Este trabalho foi apoiado pela Research Grant UWS.

1. Quitosana Preparação adesiva <ol><li> O pó de quitosano é solúvel em solução de ácido acético, para a preparação de uma solução mãe de ácido acético (2% v / v), adicionar 10 ml de ácido acético glacial e 490 ml de água desionizada (DI-H 2 O). </li><li> Para a preparação de uma solução stock de Rose Bengal (RB) (0,01% w / v) em ácido acético, pesar 5 mg de RB em um frasco envolto com folha de alumínio para evitar a fotodegradação. Adicionar 0,5 ml de O 2 DI-H para dissolver o pó em seguida, adicionar 49,5 ml da solução de ácido acético stock (descrito na secção 1.1). </li><li> Para a preparação da solução de quitosano (1,7% w / v), pesar 0,85 g de quitosano em pó (MW médio, o grau de 85% de acetilação) e adicionar 50 ml de solução de ácido acético a RB. O controlo foi preparado por dissolução da quitosana em solução de ácido acético sem RB. </li><li> Adicionar um agitador magnético à mistura de quitosano e agitou-se à temperatura ambiente. Para assegurar a dissolução completa dos componentes, agitar a quitosanaem ácido acético + RB durante duas semanas e o quitosano em ácido acético, durante 5 dias (de controlo). Quando o quitosano é dissolvido, o pH da solução aumenta de 2,6 a ~ ~ 3.9. Note-se que RB é pouco solúvel em pH ácido e que exige um aumento do pH da solução, e um tempo prolongado de agitação para dissolver. </li><li> Centrifugar as soluções a 3270 xg, durante 1 hora e recolher o sobrenadante, utilizando, por exemplo, uma seringa estéril ml 10. Certifique-se de não perturbar a impureza da pelota. Durante esta etapa, a solução é feita a partir de impurezas macroscópicas e matéria insolúvel. </li><li> Injectar a (centrifugada) solução de quitosano sobre uma laje perpex plana usando uma seringa estéril. A espessura do filme seco depende da relação entre a quantidade de solução dispensada e a área da superfície sobre a qual a solução é espalhada. Assegure-se que a solução esteja livre de bolhas de ar e uniformemente distribuído sobre a laje. Para se obter filmes finos, com ~ 13 um de espessura, injectar ~ 1 ml de solução acima de 12 cm 2 de superfícieárea (~ dimensões 3x4 centímetros, Figura 1A). </li><li> Cobrir a placa de perspex com uma tela, feita de folha de alumínio, por exemplo, para evitar a foto-branqueamento da solução. Garantir que a ventilação de ar suficiente é garantida para secar a solução sob a tela. </li><li> Deixar a solução a secar à temperatura ambiente (25 ° C) e pressão (1 atm) até que o filme resultante é insolúvel em água e não incham macroscopicamente. Isto é tipicamente alcançado em 2 semanas. </li><li> Retire cuidadosamente os filmes usando uma espátula fina e plana: esses filmes podem ser facilmente arrancado a laje Perspex sem danificá-los. </li><li> Medir a espessura das películas utilizando um micrómetro. </li><li> Corte o adesivo em tiras retangulares com uma tesoura afiada limpa e coloque-os entre lâminas de vidro estéreis para preservar sua forma plana. Enrole as lâminas em folha de alumínio para a luz de blindagem e armazená-los à temperatura ambiente. Rotular os adesivos com ou sem RB como &quot;subiu adesivo&quot; ou &quot;quitosana ADHESive &quot;, respectivamente. </li><li> A rosa películas adesivas podem ser esterilizados utilizando etanol (90%) ou com radiação gama (0,1 kGy / h durante 24 horas) 4,5. </li></ol> 2. Atenuação Optical Adhesive <ol><li> Fixar o adesivo rosa dentro de um cadinho de quartzo. </li><li> Gravar o seu espectro atenuada na gama de 400-800 nm utilizando um espectrofotómetro de duplo feixe 4. </li><li> Assumindo que a validade da lei de Beer, calcular o comprimento de atenuação do adesivo como se segue: I = I 0 e Ax-onde 0 é a intensidade do feixe incidente, 1 / A atenuação é o comprimento, e x é a espessura da película. </li><li> Repita os passos 2,1-2,3 para o adesivo de quitosano para servir como um controlo. </li></ol> 3 aplicação in vitro:. Colagem de Tecidos Laser em Intestino <ol><li> Intestino de bezerro colheita imediatamente após a eutanásia dos animais e armazenar a -80 ° C. </li><li> Antes de usar, degelo e hidratar a tissue por imersão em O 2 DI-H durante 10 min. </li><li> Bissectar o tecido em secções (2 x 1 cm) usando uma lâmina 10 sob um microscópio de operação (x10). Manter a umidade seções usando DI-H 2 O. </li><li> Traga os tocos incisão juntos (ponta a ponta). Limpe o excesso de água da superfície usando pontas de algodão. </li><li> Coloque uma tira adesiva de rosa (10 x 6 mm) através da incisão na camada serosa com microfórceps garantindo contacto total com o intestino (Figura 2). </li><li> A aderência do tecido do adesivo rosa é activado por um diodo laser bombeado de estado sólido, o qual é acoplado a uma fibra óptica multimodo (diâmetro do núcleo de 200 um) 4. </li><li> Definir o nível de potência do laser de 180 mW e irradiar o adesivo para ~ min 6 com um tamanho de mancha de feixe de cerca de 5 mm, utilizando-se os parâmetros listados na Tabela 1. Ponto irradiam-o adesivo assegurando que cada local é irradiada para ~ 5 segundos antes de mover o feixe para o local adjacente. Tseu procedimento garante que o feixe laser varre a área de superfície total dos vários tempos adesivas. </li><li> Para avaliar a resistência de ligação do tecido, uma amostra de apertar a um tensiómetro de coluna única calibrado (Instron, MA, EUA), utilizando apertos mecânicos. Mover as pegas a 22 mm / min até que os dois cotos de tecidos separados 4. </li><li> Coloque um adesivo rosa sobre o tecido, tal como descrito no passo 3.5. Não irradiar a amostra e medir a resistência de ligação do tecido, tal como descrito no passo 3.8. Este exemplo serve como um controlo. </li></ol> 4 Na Aplicação Vivo:. Anastomose do nervo tibial <ol><li> Ratos Wistar com sedar o isoflurano / S mistura 2 (4% durante a indução, 2% depois) 5. Injectar subcutaneamente Buprenex (0,03 mg / kg) para fornecer intra-operatória actividade analgésica e possível aliviar a dor ou desconforto. </li><li> O seguinte procedimento cirúrgico deve ser realizado sob um microscópio de operação (x10). Faça uma pele oblíquaincisão de cerca de 3 cm de comprimento, na parte dorso-lateral da coxa direita e expor o nervo tibial com uma abordagem músculo-divisão através dos músculos glúteos 2. </li><li> Parcialmente dissecar e aparar a adventícia do nervo tibial usando retas micro-tesoura e absorver o excesso de água com uma gaze estéril ou pontas de algodão. </li><li> Cortar o nervo tibial com micro-tesoura. </li><li> Posicionar uma tira estéril quitosana (dimensões 5 x 4 mm) abaixo do nervo tibial utilizando micro-fórceps. </li><li> Aproximar os cotos end-to-end com micro-pinças sobre a fita adesiva rosa. </li><li> A tira completamente adere ao nervo formando um colar, o que pode auxiliar na rotação do nervo durante a irradiação com laser da tira. </li><li> Activar a rosa adesivo com o laser, tal como descrito nas etapas 3.6) e 3.7). </li><li> Apare o adesivo redundante subiu de o colar do nervo operado. </li><li> Feche os músculos e pele, com cinco suturas 3-0 5. </li&gt; <li> Procedimentos padrão de recuperação pós-operatória deve ser seguido, incluindo inspeção diária para deiscência ou formação de pus e administração de analgésicos, se necessário. </li></ol> 5. Resultados representativos As películas que se obtêm são brilhantes de cor rosa, fina e tem uma superfície lisa (figura 1). Eles também são flexíveis e podem ser enroladas em tubos de pequeno diâmetro, sem lágrimas causam ou qualquer outro dano aparente (Figura 1B). O adesivo rosa tem dois picos de absorção a 530 e 562 nm, o laser verde é assim fortemente absorvida pelo adesivo e o comprimento de atenuação correspondente a 532 nm é de ~ 12 ^ m (Figura 3). Em contraste, a quitosana sem películas fracamente RB atenua a laser (1 / A ~ 162 mm), provavelmente devido à dispersão. Depreende-se que a trama espectros sem agregação significativa de RB ocorre nos filmes. A rosa ligações adesivas firmementepara o intestino, após irradiação do laser (Figura 2) a realização de uma carga máxima típica de falha de 0,9 ± 0,4 N (n = 30). A força de adesão foi estimada como sendo a carga máxima dividida pela área da superfície de colagem, isto é, de 15 ± 6 kPa (n = 30). O adesivo não irradiado rosa ligado significativamente menor para o tecido (2 ± 2 kPa, n = 30, teste t não emparelhado p &lt;10 6). A anastomose in-vivo do nervo tibial foi atingido com sucesso após a aplicação e colagem do adesivo rosa em conjunto com o laser verde. Uma semana após a cirurgia, o nervo foi operado em continuidade e a força de reparação foi de 17 ± 9 kPa (n = 10). <img alt="Figura 1" src="/files/ftp_upload/4158/4158fig1.jpg" /> Figura 1. A) A solução de quitosano-RB é fundido em uma superfície plana Perspex espessura (laje laje ~ 5 mm). Papel gráfico é colocado por baixo da laje para facilitar o passo de dry-cast. O f insolúvel em águailm é obtido de duas semanas depois de fundição. B) O adesivo rosa é flexível e pode ser enrolado num cilindro pequeno. <img alt="Figura 2" src="/files/ftp_upload/4158/4158fig2.jpg" /> Figura 2. O adesivo está ligado a rosa de intestino de bezerro após irradiação do laser. Irradiação uniforme é aplicada sobre o tecido durante adesivo RB-ligação; manchas entretanto seleccionados são mostrados nesta figura para ilustrar o efeito de foto-branqueamento do laser sobre o adesivo. <img alt="Figura 3" src="/files/ftp_upload/4158/4158fig3.jpg" /> Figura 3. O espectro de absorção (em unidades arbitrárias) do adesivo rosa mostra dois picos a 530 e 562 nm. O laser verde (λ = 532 nm) é, assim, fortemente absorvida pelo adesivo durante a exposição à radiação laser. O adesivo de quitosano sem RB absorve mal o feixe de laser. <table border="1"><tbody><tr><td></td><td> n </td><td><strong&gt; Área (mm 2) </td><td> Potência (W) </td><td> Tempo (s) </td><td> Fluência (J / cm 2) </td><td> I (W / cm 2) </td><td> Max Load / Área (kPa) </td></tr><tr><td> Adesivo + Laser </td><td> 30 </td><td> 60 ± 10 </td><td> 0,18 ± 0,03 </td><td> 365 ± 5 </td><td> ~ 110 </td><td> ~ 0,9 </td><td> 15 ± 1 </td></tr><tr><td> Adesivo </td><td> 30 </td><td> 60 ± 10 </td><td> NA </td><td> NA </td><td> NA </td><td> NA </td><td> 2 ± 2 </td></tr></tbody></table> Tabela 1. Legend. n, número da amostra; Área, área de superfície do adesivo rosa (média ± erro máximo); Power, potência do laser (média ± erro máximo); Tempo, tempo de irradiação (média ± erro máximo); Fluence, fluência laser média, eu, TIR estimadaadiance; máx / Area, carga máxima de ruptura do tecido reparado dividida pela área da superfície do adesivo, (média ± EP).

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A rosa fabricação adesivo é baseado num processo de seca-molde simples, embora a dissolução do RB em pH ácido requer agitação prolongada de solução de quitosano. É importante deixar a solução secar-se até se tornar uma película insolúvel em água. Isto acontece quando o teor em água de peso é de ~ 10%, em que o filme seco 6. Filmes insolúveis são geralmente obtidas duas semanas após a seca-casting em condições padrão de temperatura e pressão (~ 25 ° C e ~ 1 atm). O mecanismo de liga?...

<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> Rosa Bengala </td><td> Sigma-Aldrich </td><td> 632-69-9 </td><td></td></tr><tr><td> A quitosana (média MW) </td><td> Sigma-Aldrich </td><td> 10318AJ </td><td></td></tr><tr><td> Ácido acético glacial </td><td> Sigma-Aldrich </td><td> 08050051 </td><td> 2% v / v em água Dl </td></tr><tr><td> Agitador magnético </td><td> Heidolph </td><td> MR Hei-Mix S </td><td></td></tr><tr><td> Centrifugador </td><td> Beckman Coulter </td><td> Allegra X-12R </td><td></td></tr><tr><td> Espectrofotômetro </td><td> Varian </td><td> Cary 4000 UV-Visível </td><td></td></tr><tr><td> Laser </td><td> CNI Laser </td><td> MGL-532 </td><td></td></tr><tr><td> Micrômetro </td><td> Mitutoyo </td><td> Série 227 </td><td></td></tr><tr><td> Microscópio cirúrgico </td><td> Zeiss </td><td> OPMI </td><td></td></tr></tbody></table>

fabrication and application of rose bengal-chitosan films in laser tissue repair

Colagem de tecidos fotoquímica (PTB) é uma técnica de sutura para a reparação de tecidos, o que é conseguido através da aplicação de uma solução de rose bengal (RB) entre duas extremidades de tecido 1,2. Estes são depois irradiadas por um laser que é selectivamente absorvido pelo RB. As reações fotoquímicas resultantes supostamente crosslink as fibras de colágeno no tecido com 3 mínima produção de calor. Neste relatório, RB, foi incorporada em filmes finos de quitosano para o fabrico de um adesivo de tecido novo que é activado por laser. Películas adesivas, com base em quitosano e contendo ~ 0,1% em peso de LD, são fabricados e ligados ao intestino de vitelo e os nervos tibiais de ratos por um laser de estado sólido (λ = 532 nm, de fluência ~ 110 J / cm 2, tamanho do ponto ~ 0,5 cm) . Um tensiómetro de coluna única, em interface com um computador pessoal, é utilizado para testar a resistência de ligação. Os RB-quitosano ligações adesivas com firmeza para o intestino com uma força de 15 ± 6 kPa, (n = 30). A força de adesão diminui para 2 &amp;plusmn; 2 kPa (n = 30) em que o laser não é aplicada ao adesivo. A anastomose de nervos tibial pode ser igualmente concluída sem a utilização de suturas. A novel quitosana adesivo ter sido fabricado que os títulos fotoquimicamente ao tecido e não requer sutura.

Watch this Scientific Journal Video about Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair at JoVE.com

Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Suturas são normalmente necessários para reparar o tecido durante procedimentos cirúrgicos. No entanto, a sua aplicação pode ser problemática uma vez que são invasivos e pode danificar o tecido. Os métodos de fabricação e aplicação de um adesivo de tecido romance são aqui relatados. Esta película adesiva é activada por laser e não requer o uso de suturas.

Fabricação e Aplicação de Rosa Bengala-quitosana Films em Reparo de Tecidos Laser

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two ...

fabrication-application-rose-bengal-chitosan-films-laser-tissue

Research

JoVE Journal

Bioengineering

12.1K visualizações.  University of Western Sydney, NSW Australia. Suturas são normalmente necessários para reparar o tecido durante procedimentos cirúrgicos. No entanto, a sua aplicação pode ser problemática uma vez que são invasivos e pode danificar o tecido. Os métodos de fabricação e aplicação de um adesivo de tecido romance são aqui relatados. Esta película adesiva é activada por laser e não requer o uso de suturas.

Vídeo: Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

Biologicamente fabricação de materiais derivados injetável para Engenharia de Tecidos do Miocárdio

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Bioengenharia

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Campo elétrico controlado de migração dirigida de células progenitoras neurais em ambientes 2D e 3D

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

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 ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Andaimes auto-relato para cultura de células 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Cartilagem Human Tissue Fabricação Usando tridimensional Inkjet Tecnologia de Impressão

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adaptação do Processo Electrospinning para fornecer três ambientes únicos para um Tri-camadas<em&gt; In Vitro</em&gt; Modelo da parede das vias aéreas

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

O tecido triturado em Compressed Collagen: Um BioTransplant contendo celular para o reparo Reconstructive Single-encenado

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

guiado por imagem, fabricação à base de laser de Redes microfluídicos Vascular derivadas

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Fabricação e caracterização de espectros ópticos de tecido contendo Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

Imagem fotoacoustic de pequeno animal de varredura dupla raster-scaning para visualização vascular

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...