Este trabalho foi financiado em parte pela sociedade do Japão para o promoção da ciência (JSPS) subsídio para a investigação científica (26861689 e 17 K 11813). Os autores gostaria de agradecer o Sr. Nathaniel Green para revisão.

Este protocolo é projetado para criar modelos de gengivite, modelos de ferimento gengival e equivalentes de tecido gengival humano. Pele humana queratinócitos epidérmicos (HaCaT) foram gentilmente cedidos do Professor Norbert E. Fusenig do Deutsches Krebsforschungszentrum (Heidelberg, Alemanha)7. Fibroblastos gengivais humanos (HGFs) foram isolados a partir de tecidos gengivais, de acordo com os protocolos anteriormente publicados8. Foi obtido consentimento previamente, e o estudo foi aprovado de acordo com as orientações definidas pelo Comitê de ética, a Nippon Dental Universidade escola de vida Odontologia em Tóquio (número de autorização: NDU-T2012-35). Protocolo etapas 1-3 devem ser executadas em uma capa de cultura de células.
1. preparação do tecido humano 3D equivalentes da gengiva (GTE) (figura 1A)
<ol><li>Mix-colágeno tipo-A gel com 10% concentrados MEM-alfa e buffer de reconstrução de 10% (2,2 g de NaHCO3 e 4,47 g HEPES em 100 mL 0,05 N NaOH) em um tubo estéril 15 mL por pipetagem. Conservar a solução mista de colágeno no gelo até usado.</li>
	<li>Coloca uma placa de 24 inserções 24-poço de cultura (poros tamanho 3,0 µm).</li>
	<li>
Remova HGFs da cultura superfície usando a solução de EDTA-tripsina 0,25%. Suspender as células em 10 mL de meio de cultura A (alfa-MEM + 20% FBS + 1% GlutaMAX). Conte as células por um contador de célula Bürker-Türk.
	<ol>
<li>Extrair a quantidade desejada de células (1-5 x105 células/mL) e, em seguida, centrifugar durante 4 min a 190 x g.</li>
		<li>Suspenda as células na solução mista de colágeno. Manter a mistura no gelo até o próximo passo.</li>
	</ol>
</li>
	<li>Adicione 0,5 mL da mistura de célula-colágeno (0.5-2.5 x 105 células/poço) para inserir a cultura sem produzir quaisquer bolhas. Incube a mistura por 10 a 30 min em um ambiente umidificado com 5% CO2 a 37 ° C para coagular a mistura em um gel. Nota: Adicione a mistura de colágeno no centro da inserção da cultura, para evitar formação desigual de gel de colágeno.</li>
	<li>Adicione 1 mL do meio de cultura para o bem e 0,5 mL para a inserção. Incubar a placa de cultura para 24h ou durante a noite em um ambiente umidificado com 5% CO2 a 37 ° C.</li>
	<li>Remova HaCaT células da superfície da cultura usando a solução de EDTA-tripsina 0,25%. Suspender as células em 10 mL do meio B (DMEM + 10% FBS + 1% GlutaMAX) e contar as células. Extrair a quantidade necessária de células (1 – 5 x 105 células/mL) com uma pipeta, centrífuga para 4 min em x 190 g. suspender as células em média B.</li>
	<li>Remova o meio das inserções de cultura, que contêm a mistura de HGF-colágeno, por aspiração. Adicione 0,5 mL de suspensão de células HaCaT (0.5-2.5 x 105 células/poço) no topo da mistura de HGF-colágeno. Incube as culturas por 24 horas ou durante a noite. Nota: Neste ponto do tempo, o meio na cultura bem deve ser A média, enquanto a média na inserção da cultura deve ser médio B.</li>
	<li>Substituir o meio de cultura com meio KSR (meio de cultura co) (MEM-alfa + 15% de substituição de soro nocaute + 1% GlutaMAX) + 5% FBS. Adicione 1 mL do meio de cultura para a cultura bem e 0,5 mL para a inserção da cultura. Incube as culturas por 24 – 48 h.</li>
	<li>Substituir o meio de cultura com KSR médio + 1% FBS. Adicione 1 mL na cultura bem e 0,5 mL para a inserção de cultura. Incube as culturas por 24 horas ou durante a noite.</li>
	<li>Substituir o meio de cultura na cultura bem com 0,7 a 1 mL de meio de KSR. Remova completamente o meio de inserção da cultura (a superfície de cultura deve ser exposta ao ar). Incube as culturas por 1 – 2 semanas. Mude o meio na cultura bem duas vezes por semana. Nota: Não deixe que a ascensão de nível média acima da superfície da cultura (a camada superior composta por queratinócitos). Mantenha sempre a superfície de cultura seca.</li>
</ol>2. preparação do GTE ferido (figura 1B)
<ol><li>Prepare um perfurador de tecido 1 a 3 mm de diâmetro. Esterilize-a com uma autoclave a 121 ° C por 20 min.</li>
	<li>Empurre o perfurador de tecido perpendicularmente GTE sobre 300 µm profundo e um buraco no tecido para simular um ferimento.</li>
	<li>Neste passo, use o GTE ferido para investigar a regeneração de tecido e cura ferida. Alternativamente, conserte a GTE usando solução de tampão neutro de formalina 10% para realizar a análise histológica.</li>
</ol>3. preparação do GTE inflamatória (iGTE) (figura 1B)
<ol><li>Cultive células THP-1 de acordo com o relatório anterior9.</li>
	<li>Mix-colágeno tipo-A gel com 10% concentrados RPMI 1640, buffer de reconstrução de 10% (2,2 g de NaHCO3 e 4,47 g HEPES em 100 mL 0,05 N NaOH) e 10 ng/mL PMA. Manter a solução de colágeno misto no gelo até o uso.</li>
	<li>Contagem de 1 – 2 x 106 THP-1 células e centrifugar durante 4 minutos a 190 x g. suspender as células em 1 mL da solução mista de colágeno. Manter a mistura no gelo até o próximo passo.</li>
	<li>Adicione a mistura de THP-1-colágeno do 10 a 30 µ l para a área de feridos do GTE. Substituir o meio de cultura para 0,7-1 mL de meio de KSR contendo 10 ng/mL de PMA.</li>
	<li>Incube a mistura em um ambiente umidificado com 5% CO2 a 37 ° C por 72 h.</li>
	<li>Usar o modelo para investigar a gengivite ou doença periodontal (por exemplo, adicionar o potencial medicamento anti-inflamatório para iGTE para ver seus efeitos sobre a liberação de citocinas10). Alternativamente, omitir PMA-tratamento de 72 h, adicionar 2 µ g/mL de LPS na cultura e incubar o tecido em um ambiente umidificado com 5% CO2 a 37 ° C por 48 h11,12.</li>
</ol>4. fixação e toda montagem imunocoloração de GTE e iGTE culturas
<ol><li>
Fixação das culturas.
	<ol>
<li>Remova a placa de 24, o meio de cultura. Lave os tecidos 2 vezes com solução salina tamponada fosfato (PBS).</li>
		<li>Adicione 1 mL de solução-tampão neutro de formalina 10% para a inserção e 1 mL da cultura bem. Deixe a placa de 24 em um refrigerador a 4 ° C durante a noite.</li>
		<li>Remova a solução-tampão neutro de formalina 10% no dia seguinte.</li>
		<li>Para toda montagem imunocoloração, lave os tecidos 3 - 4 vezes com PBS após a fixação.</li>
		<li>Por hematoxilina e eosina (H & E) coloração e imunocoloração regular, prosseguir com a desidratação, incorporação de parafina e de corte. Nota: Para toda montagem imunocoloração, esta etapa pode ser omitida.</li>
	</ol>
</li>
	<li>
Toda montagem de imunocoloração Nota: Este protocolo foi modificado na referência 13.<ol>
<li>Lavar os tecidos 3 x em PBS 0,5 – 1% Triton, 10 – 30 min cada tempo.</li>
		<li>Incubar os tecidos duas vezes por 30 min em tampão de bloqueio (PBS 1% Triton + 10% BSA), à temperatura ambiente.</li>
		<li>Lave os tecidos 2 × em tampão de bloqueio, 5 – 10 min cada tempo.</li>
		<li>Adicione 50 µ l de solução de anticorpo primário na concentração/diluição necessária para as inserções. Use um filme de plástico plástico fino para encapsular as inserções para evitar a secagem do tecido.</li>
		<li>Incubar o tecido durante 1 a 2 dias a 4 ° C.</li>
		<li>Lavar os tecidos 3 vezes, por 30 min em PBS 1% Triton + 10% de BSA. Lave novamente 3 vezes, por 5 min em PBS 1% Triton.</li>
		<li>Adicionar 50 µ l de anticorpo secundário em tampão de bloqueio (PBS 1% Triton + 10% BSA) e 5 µ l de solução de Hoechst 33342 (mancha de NucBlue ao vivo celular) para cada inserção. Use um filme de plástico plástico fino para encapsular as inserções para evitar a secagem do tecido.</li>
		<li>Incubar durante 1 a 2 dias a 4 ° C. Embrulhe a placa de 24 em uma toalha de papel molhada e depois colocá-lo em um bloco de plástico a ser usado ao longo de incubação.</li>
		<li>Lavar os tecidos 3 vezes, por 10 min em Tritão de 1% de PBS</li>
		<li>Use uma agulha afiada para cortar a membrana da inserção da cultura para obter o tecido. Lugar da membrana para um slide. Nota: O tecido deve ser na membrana.</li>
		<li>Adicione 2 a 3 gotas de meio de montagem de fluorescência. Cubra o tecido com uma tampa de vidro e a loja a 4 ° C, no escuro até análise.</li>
		<li>Ver os tecidos com um laser confocal, microscopia (LSM).</li>
	</ol>
</li>
</ol>

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Os autores declaram não haverá conflito de interesses.

Este protocolo é baseado em métodos de criação de equivalentes de tecido gengival e equivalentes de tecido adiposo subcutâneos descritos por anteriores relatórios8,21,22. Embora este seja um método simples e fácil, algumas etapas requerem atenção especial. Por exemplo, a mistura de colágeno deve ser mantida no gelo até o uso para evitar a formação de gel na solução. Ao adicionar a mistura de colágeno para a inse...

As doenças periodontais são a principal causa de perda dentária em adultos. Gengivite e periodontite é as doenças periodontais mais comuns. Ambos apresentam alterações inflamatórias mediada por biofilme agudas ou crônicas na gengiva. A gengivite é caracterizada por inflamação aguda, Considerando que a periodontite apresenta-se geralmente como uma inflamação crônica. A nível histológico, componentes bacterianas desencadear a ativação de células do sistema imunológico, tais como macrófagos, linfócitos, plasmócitos e mastócitos1,2. Estas células imunes, especialmente os macrófagos, interagirem com as células locais (incluindo células epiteliais gengivais, fibroblastos, células endoteliais e osteoblastos) resultando em lesões inflamatórias no tecido periodontal3,4. Modelos experimentais de doenças periodontais foram estabelecidos em vários tipos de animais, como ratos, hamsters, coelhos, furões, caninos e primatas. No entanto, a fisiopatologia de modelos animais é diferente dos seres humanos, dificultando a mecanismos celulares e moleculares de analisar e avaliar novos medicamentos de doenças periodontais5. Co de cultivo de bactérias periodontais e células epiteliais oral humanas monocamada serviu para investigar o mecanismo das infecções periodontais6. No entanto, culturas de monocamadas de células orais faltam a arquitetura de celulares tridimensional (3D) do tecido intacto; Portanto, eles não podem imitar a situação em vitro .
Aqui, equivalentes 3D tecido humano inflamatória da gengiva (iGTE) são criados para representar doenças periodontais em vitro. Este modelo 3D das doenças periodontais ocupa uma posição intermediária entre as culturas de células monocamadas e modelos animais. Três tipos de células humanas, incluindo HaCaT queratinócitos, fibroblastos gengivais e THP-1 macrófagos, são co cultivados em gel de colágeno e estimulados pela inflamatórios iniciadores para construir iGTE. IGTE intimamente simula as condições na vivo de diferenciação celular, interação célula-célula e ativação de macrófagos na gengiva. Este modelo tem muitas aplicações possíveis drogas triagem e testando novas abordagens farmacológicas em doenças periodontais, bem como para analisar os mecanismos celulares e moleculares na cicatrização de feridas, inflamação e regeneração de tecidos.

<table><tbody><tr><td>Collagen type I-A</td><td>Nitta Gelatin Inc</td><td></td><td>For making dermis of GTE</td></tr><tr><td>MEM-alpha</td><td>Thermo Fisher Scientific</td><td>11900073</td><td>Cell culture medium</td></tr><tr><td>Cell Culture Insert (for 24-well plate), pore size 3.0 &amp;mu;m</td><td>Corning, Inc.</td><td>353096</td><td>For tissue culture</td></tr><tr><td>GlutaMAX</td><td>Thermo Fisher Scientific</td><td>35050061</td><td>Cell culture reagent</td></tr><tr><td>DMEM</td><td>Thermo Fisher Scientific</td><td>31600034</td><td>Cell culture medium</td></tr><tr><td>KnockOut Serum Replacement</td><td>Thermo Fisher Scientific</td><td>10828028</td><td>Cell culture reagent</td></tr><tr><td>Tissue puncher</td><td>Shibata system service co., LTD</td><td>SP-703</td><td>For punching holes in GTE</td></tr><tr><td>RPMI 1640</td><td>Thermo Fisher Scientific</td><td>31800022</td><td>Cell culture medium</td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A3294</td><td>For immunostaining</td></tr><tr><td>Hoechst 33342 (NucBlue Live Cell stain)</td><td>Thermo Fisher Scientific</td><td>R37605</td><td>For labeling nuclei</td></tr><tr><td>Fluorescence mount medium</td><td>Dako</td><td></td><td>For mounting samples after immunostaining</td></tr><tr><td>Anti-Cytokeratin 8+18 antibody [5D3]</td><td>abcam</td><td>ab17139</td><td>For identifying epithelium</td></tr><tr><td>Scaning electron microscope</td><td>Hitachi, Ltd.</td><td>HITACHI S-4000</td><td>For observing samples&#039; surface topography and composition</td></tr><tr><td>Confocal laser scanning microscopy</td><td>LSM 700; Carl Zeiss Microscopy Co., Ltd.</td><td>LSM 700</td><td>For observing samples&#039; immunofluorescence staining</td></tr><tr><td>Anti-Cytokeratin 19 antibody</td><td>abcam</td><td>ab52625</td><td>For identifying epithelium</td></tr><tr><td>Anti-vimentin antibody</td><td>abcam</td><td>ab92547</td><td>For identifying fibroblasts and activated macrophages</td></tr><tr><td>Anti-TE-7 antibody</td><td>Millipore</td><td>CBL271</td><td>For identifying fibroblasts in the dermis</td></tr><tr><td>Anti-CD68 antibody</td><td>Sigma-Aldrich</td><td>SAB2700244</td><td>For identifying macrophages</td></tr><tr><td>Human CD14 Antibody</td><td>R&amp;amp;D Systems</td><td>MAB3832-SP</td><td>For identifying macrophages</td></tr><tr><td>Alexa Fluor 594-conjugated secondary goat anti-rabbit antibody</td><td>Thermo Fisher Scientific</td><td>A11012</td><td>For immunofluorescence staining</td></tr><tr><td>Alexa Fluor 488-conjugated secondary goat anti-mouse antibody</td><td>Thermo Fisher Scientific</td><td>A11001</td><td>For immunofluorescence staining</td></tr><tr><td>EVOS FL Cell Imaging System</td><td>Thermo Fisher Scientific</td><td></td><td>For observing the sample&#039;s immunofluorescence staining</td></tr><tr><td>THP-1 cells</td><td>Riken BRC cell bank</td><td>RCB1189</td><td>For making iGTE</td></tr><tr><td>PMA(Phorbol 12-myristate 13-acetate)</td><td>Sigma-Aldrich</td><td>P8139</td><td>For differentiatting THP-1 cells</td></tr></tbody></table>

three-dimensional inflammatory human tissue equivalents of gingiva

Doenças periodontais (tais como gengivite e periodontite) são as principais causas de perda dentária em adultos. Inflamação na gengiva é fundamental fisiopatologia das doenças periodontais. Atuais modelos experimentais de doenças periodontais foram estabelecidos em vários tipos de animais. No entanto, a fisiopatologia de modelos animais é diferente dos seres humanos, dificultando a mecanismos celulares e moleculares de analisar e avaliar novos medicamentos para doenças periodontais. Aqui, apresentamos um protocolo detalhado para reconstruir equivalentes de tecido humano inflamatória da gengiva (iGTE) em vitro. Primeiro construímos equivalentes de tecido humano da gengiva (GTE), utilizando dois tipos de células humanas, incluindo fibroblastos gengivais humanos (HGF) e pele humana queratinócitos epidérmicos (HaCaT), sob condições tridimensionais. Criamos um modelo de ferida usando um furador de tecido para fazer um buraco na GTE. Em seguida, humanos monócitos THP-1 misturados com gel de colágeno são injetados no furo do GTE. Por adimistration de 10 ng/mL phorbol myristate 12 13-acetato (PMA) para 72 h, THP-1 células diferenciadas em macrófagos aos focos inflamatórios da forma no GTE (iGTE) (IGTE também pode ser stumilated com 2 µ g/mL de lipopolissacarídeos (LPS) para 48 h iniciar a inflamação ). IGTE é o primeiro em vitro modelo da gengiva inflamatória usando células humanas com uma arquitetura tridimensional. IGTE reflete grandes mudanças patológicas (immunocytes activition, interações intracelulares, entre fibryoblasts, células epiteliais, monócitos e macrófagos) em doenças periodontais. GTE, GTE ferido e iGTE podem ser usados como ferramentas versáteis para estudar a cicatrização de feridas, regeneração tecidual, inflamação, interação célula-célula e tela potenciais medicamentos para as doenças periodontais.

As células HaCaT exibidas morfologia típica queratinócito sob observação microscópica de contraste de fase (Figura 2A). Scanner elétron microscópico (SEM) imagens mostrou que as superfícies de célula HaCaT estavam cobertas por muitos microvilli. Conexões intercelulares entre células de HaCaT foram mediadas por processos de membrana (Figura 2B). HaCaT células expressaram epitélio gengival marcador K8/18<sup class="xr...

Watch this Scientific Journal Video about Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva at JoVE.com

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

O objetivo do protocolo é construir um modelo de gengiva humana inflamatória em vitro. Este modelo de tecido co cultiva três tipos de células humanas, HaCaT queratinócitos, fibroblastos gengivais e macrófagos THP-1, sob condições tridimensionais. Este modelo pode ser aplicado para investigar doenças periodontais, como gengivite e periodontite.

Equivalentes de tridimensional tecido humano inflamatória da gengiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

three-dimensional-inflammatory-human-tissue-equivalents-of-gingiva

Research

JoVE Journal

Developmental Biology

9.8K visualizações.  The Nippon Dental University. O objetivo do protocolo é construir um modelo de gengiva humana inflamatória em vitro. Este modelo de tecido co cultiva três tipos de células humanas, HaCaT queratinócitos, fibroblastos gengivais e macrófagos THP-1, sob condições tridimensionais. Este modelo pode ser aplicado para investigar doenças periodontais, como gengivite e periodontite.

Vídeo: Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

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 ...

Bioengenharia

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

Isolamento, processamento e análise de células gengivais murino

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Imunologia e Infecção

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 Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Engenharia tridimensionais tecidos epiteliais incorporado dentro Matriz Extracelular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Isolamento, caracterização e exploração funcional da célula de rede gengival Imune

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Tecido conjuntivo de engenharia humana derivada do fibroblasto para aplicações de triagem

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

Uma nova abordagem para o monitoramento de neovascularização de enxerto na gengiva humana

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

Medicina

Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While in-house grown 2D epithelial monolayers lack true representation of the in vivo situation, commercially available tissue models are often overlooked due to their cost and practicality. However, with careful planning, such models provide reproducible platforms for a vast array of different applications. Here, we report the use of epithelial models that can be utilized for a wide variety of experimental purposes to investigate host-pathogen interactions in various ecological niches, such as the oral cavity, skin, and vaginal mucosa. From simple planktonic cells to complex biofilm co-culture, epithelial models are used to assess microbial adherence and invasion, and to evaluate the host response at a transcriptional and/or protein level, with scope for more detailed profiling using different omics approaches. Furthermore, these biological systems can be used as more accurate test beds for evaluating conventional and novel antimicrobial activity in a complex host-pathogen microenvironment in vitro. The protocols described herein document how models are handled upon arrival and prepared in the laboratory for co-culture stimulation with biofilm communities. The methods detail how experimental outputs are achieved from the model systems, including the processing of tissue, the co-culture setup, and data generation. These experiments include host gene expression through single- and multiplex qPCR analyses and inflammatory protein detection using ELISAs. In conclusion, epithelial models provide useful in vitro systems for preclinical investigatory studies into simple or complex host-pathogen interactions.

The present protocol outlines the application of organotypic tissue models for investigating host-pathogen interactions in vitro. Specifically, the model system described uses a multi-layered oral epithelium exposed to microorganisms associated with biofilms (dental plaque) within the context of oral health versus disease.

Sistemas de Modelo de Tecido Organotípico para Investigação de Interações Patógeno-Hospedeiro In Vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While ...

Criando um modelo de camundongo de inflamação pulpar usando uma nova mordaça na boca 

Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms underlying pulpitis and its efficient treatment is an ongoing focus of endodontic research. Therefore, understanding the inflammatory process within the dental pulp is vital for improving pulp preservation. Compared to other in vitro experiments, a murine pulpitis model offers a more authentic and genetically diverse context to observe the pathological progression of pulpitis. However, using mice, despite their cost-effectiveness and accessibility, poses difficulties due to their small size, poor coordination, and low tolerance, complicating intraoral and dental procedures. This protocol introduces a novel design and application of a mouth-gag to expose mouse pulp, facilitating more efficient intraoral procedures. The mouth-gag, comprised of a dental arch readily available to most dentists and can significantly expedite surgical preparation, even for first-time procedures. Micro-CT, hematoxylin-eosin (HE) staining, and immunofluorescence staining were used to identify changes in morphology and cell expression. The aim of this article is to help researchers establish a more reproducible and less demanding procedure for creating a pulp inflammation model using this novel mouth-gag.

Autor em destaque: aprimorando a pesquisa da polpa dentária com modelos de camundongos aprimorados

This article presents a streamlined protocol for establishing a pulpitis model in mice using an innovative mouth-gag, followed by subsequent histological analysis.

Equivalentes de tridimensional tecido humano inflamatória da gengiva

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Engenharia de Tecidos de um 3D Humano