. Por um lado, compacto de testes convencionais, tais como três pontos de flexão, tensão, e testes de flexão são comumente realizadas em bovino fêmur e tíbia para caracterização de fratura em escala macroscópica6,7, 8. por outro lado, para medir a tenacidade à fratura em escala microscópica, fratura de recuo do Vicker foi proposto9. Micro recuo foi executado usando do indenter da Vicker para gerar rachaduras radiais. Além disso, o método de dureza Oliver Pharr nanoindentação fratura foi realizado usando um cubo afiada canto do indenter10.
Os estudos de resistência de fratura nanoindentação baseado acima, os comprimentos das fissuras gerados foram medidos pelo observador e um modelo semi-empíricos foi utilizado para calcular a tenacidade à fratura. No entanto, esses métodos são reprodutíveis, subjetivo, e os resultados são altamente dependentes da habilidade do observador, devido à necessidade de medir os comprimentos de rachadura usando microscopia ótica ou microscopia eletrônica. Além disso, zero testes foram conduzidos em escala nanométrica, mas o modelo matemático subjacente não é baseado em física que não leva em conta a redução da resistência devido a rachaduras e defeitos11. Assim, existe uma lacuna de conhecimento: um método para avaliação de fratura em nível microscópico com base em um modelo mecanicista baseado em física. Esta lacuna de conhecimento motivado a aplicação de micro arranhão testes para compactar osso focando primeiro espécimes suínos5. O estudo foi ainda mais estendido agora para entender o osso cortical bovino.
Duas orientações diferentes dos espécimes são possíveis: transversal longitudinal e a curta distância longitudinal. Longitudinal transversal corresponde a fratura Propriedades perpendiculares ao eixo longitudinal do fêmur. Considerando que, a curto, longitudinal corresponde às propriedades fratura ao longo do eixo longitudinal do fêmur5. Neste estudo, aplicamos testes zero de ossos corticais bovina para caracterizar a resistência de fratura do osso no sentido longitudinal curta.

<table>
	<tbody>
		<tr>
			<td>Table Top Diamond Band Saw</td>
			<td>McMaster Carr, Elmhurst, IL</td>
			<td>Model&#160; C-40</td>
			<td>Blade speed of 40 mph; Blade dimensions: 37 inch in diameter, 0.02 inch wide and 0.14 inch deep</td>
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			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>112780</td>
			<td>Blade speed in the range of 200-5000 rpm in 50 rpm incrments; 8 inch diamond wafering blade</td>
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			<td>Branson 5800 Ultrasonic Cleanser</td>
			<td>(Through) Grainger, Peoria, Illinois</td>
			<td>39J365</td>
			<td>Bransonic CPXH ultrasonic bath has a tank capacity of 2.5 gal</td>
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			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>497250</td>
			<td>8 inch base plate with a speed range from 10-500 rpm</td>
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			<td>Anton Paar Switzerland AG</td>
			<td>163251</td>
			<td>Compact Platform, Acoutstic Emission Sensor</td>
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			<td>JEOL 6060LV general purpose scanning electron microscope</td>
			<td>JEOL USA, Inc., Peabody, MA</td>
			<td></td>
			<td>Environmental scanning electron microscope which enables imaging at low vacuum levels.</td>
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			<td>Philips XL30 ESEM FEG&#160;</td>
			<td>FEI Company</td>
			<td></td>
			<td>Wet mode working of the instrument enables imaging of non conductive samples without altering them&#160;</td>
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			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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			<td>Consumables</td>
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			<td>Bovine Femur</td>
			<td>L&#38;M Slaughter house, Georgetown, IL</td>
			<td></td>
			<td>Corn fed, 24-30 month old mature bovine specimens.</td>
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			<td>Alconox Powdered Precision Cleaner</td>
			<td>Alconox, Inc., 30 Glenn St., Ste. 309, White Plains, NY, 10603</td>
			<td>1104-1</td>
			<td>Biodegradable, Non caustic, Interfering-residue free</td>
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			<td>Electron Microscopy Sciences</td>
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			<td>CarbiMet SiC Abrasive Paper 400 grit, 8 inch, PSA backed</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>36080400</td>
			<td>Grinding - Abrasive Papers</td>
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			<td>CarbiMet SiC Abrasive Paper 600 grit, 8 inch, PSA backed</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>36080600</td>
			<td>Grinding - Abrasive Papers</td>
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			<td>MicroCut Discs 800 grit, 8 inch, PSA backed</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>36080800</td>
			<td>Grinding - Abrasive Papers</td>
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			<td>MicroCut Discs 800 grit, 8 inch, PSA backed</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>16081200</td>
			<td>Grinding - Abrasive Papers</td>
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			<td>Texmet P For 8&#39;&#39; Wheel PSA</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>407638</td>
			<td>Polishing Cloth</td>
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			<td>8&#39;&#39; Microcloth PSA</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>407518</td>
			<td>Polishing Cloth</td>
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			<td>Meta Di Supreme Polycrystalline Diamond Suspension, 3 &#181;m</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
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			<td>Polishing suspension</td>
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			<td>Meta Di Supreme Polycrystalline Diamond Suspension, 1 &#181;m</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>406630</td>
			<td>Polishing suspension</td>
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			<td>Meta Di Supreme Polycrystalline Diamond Suspension, 0.25 &#181;m</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>406629</td>
			<td>Polishing suspension</td>
		</tr>
		<tr>
			<td>MasterPrep Polishing Suspension, 0.05&#181;m</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>40-6377-032</td>
			<td>Polishing suspension</td>
		</tr>
		<tr>
			<td>HBSS, calcium, magnesium, no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>14025126</td>
			<td>Buffer Solution</td>
		</tr>
	</tbody>
</table>

fragility assessment of bovine cortical bone using scratch tests

Osso é um material complexo hierárquico com cinco níveis distintos de organização. Fatores como o envelhecimento e doenças como a osteoporose aumentam a fragilidade do osso, tornando-se sujeito a fratura. Devido ao grande impacto sócio-económico de fratura óssea na nossa sociedade, há uma necessidade de novas formas de avaliar o desempenho mecânico de cada nível hierárquico do osso. Embora a rigidez e resistência podem ser analisados em todas as escalas – nano, micro, meso-, e macroscópica – avaliação de fratura está até agora confinada aos ensaios macroscópicos. Essa limitação restringe a nossa compreensão de fratura óssea e restringe o escopo de laboratório e estudos clínicos. Nesta pesquisa, investigamos a resistência da fratura de osso do microscópico para as escalas de comprimento mesoscópica usando micro arranhão testes, combinadas com a mecânica da fratura linear. Os testes são realizados na orientação longitudinal curta em espécimes de osso cortical bovino. Um protocolo experimental meticuloso é desenvolvido e um grande número (102) de testes são realizados para avaliar a tenacidade à fratura de espécimes de osso cortical, enquanto a contabilidade para a heterogeneidade associada com a microestrutura do osso.

Microscopia de força atômica foi usada para medir a rugosidade da superfície polida. Como regra geral, o espécime qualifica-se como um bem polido se a aspereza de superfície é uma ordem de magnitude menor do que as características da superfície de interesse. Neste caso, a aspereza de superfície medida de 60 nm sobre uma área de µm 40 µm x 40 inscreve-se claramente este critério.
A Figura 4</stro...

Watch this Scientific Journal Video about Fragility Assessment of Bovine Cortical Bone Using Scratch Tests at JoVE.com

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Avaliação da fragilidade do osso Cortical bovino, usando testes de zero

Este estudo avalia a tenacidade à fratura de osso cortical bovino a nível sub-meso usando exames microscópicos de zero. Este é um original, objetivo, rigoroso, e reprodutível método proposto para sondar a tenacidade à fratura abaixo a escala macroscópica. Potenciais aplicações estão a estudar alterações fragilidade óssea devido a doenças como a osteoporose.

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the ...

fragility-assessment-of-bovine-cortical-bone-using-scratch-tests

Research

JoVE Journal

Bioengineering

9.2K visualizações.  University of Illinois at Urbana Champaign. Este estudo avalia a tenacidade à fratura de osso cortical bovino a nível sub-meso usando exames microscópicos de zero. Este é um original, objetivo, rigoroso, e reprodutível método proposto para sondar a tenacidade à fratura abaixo a escala macroscópica. Potenciais aplicações estão a estudar alterações fragilidade óssea devido a doenças como a osteoporose.

Vídeo: Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Uso de Células-tronco humanas perivasculares para Regeneração Óssea

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Bioengenharia

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

Preparação de Intact Discos cauda bovina Intervertebral de Cultura de Órgãos

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

In Vivo quantitativa Avaliação da Estrutura do miocárdio, função, perfusão e viabilidade Usando Cardiac Micro-Tomografia Computadorizada

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Modified Drop Tower Impact Tests for American Football Helmets

A modified National Operating Committee on Standards for Athletic Equipment (NOCSAE) test method for American football helmet drop impact test standards is presented that would provide better assessment of a helmet's on-field impact performance by including a faceguard on the helmet. In this study, a merger of faceguard and helmet test standards is proposed. The need for a more robust systematic approach to football helmet testing procedures is emphasized by comparing representative results of the Head Injury Criterion (HIC), Severity Index (SI), and peak acceleration values for different helmets at different helmet locations under modified NOCSAE standard drop tower tests. Essentially, these comparative drop test results revealed that the faceguard adds a stiffening kinematic constraint to the shell that lessens total energy absorption. The current NOCSAE standard test methods can be improved to represent on-field helmet hits by attaching the faceguards to helmets and by including two new helmet impact locations (Front Top and Front Top Boss). The reported football helmet test method gives a more accurate representation of a helmet's performance and its ability to mitigate on-field impacts while promoting safer football helmets.

This article provides a novel technique to assess the performance characteristics of American football helmets by inclusion of faceguards during NOCSAE Standard drop tests. Additionally, two more impact locations are proposed to be added to the NOCSAE certification.

Testes de queda Torre de impacto modificadas para American Football Helmets

A modified National Operating Committee on Standards for Athletic Equipment (NOCSAE) test method for American football helmet drop impact test standards ...

A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral surface obtained from mechanical testing. This optical method requires a texture of many contrasting fiduciary marks on a solid white background for accurate tracking of surface deformation as loading is applied to the specimen. Immediately prior to testing, the surface of interest in the camera view is painted with a water-based white primer and allowed to dry for several minutes. Then, a black paint is speckled carefully over the white background with special consideration for the even size and shape of the droplets. Illumination is carefully designed and set such that there is optimal contrast of these marks while minimizing reflections through the use of filters. Images were obtained through high speed video capture at up to 12,000 frames/s. The key images prior to and including the fracture event are extracted and deformations are estimated between successive frames in carefully sized interrogation windows over a specified region of interest. These deformations are then used to compute surface strain temporally during the fracture test. The strain data is very useful for identifying fracture initiation within the femur, and for eventual validation of proximal femur fracture strength models derived from Quantitative Computed Tomography-based Finite Element Analysis (QCT/FEA).

In this protocol, the femur surface strains are estimated during fracture testing using the digital image correlation technique. The novelty of the method involves application of a high-contrast stochastic speckle pattern on the femur surface, carefully specified illumination, high speed video capture, and digital image correlation analysis for strain calculations.

Um método para estimar cepas Cortical de fêmur cadavérico durante fratura testes usando a correlação de imagem Digital

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

Perfil de compatibilidade biológica em biomateriais para regeneração óssea

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

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

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

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

Modelo calvarial de aumento ósseo em coelho para avaliação do crescimento ósseo e neovascularização em materiais de substituição óssea

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

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

Medindo kinemática de ombro 3D in vivo usando videoradiografia biplanar

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Bioimpressão 3D de astrócitos cortical de murina para engenharia de tecido neural

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

Ensaio de co-cultura 3D: nichos osteogênicos vascularizados para triagem de drogas contra o câncer

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise&#160;would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

Autor em destaque: Estabelecimento simples de um nicho osteogênico vascularizado da medula óssea usando hidrogéis de poli (etilenoglicol) (PEG) pré-moldados em uma microplaca de imagem  

An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

Estabelecimento simples de um nicho osteogênico vascularizado da medula óssea usando hidrogéis pré-moldados de poli(etilenoglicol) (PEG) em uma microplaca de imagem

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...