Este trabalho foi financiado por fundos da American Heart Association (AM), Departamento de Defesa dos EUA (BC074986), e do Boston Children Hospital Faculdade Career Development Fellowship (TM, AM). Os autores agradecem a Amanda Jiang e Elisabeth Jiang para assistência técnica.

NOTA: O estudo in vivo em animais foi realizado em estrita conformidade com as recomendações do Guia para o Cuidado e Uso de Animais de Laboratório do Instituto Nacional de Saúde. O protocolo foi revisto e aprovado pelo Comitê de Cuidados e Uso Animal do Hospital Infantil de Boston (números de protocolo: 13-10-2526R, 14-02-2568R). Todos os medicamentos utilizados neste protocolo são de grau farmacêutico e esses medicamentos são preparados em condições estéreis. 1. Preparação de fibrina Gel <ol><li> Prepare gel de fibrina que contém o VEGF e bFGF. <ol><li> Descongelar as soluções estoque de fibrinogénio e trombina que são armazenadas a -80 ° C até à temperatura ambiente (25 ° C). </li><li> Adicionar trombina (concentração final: 2,5 U / ml), CaCl2 (concentração final: 45 mM), de VEGF (concentração final: 0-100 ng / ml) e bFGF (concentração final: 0-100 ng / ml) para o fibrinogénio solução (concentração final: 12,5 mg / ml em 0,9% sodium uma solução de cloreto 43-45) em um tubo de 1,5 ml. </li><li> Misture delicadamente por pipetagem. </li><li> Pipeta suavemente 200 ul da mistura num prato de plástico estéril de uma forma gota a gota, usando uma pipeta de ponta p200. </li><li> Incubar as gotas a 37 ° C durante 30-60 minutos até se solidificar. NOTA: O gel solidificado pode ser mantido no prato de plástico selado à temperatura ambiente (25 ° C) durante várias horas antes da implantação (Figura 1a). </li></ol></li><li> Apare o gel de fibrina em aproximadamente 3 x 3 x 3 cubos mm utilizando pequenas tesouras cirúrgicas antes da implantação. </li></ol> 2. Preparação do rato <ol><li> Anestesiar rato adulto (8-12 semanas) por injecção intraperitoneal (IP) de injecção de cetamina (100 mg / kg) e xilazina (10 mg / kg) e confirmam que o rato é adequadamente anestesiados por beliscar dedo do pé do rato. <ol><li> Use vet pomada nos olhos do mouse para prevenir o ressecamento durante o experimento. </li></ol></li><li> Shave fur sobre o lado esquerdo da caixa torácica do mouse. </li><li> Execute entubação endotraqueal do mouse. </li><li> Posicione o mouse sobre a intubação ficar em ângulo de 70 ° e segure o mouse no lugar ligando seus incisivos superiores ao longo de um pequeno anel de borracha localizado na parte superior do suporte. </li><li> Gentilmente retrair a língua de um lado usando uma pinça sem corte. </li><li> Visualizar a laringe, com o auxílio de um microscópio de pescoço de ganso iluminador de fibra óptica. </li><li> Insira endotraqueal cateter elástico (21 G) na traquéia. </li><li> Confirme se o mouse está respirando espontaneamente de uma forma suave (regular 100-150 ciclos / min, sem respiração paradoxal ou superficial). </li><li> Posicione o mouse em posição frontal sob o microscópio de dissecação. </li><li> Mecanicamente ventilar o mouse utilizando um ventilador de roedor (150 ciclos / min e 7 ml / kg volume corrente). </li><li> Contagem costelas para localizar espaço intercostal entre 4ª e 5ª costela. </li><li> Criar um campo estéril sobre a área by completamente limpando com álcool e iodopovidona. Cubra o campo cirúrgico adequadamente com um campo cirúrgico estéril. </li></ol> 3. Rato Cirurgia <ol><li> Após a injecção local de bupivacaína a 0,25% (200 ul) na pele, fazer uma incisão cutânea transversal (cerca de 1 cm de comprimento) sobre o espaço intercostal com tesouras de dissecção. </li><li> Após a injeção de bupivacaína a 0,25% (200 mL) no músculo intercostal, fazer uma incisão do músculo entre a 4ª e 5ª costela usando uma tesoura pequena multa. </li><li> Insira um afastador dissecando entre as costelas para visualizar totalmente o pulmão esquerdo. <ol><li> Raspe uma pequena área (1 x 1 mm quadrados) de pleura visceral do centro de pulmão esquerdo usando uma pinça fina. </li><li> Aplique uma leve pressão sobre a área usando um cotonete estéril até que o sangramento e vazamentos são totalmente controlados. </li><li> Coloque pequena quantidade de mistura fresca de fibrinogénio / trombina (cola de fibrina) (passo 1.1.2. 20 &amp; #181; l) sobre a área usando ponta p200 pipeta. </li></ol></li><li> Suavemente colocar um gel de fibrina (passo 1.2), utilizando pequenas pinças sobre a área (Figura 1b). <ol><li> Confirmar que o gel é bem fixo na área durante os movimentos respiratórios dos pulmões. </li></ol></li><li> Certifique-se de que não existe nem enorme vazamento de ar, nem hemorragia do pulmão. </li><li> Fechar incisões (camadas de músculo e pele) com sutura absorvível, que não tem de ser removido. </li><li> Aspirar a cavidade torácica com 27 G agulha e seringa de 1 ml para evitar pneumotórax. </li><li> Terminar ventilação mecânica. </li></ol> 4. Rato Recovery <ol><li> Verifique se o mouse está respirando espontaneamente de uma forma suave (regular 100-150 ciclos / min, sem respiração paradoxal ou superficial). </li><li> IP injectar 1 ml de pré-aquecido 0,9% de NaCl para evitar a desidratação. </li><li> Permitir mouse para recuperar na almofada de água quente circulante. </li><li> Remover o endotrachetubo al depois de confirmar que o rato tem respiração estável. </li><li> Injectar meloxicam (5 mg / kg, injecção subcutânea (SC), durante 3 dias como analgésico pós-operatório. </li><li> Monitorar os movimentos do mouse com cuidado em um mínimo de 15 minutos de intervalo até que seja esternal (capaz de rolar sua estômago e permanecer em pé) e consciente. </li><li> Após a recuperação, o retorno do mouse para uma nova gaiola isolada de ratos sem cirurgia. </li><li> Monitorar o local da cirurgia para sinais de infecção (vermelhidão, inchaço, secreção), funções biológicas básicas do animal (alimentos e água ingestão, urinar, defecar, ganho de peso corporal), bem como sinais clínicos de angústia (piloerection, reduziu a locomoção) diariamente após a procedimento cirúrgico. </li></ol> 5. A colheita do Pulmão <ol><li> 7 a 30 dias após a implantação, eutanásia do rato usando CO 2 através fonte de gás comprimido. </li><li> Faça uma incisão entre a ponta do apêndice xifóide eo esternoentalhe (esternotomia mediana) e toda a colheita de pulmão com o gel implantado para análise histológica e bioquímica, cortando a traqueia e dissecar todas as conexões com o coração, pulmões e traquéia. </li><li> Corrigir gel implantado com pulmão com solução de paraformaldeído a 4% durante a noite a 4 ºC, incorporar em composto OCT, e tomar seções passo em série de 30 mm de espessura. </li><li> Execute histológico (hematoxilina e eosina) e análises de imuno-histoquímica (marcador endotelial: CD31, marcador epitelial: aquaporin (AQP) 5 e surfactante proteína (SP) -B) usando microscópio confocal 22,37,40. </li><li> Compilar pilhas de secções ópticas (30 mm de espessura) para formar imagens tridimensionais de endotelial pulmão e células epiteliais utilizando imagem 3D software de análise de 37. </li><li> Quantificar áreas projectadas dos vasos sanguíneos recém-formados usando software de análise de imagem 46. </li></ol>

The authors declare that they have no competing financial interests.

Este artigo apresenta um novo método para implantar biomateriais na superfície do pulmão de vida do rato adulto. Com este sistema, a angiogénese derivados de pulmão hospedeiro com sucesso, se condensa no interior do material. Este sistema permite que os investigadores para explorar a interferência entre as células endoteliais, outras células (por exemplo, células epiteliais, células mesenquimais, células imunes), e vários componentes da matriz extracelular que são necessárias para a angiogénese l...

O objetivo geral deste protocolo é a introdução de um método para implantar gel de fibrina na superfície do pulmão de rato adulto, o que permite aos pesquisadores para caracterizar os mecanismos moleculares da vascular pulmonar e desenvolvimento alveolar, e para aproveitar esse conhecimento, a fim de desenvolver materiais biomiméticos capaz de recapitular vascular pulmonar fisiológico e formação alveolar para o tratamento de várias doenças pulmonares. Mais de 35 milhões de americanos sofrem de doenças pulmonares crônicas, incluindo doença pulmonar obstrutiva crônica e fibrose pulmonar. Esses pacientes têm sintomas crônicos de longa duração respiratórios, como falta de ar, aperto no peito, tosse persistente e cansaço, que prejudiquem significativamente a sua vida diária 1-3. Apesar de uma grande quantidade de esforço para desenvolver terapias eficazes para essas doenças pulmonares, atualmente não há cura; portanto, a qualidade de vida para estes pacientes é pobre e econômica e os custos humanos são oigh 4-7. Atualmente, o transplante de pulmão é a única maneira de salvar os pacientes em fase terminal de doenças pulmonares crônicas. No entanto, por causa da escassez de doadores de transplante, de alto custo, complicações graves, e baixa taxa de sobrevivência 11/08, o transplante não é uma abordagem ideal. Rápido progresso recente em técnicas de engenharia de tecidos tem possibilitado aos pesquisadores modificar geneticamente pulmão implantável por repovoamento todo descelulada pulmão com vários tipos de células progenitoras ou células-tronco pluripotentes induzidas (iPS) células 12,13. No entanto, esses pulmões bioengenharia são funcionais apenas em animais hospedeiros, por várias horas após a implantação 12,14,15. Utilizando biomateriais para regenerar as estruturas complexas e funções dos pulmões também tem sido bastante bem sucedida. Isso pode ser porque os processos biológicos fundamentais que regem a regeneração do pulmão adulto não têm sido bem explorado. No pulmão, a formação do sistema vascular é um dos acontecimentos mais precoces e mais importantes during desenvolvimento e regeneração 16-21. Vasculaturas recém-formados no pulmão não só fornecer oxigénio, nutrientes e os vários componentes celulares necessários para a formação de órgãos, mas também fornecem sinais reguladores instrutivos para células vizinhas 22-25. Assim, a angiogênese desempenha papel-chave na alveolarização regenerativo no pulmão adulto 24,26,27. Além disso, contribui para a angiogênese desregulamentado doenças pulmonares crônicas, como doença pulmonar obstrutiva crônica (DPOC) 28, displasia broncopulmonar (DBP) 21-23, e fibrose pulmonar 29. Assim, o desenvolvimento de estratégias mais eficientes para a engenharia pulmões ou o tratamento de doenças pulmonares crônicas, é necessário entender os mecanismos fundamentais da angiogênese específico do pulmão. Cada órgão apresenta propriedades mecânicas e químicas únicas, que podem diferir entre as condições fisiológicas e patológicas 30-33. Estes microenviron órgão-específicamentos regular comportamentos de células endoteliais e orquestrar a formação da rede vascular de forma específica do órgão 24,34-36. Assim, o desenvolvimento de estratégias mais eficazes para a regeneração do pulmão, o mecanismo subjacente a angiogênese específico do pulmão precisa ser entendido. Embora os ensaios de angiogénese in vivo convencionais, tais como implantação subcutânea de hidrogel têm sido amplamente utilizados para pesquisa da angiogênese 37-39, esses métodos não recapitular angiogénese específica de órgão. Recentemente, um novo método para implante de Matrigel em um molde elástico no pulmão do rato tem sido desenvolvido e mostrado para recrutar com sucesso os vasos sanguíneos e células epiteliais do pulmão em 22 géis. Esta abordagem única permitirá que os investigadores para explorar o mecanismo de angiogênese específico do pulmão, bem como as interações entre os vasos sanguíneos e células pulmonares não-vasculares em condições fisiológicas e patológicas. Desde 1) Matrigel não é adequado para a aplicação clínica; 2) o emolde lastic usado para lançar o gel pode afetar interações entre hidrogéis e tecido pulmonar de acolhimento e 3) o molde elástico no pulmão potencialmente causa comprometimento da função pulmonar e dor durante a respiração, como uma abordagem mais clinicamente relevante, uma matriz de fibrina 3D contendo fatores angiogênicos (factor de crescimento endotelial vascular (VEGF) / factor de crescimento de fibroblastos básico (bFGF)) foi implantado no pulmão do rato sem vazamento no molde elástico, e tem sucesso recapitulado hospedeiro angiogénese derivados de pulmão. Gel de fibrina, fibrilas de polímero gerados a partir de fibrinogénio clivada pela trombina, a armadilha é conhecida uma variedade de factores angiogénicos, tais como bFGF e VEGF para acelerar a angiogénese in vivo 40,41. Devido à sua capacidade regenerativa e natureza biodegradável 42, gel de fibrina é amplamente utilizada no campo da engenharia de tecidos. Este artigo apresenta uma abordagem inovadora e única para implantar gel de fibrina na superfície do pulmão de Adul vivot mouse e demonstra que o anfitrião angiogênese derivado do pulmão é recapitulada no interior dos géis in vivo. Este método, o qual permite aos investigadores a estudar angiogénese específica do pulmão, provavelmente irá levar ao desenvolvimento de novas abordagens terapêuticas para vários tipos de doenças pulmonares e avançar significativamente os esforços para regenerar com sucesso adulto pulmão.

<table border="0" cellpadding="0" cellspacing="0" width="956">
	<tbody>
		<tr height="48">
			<td height="48" style="height:48px;width:343px;">Name of Material/ Equipment</td>
			<td style="width:177px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td style="width:289px;">Comments/Description</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fibrinogen from human placenta</td>
			<td>Sigma</td>
			<td>F4883</td>
			<td>For fabrication of fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Thrombin from bovine plasma</td>
			<td>Sigma</td>
			<td>T9549</td>
			<td>For fabrication of fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Recombinant mouse VEGF 164</td>
			<td>R&#38;D</td>
			<td>493-MV</td>
			<td>For supplementation to fibrin gel</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Recombinant mouse bFGF</td>
			<td>R&#38;D</td>
			<td>3139-FB</td>
			<td>For supplementation to fibrin gel</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:343px;">Rodent Intubation Stand</td>
			<td style="width:177px;">Braintree Scientific INC</td>
			<td style="width:147px;">RIS 100</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Fiber-Optic Light Source</td>
			<td style="width:177px;">Fisher Scientific</td>
			<td>12-565-35</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">20G Elastic catheter</td>
			<td>B.Braun</td>
			<td>4251652-02</td>
			<td>For intubation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:343px;">MiniVent Ventilator</td>
			<td style="width:177px;">Harvard Apparatus</td>
			<td style="width:147px;">CGS-8009</td>
			<td>For ventilation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Stemi DV4 Steromicroscope</td>
			<td style="width:177px;">Fisher Scientific</td>
			<td>12-070-515</td>
			<td>For surgey</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Absobable suture</td>
			<td>Ethicon</td>
			<td>PDP304</td>
			<td>Surgical suture</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against CD31</td>
			<td>BD Biosciences</td>
			<td align="right">553370</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against AQP5</td>
			<td style="width:177px;">Abcam</td>
			<td>AB78486</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Antibody against SP-B</td>
			<td style="width:177px;">Millipore</td>
			<td>AB40876</td>
			<td>Immunohistochemistry</td>
		</tr>
	</tbody>
</table>

implantation of fibrin gel on mouse lung to study lung-specific angiogenesis

Os recentes avanços significativos nas técnicas de pesquisa com células-tronco e bioengenharia têm feito grandes progressos na utilização de biomateriais para regenerar e reparar danos em tecidos simples nas áreas de ortopedia e periodontais. No entanto, as tentativas para regenerar as estruturas e as funções dos órgãos (3D) mais complexas tridimensionais, tais como os pulmões não têm sido muito bem sucedida, porque os processos biológicos de regeneração de órgãos não foram bem exploradas. Torna-se claro que a angiogênese, a formação de novos vasos sanguíneos, desempenha papel fundamental na regeneração de órgãos. Recentemente formado vasculaturas não só fornecer oxigénio, nutrientes e os vários componentes celulares que são necessários para a regeneração de órgãos, mas também fornecem sinais instrutivos para os tecidos em regeneração locais. Portanto, para regenerar com sucesso em pulmões de um adulto, que é necessário para recapitular os microambientes específicos do pulmão em que a angiogénese unidades de regeneração dos tecidos pulmonares locais. Embora conventional ensaios in vivo da angiogénese, tais como a implantação subcutânea de matriz extracelular (ECM) rico em hidrogéis (por exemplo, colagénio ou fibrina géis ou Matrigel - mistura de proteínas de ECM segregado por células de sarcoma de Engelbreth-Holm-Swarm rato), são amplamente utilizados para explorar a mecanismos gerais de angiogénese, a angiogénese específica do pulmão não tem sido bem caracterizada porque os métodos para o implante ortotópico de biomateriais no pulmão não foram bem estabelecidos. O objetivo deste protocolo é a introdução de um método único para implantar gel de fibrina na superfície do pulmão de vida do rato adulto, permitindo a recapitulação bem sucedida de acolhimento angiogênese derivado do pulmão dentro do gel. Esta abordagem permite aos investigadores para explorar os mecanismos pelos quais o microambiente-pulmão específico controla a angiogénese e regeneração alveolar em condições normais e patológicas. Desde o lançamento biomateriais implantados e fornecer sinais físicos e químicos para adjacente ltecidos ung, implantação destes biomateriais no pulmão doente pode potencialmente normalizar os tecidos doentes adjacentes, permitindo que pesquisadores a desenvolver novas abordagens terapêuticas para vários tipos de doenças pulmonares.

Para examinar se a formação vascular pulmonar derivados do hospedeiro, se condensa no interior dos biomateriais implantados no pulmão, geles de fibrina suplementado com grandes factores angiogénicos de VEGF e bFGF (0, 10 e 100 ng / ml cada) foram implantados na superfície das que vivem nos pulmões de ratinho quanto relataram o uso de Matrigel 22. 47 geles de fibrina que contêm estes factores de crescimento angiogénicos foram fabricados como se mostra na Figura 1a. Após toracotomia, u...

Watch this Scientific Journal Video about Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis at JoVE.com

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Implantação de fibrina Gel no tapete do pulmão para o Estudo de angiogênese Lung-specific

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

implantation-fibrin-gel-on-mouse-lung-to-study-lung-specific

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Medicine

9.9K visualizações.  Boston Children's Hospital and Harvard Medical School. Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse. 

Vídeo: Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

Um modelo do rato do Ensaio de bolso Córnea para o Estudo da angiogênese

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Medicina

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

Treatment of Osteochondral Defects in the Rabbit&#039;s Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

Tratamento de defeitos osteocondrais no Joelho do Coelho por Implantação de alogênico Células-Tronco Mesenquimais em coágulos de fibrina

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

Angiogênese no pulmão de rato Isquêmica

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Um método simples de intubação Lung Rato

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

Não invasiva Intratraqueal intubação para estudar a Patologia e Fisiologia do Rato Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Descolamento de Retina modelo em roedores por injeção sub-retiniana de hialuronato de sódio

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

Intratraqueal (IMIT) instilação mediada por intubação: A não-invasiva, sistema de entrega Lung-specific

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

Rato Pneumonectomia modelo de crescimento compensatório pulmonar

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

Tumor primário e isolamento de células MEF para o Estudo de metástases pulmonares

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Criação de um Modelo de Xenoenxerto Humano mieloma múltiplo na galinha para estudar o crescimento tumoral, invasão e angiogênese

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...