Tracy C. Grikscheit, Erik R. Barthel, e Frédéric G. Sala são suportados pelo Instituto de Medicina Regenerativa da Califórnia (CIRM), números de subsídios RN2-00946-1 (TCG) e TG2-01168 (ERB, FGS). Allison L. Speer é uma Sociedade de Cirurgiões Universidade Ethicon estudioso. Yashuhiro Torashima é financiado pelo Los Angeles de Crianças Hospital Saban Research Institute Fellowship Desenvolvimento de Carreira.

1. Preparação Unidades organóides <ol><li> Instrumentos adequados para dissecção mouse (tesouras e pinças) devem ser esterilizados por autoclave. </li><li> Humanamente eutanásia do camundongo doador de acordo com os protocolos locais IACUC. Certifique-se que o animal está morto antes de prosseguir. </li><li> Fazer uma incisão na linha média para obter acesso ao interior da cavidade peritoneal. Retalhos de pele pode ser refletido como necessário para melhorar a exposição. </li><li> Estripar o intestino delgado e dividi-lo apenas distal do ligamento de Treitz. Separe o intestino delgado de seu mesentério usando afiada e dissecção romba suave. Identificar a junção ileocecal e dividir o intestino delgado 5 mm proximal ao presente. </li><li> Com uma tesoura, abrir o intestino longitudinalmente ao longo da borda antimesentérica num prato de Petri com 10 ml de 4 ° C, a solução salina tamponada estéril de Hanks (HBSS, Invitrogen, Carlsbad CA) / 1X antibiótico-antifúngico (Anti-Anti, Invitrogen) de solução. Limpar a matéria fecal no intestino aberto comagitação suave, em seguida, transferir intestino aberto para um tubo de centrífuga de 15 ml com 10 ml de 4 ° C, HBSS estéril / 1X anti-anti. </li><li> Lavar o intestino aberto três vezes em 10 ml de 4 ° C, HBSS estéril 1X / anti-anti num tubo de ensaio. Cada lavagem pode ser realizada com agitação suave do tubo de 15 mL durante 30 seg. Após agitação, o tecido intestinal afunda para o fundo do tubo. Descartar o material flutuante, a qual é detritos mesenquimal. Remover a solução de lavagem cuidadosamente com uma pipeta. </li><li> Picar o intestino lavado numa placa de Petri com 10 mL de 4 ° C, HBSS estéril 1X / anti-anti-para menos do que 1 milímetro pedaços quadrados usando uma tesoura. Reúna o material picada com uma pipeta automática e colocá-lo em um tubo de ensaio. </li><li> Centrifuga-se o tubo a 500 rpm durante 8 minutos. Rejeitar o sobrenadante, que contém a gordura e mesênquima. </li><li> Digerir o material, picada lavada com 10 ml de HBSS estéril 1X / anti-anti mais 0,125 mg / ml de dispase (Invitrogen) e 800 unidades / ml colagenase tipo 1 (Worthington, Lakewood NJ). Para preparar 40 ml da solução de digestão, pesam-se 5 mg de dispase, 142 mg de colagenase, e adicione HBSS estéril até um volume de 40 ml. Preparar esta solução recentemente cada unidade de tempo organóides são preparados, e manter a 4 ° C até estar pronto para usar. Adicionar a solução de digestão directamente para a pellet a partir do passo 1.8. </li><li> Incubar o tubo do teste contendo o material, picada lavado com a solução de digestão a 37 ° C durante 20 min. </li><li> Recuperar o tubo de ensaio e perturbar ainda mais o tecido digerido através de trituração com uma pipeta de 10 ml. Repita entre 20 a 50 vezes, até que uma aparência uniforme é obtido. </li><li> Centrifuga-se o tubo de ensaio durante 5 minutos a 800 rpm. Rejeitar o sobrenadante, que contém uma única célula. </li><li> Parar a reacção de digestão com 10 ml de 4 ° C, Modificado de Dulbecco estéril Eagle Médium (DMEM, Invitrogen) mais 10% v / v inactivado pelo calor de soro fetal bovino (HI-FBS, Invitrogen). Ressuspender o pellet e agitar o tubo. </li><li> Centrifuga-se o tubo de ensaio durante 5 minutos a 800 rpm. Remover o sobrenadante cuidadosamente com uma pipeta automática, até as últimas gotas. Usar uma pipeta de plástico descartável para as últimas gotas, para evitar de ressuspender o pellet. </li></ol> 2. Carregamento de ácido poliglicólico Andaime <ol><li> Forma de 2 mm de comprimento, 5 mm de diâmetro exterior andaimes cilíndricos de ácido poliglicólico não tecido (2 mm de espessura da folha, a densidade de 60 mg cm-3; porosidade%&gt; 95, Fibras Concordia, RI Coventry), como descrito na ref. 4. </li><li> Aparar a 2 mm distal de uma pipeta de microlitro descartável 1000 ponta com uma tesoura preparadas com 70% de etanol em água destilada. </li><li> Coloque o andaime em uma placa de cultura de quatro poços. Carregar as unidades organóides sobre o andaime com a pipeta de microlitro 1000, em primeiro lugar para dentro do lúmen e, em seguida, sobre a superfície exterior. Use uma pinça para assegurar o revestimento do lúmen. Não perturbem ou romper a forma cilíndrica do polímero. </li&gt; </ol> 3. Implantação em Rato Anfitrião <ol><li> Utilizar um rato hospedeiro singeneico no fundo mesmo que o doador se disponível. Caso contrário, empregam um diabético não obesos imunocomprometidos / grave imunodeficiência combinada ou NOD / SCID animal (Jackson Laboratories, Sacramento CA). </li><li> Induzir a anestesia geral com isoflurano. Barbear, preparação e armar o abdômen do rato. </li><li> Fazer uma incisão na linha média 5 milímetros para ganhar entrada para a cavidade peritoneal. Identificar e cuidadosamente estripar o omento maior. Coloque o polímero carregado no omento e envolvê-la com o tecido. Não rasgue o omento. </li><li> Fixar o polímero para o omento, com uma sutura monocryl 5-0. Suavemente o omento com o polímero enrolado na sua posição anatómica. </li><li> Feche a incisão abdominal em camadas usando suturas vicryl 4-0. Executar o fechamento muscular e tomar cuidado para não ferir as vísceras abdominais abaixo da incisão. Use suturas interrompidas para a pele. </li><li> Administrar analgesia pós-operatória, com 2 mg / kg de cetoprofeno (Ketofen, Fort Dodge Animal Health) em água estéril como uma pápula subcutânea adjacente à incisão. Os animais devem ser avaliados diariamente e se o animal está a demonstrar sinais de dor ou desconforto, uma dose adicional de cetoprofeno pode ser administrada no dia 2 pós-operatórios. No terceiro dia pós-operatório, o animal deve ser totalmente recuperado, sem evidência de dor ou sofrimento. Se a dor ou sofrimento continua no dia pós-operatório 3, este é considerado anormal e deve ser tratado de acordo com os protocolos de instalação IACUC e animal de cuidados. </li><li> Permitir o mouse para recuperar e no intestino o tecido de crescer por quatro semanas. Dar ao animal o acesso ad libitum a roedores (Lab Diet 5001, PMI Nutrition, St. Louis MO) e água com Septra mg a 200 mg / 5 ml 40 (Hi-Tech Pharmacal, Amityville NY) a uma diluição de 1:100. </li></ol> 4. Colheita <ol><li> Humanely eutanásia do animal hospedeiro quatro semanas após a implantação. </li><li> Reabrir a incisão inicial e reflectem a pele cefálica para facilitar o acesso à cavidade peritoneal. </li><li> Abrir a camada muscular e identificar a construção de tecido de engenharia como um globo de tecido. </li><li> Tome a aderência à construção a partir de vísceras intra-abdominal utilizando dissecção aguda. </li><li> Fixar o construto em formalina para posterior montagem de parafina, ou usar o tecido fresco para os ensaios bioquímicos, tais como PCR em tempo real ou de isolamento de proteínas. </li></ol>

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Nós apresentamos um protocolo para a produção de tecidos de engenharia intestino no rato usando um organóides abordagem unidades-em-andaime. Os passos mais críticos são os da preparação de unidades organóide. Cuidados devem ser tomados para limpar adequadamente e mecanicamente processar o tecido, mas o mesmo cuidado deve ser tomado para não overdigest ou overtriturate as unidades organóides após a digestão é realizada (passo 1.11). Se isto é feito, as unidades organóides pode ser reduzida a uma única célula, que pode ser perdido no sobrenadante do passo de 1,12, e não são susceptíveis de sobreviver para produzir o tecido de intestino, como o nicho intestinal células estaminais não iria ser preservada neste caso. O pequeno tamanho do rato torna difícil a anastomose directamente a construção de tecido de engenharia para o intestino do animal hospedeiro para testar a sua função in vivo. Alternativamente, o rato representa um modelo maior para o crescimento TESI que facilita na anastomose e in vivojá foi realizado, por essa razão 1. No entanto, o tamanho do rato não é um obstáculo insuperável, como outros têm sido capazes de executar a ressecção do intestino delgado e anastomose, 6 ou cirurgia anorretal, 7 nesses animais. Para responder a estas questões, experimentos para caracterizar a função de nossos tecidos de engenharia construções intestinais em um de forma vitro estão em andamento. Limitações adicionais a esta técnica incluem uma taxa de sucesso de 89% da geração de TESI 4. Ou seja, 89% dos andaimes OU carregados com sucesso gerar TESI. A aplicação desta técnica por outros podem ajudar a refinar e melhorar esta técnica o aumento do rendimento global de 100%. Além disso, esta técnica pode ser melhorada se a massa de tecido total gerada pode ser aumentada. Actualmente, a citometria de fluxo mostrou que o número total de células presentes no constructo TESI colhida é 3 vezes maior do que o número presente no implante(3,07 x 10 6 ± 0,5 x 10 6) 4. Melhorar o volume de produção TESI é um passo importante em direção a tradução para a terapia. Observamos também que o intestino do rato, sendo de paredes finas e de pequeno calibre, é particularmente fácil de processar em comparação com a de outras espécies, tais como o porco ou rato. Nos seres humanos, espera-se que alguns dos detalhes dos passos organóides unidades de preparação teria de ser modificado para ambos adequadamente digerir o tecido e evitar overdigestion para células individuais, em particular as concentrações de dispase e colagenase na solução de digestão e tempo de digestão a 37 ° C. O objectivo final desta técnica é uma transição para a terapia humana. A engenharia de tecidos do intestino humano a partir de tecido do próprio paciente poderia oferecer um bem durável, cura a longo prazo para a síndrome do intestino curto (SIC) com nenhum dos inconvenientes das terapias existentes. Intestino curto syndrome é uma condição mórbida causada por ressecção de uma fracção significativa do comprimento total do intestino delgado, geralmente maior do que 50-75 por cento, de tal modo que a sua capacidade de absorção é muito reduzida e o paciente não pode obter alimento suficiente de nutrição entérica. 2 Em crianças, as causas mais comuns de SBS são ressecção intestinal maciça pequeno secundária à enterocolite necrosante ou má rotação com intestino volvulus. 8,9 Além disso, embora menos comum, SBS pode ocorrer em adultos por causa de ressecções múltiplas na definição da doença de Crohn, ou com isquemia mesentérica secundária a doença vascular. 10,11 Como os pacientes SBS não pode manter a nutrição suficiente com a ingestão enteral, podem exigir a longo prazo de nutrição parenteral total, o que em si pode ser complicado em crianças por insuficiência hepática e cirrose. SBS 12 pacientes, portanto, suportar significativa custos de saúde, recentemente estimou a ser da ordem de US $ 1,6 milhões por pacientemais de 5 anos. 13 O padrão actual de cuidados para a insuficiência intestinal secundária a SBS é fígado, intestino / intestinal, ou outro transplante multivisceral, mas isto confere apenas uma sobrevivência de 60% ​​em 5 anos e consigna o paciente a um curso ao longo da vida da terapia imunossupressora 14. Além disso, limitados doadores de órgãos resultados de disponibilidade em um desfasamento inevitável da demanda e oferta e longos tempos de espera. 15 Portanto, a engenharia de tecidos a partir de tecido autólogo, o paciente seria uma alternativa atraente.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> HBSS </td><td> Gibco </td><td> 114170-112 </td><td></td></tr><tr><td> Antibiótico-antimicótico 100X </td><td> Invitrogen </td><td> 15240-062 </td><td></td></tr><tr><td> Dispase </td><td> Gibco </td><td> 17105-041 </td><td></td></tr><tr><td> A colagenase de tipo 1 </td><td> Worthington </td><td> LS004194 </td><td></td></tr><tr><td> DMEM alta glicose 1X </td><td> Gibco </td><td> 11995-065 </td><td></td></tr><tr><td> FBS inactivado pelo calor </td><td> Invitrogen </td><td> 16140-071 </td><td></td></tr><tr><td> Biofelt PGA 100% </td><td> Médico Concordia </td><td> FELT01-1005 </td><td> Para a preparação de polímeros como na Ref.4 </td></tr><tr><td> Poli-L-láctico </td><td> Durect </td><td> B6002-1 </td><td> Para a preparação de polímeros como na Ref. 4 </td></tr><tr><td> Colágeno tipo I cauda de rato, </td><td> Sigma-Aldrich </td><td> C3867-1VL </td><td> Para a preparação de polímeros como na Ref. 4 </td></tr><tr><td> Cetoprofeno 100 mg / ml </td><td> Fort Dodge Animal Health </td><td> 71-ceto-100-50 </td><td></td></tr><tr><td> LabDiet 5001 roedores </td><td> LabDiet </td><td> 5001 </td><td></td></tr><tr><td> Septra 200 mg / 40 mg por 5 ml, USP </td><td> Oi-Tech Pharmacal </td><td> 50383-824-16 </td><td></td></tr><tr><td> USP, isoflurano </td><td> Phoenix Pharmaceuticals </td><td> 57319-507-06 </td><td></td></tr></tbody></table>

tissue engineering of the intestine in a murine model

O tecido de intestino delgado (TESI) tem sido usados ​​com sucesso para resgatar ratos Lewis após a ressecção intestinal maciça pequeno, resultando em retorno aos pesos pré-operatórios dentro de 40 dias. 1 em humanos, a ressecção intestinal maciça pequeno pode resultar em síndrome do intestino curto, uma má absorção funcional Estado que confere significativa morbidade, mortalidade e custos de saúde, incluindo a dependência da nutrição parenteral, insuficiência hepática e cirrose, e da necessidade de transplante de órgãos multivisceral. 2 Neste trabalho, descrever e documentar o nosso protocolo para criar o tecido de intestino em um modelo de camundongo com um multicelular organóides abordagem unidades-em-andaime. Unidades organóides são agregados multicelulares derivadas do intestino que contêm elementos e mucosas mesenquimal, 3 a relação entre o que preserva o nicho de células-tronco intestinal. 4 Na investigação em curso e futuras, a transição da nossa técnica para orato irá permitir uma investigação dos processos envolvidos na formação TESI utilizando as ferramentas transgénicas disponíveis nesta espécie. 5 A disponibilidade de estirpes de ratos imunodeficientes também nos permite aplicar a técnica de tecido intestinal humano e optimizar a formação de TESI humano como um xenoenxerto mouse antes de sua transição para os seres humanos. Nosso método emprega boas práticas de fabricação (BPF) reagentes e materiais que já foram aprovadas para o uso em pacientes humanos, e, portanto, oferece uma vantagem significativa sobre as abordagens que dependem de tecidos animais descelularizados. O objetivo final deste método é a sua tradução para os seres humanos como uma estratégia de medicina regenerativa terapêutica para a síndrome do intestino curto.

A Figura 1 mostra um esquema geral para o protocolo aqui documentado. O resultado final deste protocolo é uma estrutura globo esférico ou de intestino de engenharia de tecidos de murino com um lúmen, mucosa, submucosa, e rodeando 2A. Muscularis Figura mostra um globo típica em comparação com o polímero de partida de um andaime. Figura 2B mostra a mesma construção acentuadamente bivalvadas para revelar seu lúmen. Figura 3 demonstra um hematoxilina / eosina manchado de parafina montado secção transversal de uma construção típica de sucesso após 4 semanas de incubação. Na Ref. 4, que foram capazes de produzir o tecido de intestino com sucesso 89% do tempo (39 de 44 implantes). Uma construção vencida uma em que há formas de mucosa. É impossível julgar com base na aparência bruta no momento da colheita se o mundo terão mucosa na análise histológica final. Portanto, se os ensaios bioquímicos são performed no tecido fresco construto, metade de cada globo deve ser fixada e montada parafina para análise histológica para confirmar que a mucosa está presente em cada amostra. Uma construção vencida demonstrará estroma só e fibrose em hematoxilina / eosina. <img src="/files/ftp_upload/4279/4279fig1.jpg" alt="Figura 1" /> Figura 1. Esquema para a produção de tecido de engenharia intestino em ratos. Em resumo, o tecido do dador é colhida e processada em unidades organóide. As unidades organóides sejam carregados num poroso ácido poliglicólico andaime, o qual é, em seguida, implantada num hospedeiro e deixou-se incubar durante 4 semanas. A construção de engenharia é então recuperado e pode ser caracterizado por meio de histologia ou ensaios bioquímicos. <img src="/files/ftp_upload/4279/4279fig2.jpg" alt="Figura 2" /> Figura 2. Exemplo de o tecido de intestino construto colhidas em 4 semanas. A: Construir em compaRison de iniciar polímero andaime. B: A mesma construção, bivalvadas para revelar seu lúmen. <img src="/files/ftp_upload/4279/4279fig3.jpg" alt="Figura 3" /> Figura 3. Baixa potência hematoxilina / eosina micrografia de uma construção bem sucedida intestino típico tecido de engenharia. As etiquetas e as setas indicam o lúmen com mucosa intestinal e pâncreas hospedeiro aderente.

Watch this Scientific Journal Video about Tissue Engineering of the Intestine in a Murine Model at JoVE.com

Tissue Engineering of the Intestine in a Murine Model

Este artigo eo vídeo que acompanha apresentar o nosso protocolo para gerar o tecido de intestino no mouse, utilizando uma abordagem de unidades-em-andaime organóide.

Engenharia de Tecidos do Intestino num modelo murino

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

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14.8K visualizações.  Keck School of Medicine of the University of Southern California. Este artigo eo vídeo que acompanha apresentar o nosso protocolo para gerar o tecido de intestino no mouse, utilizando uma abordagem de unidades-em-andaime organóide.

Vídeo: Tissue Engineering of the Intestine in a Murine Model

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Processamento de pós-produção de Fibras electrospun para Engenharia de Tecidos

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Bioengenharia

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Os tecidos músculo esquelético de engenharia a partir de células progenitoras mioblastos murinos e Aplicação da estimulação elétrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

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  ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Engenharia de Tecidos de um 3D Humano<em&gt; In vitro</em&gt; Sistema de Teste de Tumor

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Geração de espessura completa tridimensional Pele Equivalente e Automated Ferimento

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Produção, Caracterização e usos potenciais de uma mucosa esofágica Modelo 3D Human Tissue-engenharia

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Engenharia de Tecidos por Intrínseca Vascularização numa<em&gt; In Vivo</em&gt; Câmara Engenharia de Tecidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

The Loop arteriovenosa (AV) em um modelo animal pequeno para estudar angiogénese e Engenharia de tecido vascularizado

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

Tratamento de Estruturas Ligamentares com Soro Condicionado por Exercício: Um Modelo Translacional de Engenharia de Tecidos

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...