Este trabalho foi financiado pelo NIH conceder R01AI077610.

Todos os procedimentos foram realizados de acordo com o Guia para o Cuidado e Uso de Animais de Laboratório do Instituto Nacional de Saúde (NIH) e foram aprovados pela Comissão de Johns Hopkins University animal Cuidado e Uso (JHUACUC). Os procedimentos específicos foram realizados sob a MO13M292 protocolos ACUC aprovado e MO13M370. 1. Donor Colheita da pele <ol><li> Anestesiar o rato doador com isoflurano (vaporizador de indução a 4%, manutenção a 1 - 2% através do cone mouse). Use o dedo retirada pitada reflexo para monitorizar a profundidade de anestesia. </li><li> Usando uma máquina de barbear, a barba do dorso do animal e desinfecção com 10% de iodo de povidona (por exemplo, Betadine). </li><li> Com uma tesoura estéril, luvas e técnica asséptica, colheita doador de pele de volta a partir do quadril para o pescoço, com dissecção romba ao nível do tecido conjuntivo areolar. </li><li> Eutanásia do animal por deslocamento cervical após a colheita daenxerto de pele. </li><li> Sob um microscópio, separar o tecido conjuntivo, tecido adiposo, e panículo carnoso da pele do dorso utilizando tesouras finas Tenotomia. O panículo carnoso é um músculo fino e transparente responsáveis ​​por movimentos espasmos pele. </li><li> Utilizando uma técnica estéril instrumentos, cortar enxertos de 15 mm x 15 mm a partir da pele de volta para uma de 10 mm x 10 mm para a cama enxerto de 15 mm x 15 mm. </li><li> Armazenar os enxertos em gazes impregnadas com solução salina estéril tamponada com fosfato (PBS) numa placa de petri em gelo. NOTA: 8 - 10 enxertos pode ser obtido a partir de um rato dador. </li></ol> Transplante de pele 2. Destinatário <ol><li> Anestesiar o mouse destinatário com isoflurano (vaporizador de indução a 4%, manutenção a 1 - 2% através do cone mouse). Use o dedo retirada pitada reflexo para monitorizar a profundidade de anestesia. </li><li> Administrar 0,02 mg / kg de peso corporal de buprenorfina para o alívio da dor pós-operatória. </li><li> Raspar o lado das costas do animal, onde o wi enxertoll ser inserido e desinfectar com 10% de iodopovidona. </li><li> Com uma tesoura, corte de 10 mm x 10 mm a 15 mm x 15 mm quadrado de pele. O tamanho de defeito deve ser ligeiramente maior (10%) do que o enxerto. Corte o mais superficialmente possível. Tome cuidado para preservar o panículo carnoso e vasos. O panículo carnoso pode ser distinguida da fáscia subjacente por sua mobilidade e os vasos sanguíneos superficiais que executam a ele. </li><li> Posicionar o enxerto no leito de enxerto, evitando dobras ao longo das bordas. </li><li> Coloque 8 suturas nos cantos e no meio de cada borda. Para cada fio de sutura, a agulha passar através do enxerto e, em seguida, através do panículo carnoso do enxerto de leito inferior a pele circundante destinatário. </li><li> Retire a máscara de anestésico e deixar o animal recuperar parcialmente da anestesia antes de aplicar a bandagem adesiva. </li><li> Enrole o mouse destinatário em uma bandagem adesiva com gaze dobrada sobre o enxerto. Faça o curativo pela combinação de duas ataduras, cortandoa parte adesiva de um e colocando as duas almofadas absorventes em conjunto. </li><li> Monitorar o mouse de perto durante a recuperação para garantir que o curativo não está restringindo tórax excursão e respirar. Remover o curativo, se a taxa respiratória diminui ou o animal começa a ofegar ou respiração superficial. </li></ol> 3. Cuidados Pós-Operatórios <ol><li> Administrar enrofloxacina 5 mg / kg após a cirurgia para a profilaxia da infecção. </li><li> Posicione o mouse transplantado em uma gaiola limpa sobre uma almofada de aquecimento no forno até que se recupere totalmente da anestesia. </li><li> Observe o mouse por 1 hora de pós-operatório antes de retornar para a instalação de habitação. </li><li> Sete dias após a cirurgia, anestesiar o rato como no Passo 2.1. Remover o curativo, cortando apenas através do lado ventral do curativo. </li><li> Observar e palpar o enxerto no dia seguinte para sinais de formação de crostas, contração ou dureza. Se estiver presente, o enxerto pode não ter alcançado vasculariza adequadação e deve ser considerada uma falha técnica. </li><li> Monitorizar diariamente quanto a sinais de rejeição. Considere enxertos rejeitada quando ≥90% do tecido do enxerto torna-se necrótica. </li><li> Eutanásia dos animais rejeitados e colhê-las para análise. </li></ol>

<ol>
	<li>Furtmuller, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+Hind+Limb+Transplantation+in+the+Mouse.">Orthotopic Hind Limb Transplantation in the Mouse.</a> J Vis Exp. , (2016).</li><li>Oh, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Microsurgical+Model+for+Heterotopic,+En+Bloc+Chest+Wall,+Thymus,+and+Heart+Transplantation+in+Mice.">A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice.</a> J Vis Exp. , (2016).</li><li>Oberhuber, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+cervical+heart+transplantation+model+using+a+modified+cuff+technique.">Murine cervical heart transplantation model using a modified cuff technique.</a> J Vis Exp. , e50753 (2014).</li><li>Jones, T. R., Shirasugi, N., Adams, A. B., Pearson, T. C., Larsen, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+identifies+selectins+that+regulate+T+cell+traffic+into+allografts.">Intravital microscopy identifies selectins that regulate T cell traffic into allografts.</a> J Clin Invest. 112, 1714-1723 (2003).</li><li>Celli, S., Albert, M. L., Bousso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+the+innate+and+adaptive+immune+responses+underlying+allograft+rejection+by+two-photon+microscopy.">Visualizing the innate and adaptive immune responses underlying allograft rejection by two-photon microscopy.</a> Nat Med. 17, 744-749 (2011).</li><li>Medawar, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+and+fate+of+skin+autografts+and+skin+homografts+in+rabbits:+A+report+to+the+War+Wounds+Committee+of+the+Medical+Research+Council.">The behaviour and fate of skin autografts and skin homografts in rabbits: A report to the War Wounds Committee of the Medical Research Council.</a> J Anat. 78, 176-199 (1944).</li><li>Billingham, R. E., Brent, L., Medawar, P. B., Sparrow, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+studies+on+tissue+transplantation+immunity.+I.+The+survival+times+of+skin+homografts+exchanged+between+members+of+different+inbred+strains+of+mice.">Quantitative studies on tissue transplantation immunity. I. The survival times of skin homografts exchanged between members of different inbred strains of mice.</a> Proc R Soc Lond B Biol Sci. 143, 43-58 (1954).</li><li>Garrod, K. R., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+skin+transplantation.">Murine skin transplantation.</a> J Vis Exp. , (2008).</li><li>Schmaler, M., Broggi, M. A., Rossi, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+tail+skin+to+study+allogeneic+CD4+T+cell+responses+in+mice.">Transplantation of tail skin to study allogeneic CD4 T cell responses in mice.</a> J Vis Exp. , e51724 (2014).</li><li>Bergstresser, P. R., Toews, G. B., Gilliam, J. N., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+numbers+and+distribution+of+Langerhans+cells+in+skin+with+unique+immunologic+properties.">Unusual numbers and distribution of Langerhans cells in skin with unique immunologic properties.</a> J Invest Dermatol. 74, 312-314 (1980).</li><li>Chen, H. D., Silvers, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Langerhans+cells+on+the+survival+of+H-Y+incompatible+skin+grafts+in+rats.">Influence of Langerhans cells on the survival of H-Y incompatible skin grafts in rats.</a> J Invest Dermatol. 81, 20-23 (1983).</li><li>Chong, A. S., Alegre, M. L., Miller, M. L., Fairchild, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+and+limits+of+mouse+models.">Lessons and limits of mouse models.</a> Cold Spring Harb Perspect Med. 3, a015495 (2013).</li><li>Mayumi, H., Nomoto, K., Good, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surgical+technique+for+experimental+free+skin+grafting+in+mice.">A surgical technique for experimental free skin grafting in mice.</a> Jpn J Surg. 18, 548-557 (1988).</li><li>McFarland, H. I., Rosenberg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+allograft+rejection.">Skin allograft rejection.</a> Curr Protoc Immunol. Chapter 4, (2009).</li><li>Lee, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+Allograft+Rejection+by+Targeting+Immune+Metabolism.">Preventing Allograft Rejection by Targeting Immune Metabolism.</a> Cell reports. 13, 760-770 (2015).</li><li>Pollizzi, K. N., Powell, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+canonical+and+metabolic+signalling+programmes+in+the+regulation+of+T+cell+responses.">Integrating canonical and metabolic signalling programmes in the regulation of T cell responses.</a> Nat Rev Immunol. 14, 435-446 (2014).</li><li>Pearce, E. L., Poffenberger, M. C., Chang, C. H., Jones, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fueling+immunity:+insights+into+metabolism+and+lymphocyte+function.">Fueling immunity: insights into metabolism and lymphocyte function.</a> Science. 342, 1242454 (2013).</li></ol>

Os autores não têm nada para revelar.

Desde a sua introdução por Medawar, primeiro em estudos humanos e, em seguida, em coelhos e ratinhos, o transplante de pele tem sido um modelo de valor inestimável para o estudo das respostas imunitárias alogénicas 6,7. Neste artigo, apresentamos um modelo de grande escala, o transplante de pele não-vascularizado de espessura total com a pele para trás superior e inferior. Vários métodos alternativos, incluindo o uso da pele da cauda ou da pele da orelha de rato como fonte de tecido do enxerto, foram...

O transplante de órgãos é o tratamento de escolha para pacientes com casos de insuficiência terminal de órgãos, e os resultados têm melhorado notavelmente com os avanços em procedimentos cirúrgicos e protocolos de imunossupressão. No entanto, a imunossupressão a longo prazo está associada a efeitos colaterais significativos, e o desenvolvimento de novas estratégias que promovem a tolerância continua a ser o objectivo da investigação moderna transplante. Numerosos modelos animais têm sido desenvolvidos para a pesquisa básica em transplante, para estudar os mecanismos da rejeição do enxerto e para testar se aproxima de imunossupressão para a prevenção da rejeição de enxertos e para promover a tolerância a longo prazo 1-3. mouse modelos tornaram-se o esteio da pesquisa imunológica devido à disponibilidade exclusiva e vasta de anticorpos diagnósticos e terapêuticos e estirpes puras e transgênicas bem definidas. transplante de pele é um procedimento simples que não requer habilidades especiais de microcirurgia e podeser facilmente monitorizado no pós-operatório. Tomados em conjunto, o transplante de pele de ratinho tem sido uma ferramenta excepcional para estudar muitos aspectos envolvidos na resposta aloimune, incluindo a entrega de antigénio, o tráfico de células, e a destruição do tecido durante a rejeição de enxerto de 4,5. Aqui, mostramos o procedimento passo-a-passo para o transplante de pele de espessura total utilizando o modelo de rato, e descrevemos as etapas importantes necessárias para um enxerto bem-sucedida da pele transplantada.

<table><tbody><tr><td>Straight micro forceps</td><td>Sigma</td><td>F4017</td><td></td></tr><tr><td>Curved micro forceps</td><td>Aesculap</td><td>BD333R</td><td></td></tr><tr><td>Curved Stevens tenotomy scissors</td><td>Aesculap</td><td>BC905R</td><td></td></tr><tr><td>Mayo dissecting scissors</td><td>Sigma</td><td>S3146</td><td></td></tr><tr><td>Micro needle holder</td><td>Aesculap</td><td>BM563R</td><td></td></tr><tr><td>Sterile gauze</td><td>Covidien</td><td>441218</td><td></td></tr><tr><td>6-0 Nylon suture</td><td>MWI</td><td>31849</td><td></td></tr><tr><td>Plastic Strips Band&amp;#45;Aid</td><td>Johnson &amp;amp; Johnson</td><td></td><td>Obtained from pharmacy</td></tr><tr><td>10 cm Petri dish</td><td>Fisherbrand</td><td>FB0875712</td><td></td></tr><tr><td>PBS</td><td>Quality Biological</td><td>119069131</td><td></td></tr><tr><td>Buprenorphine</td><td></td><td></td><td>DEA Number required; Obtained from hospital pharmacy</td></tr><tr><td>Enrofloxacin</td><td>Bayer Health Care</td><td>186599</td><td></td></tr></tbody></table>

murine full-thickness skin transplantation

O transplante de pele de espessura total de murino é um modelo bem estabelecido para o estudo in vivo de resposta aloimune e rejeição de enxerto. Apesar da sua aplicação limitada a seres humanos, o transplante de pele em ratinhos tem sido amplamente utilizada para a pesquisa em transplantes. O procedimento é fácil de aprender e executar, e não requer técnicas de microcirurgia delicada, nem treinamento extensivo. Além disso, a rejeição do enxerto neste modelo ocorre em uma reacção imunológica muito reprodutível e é facilmente monitorizado por inspecção e palpação directa. Além disso, o transplante de pele secundário combinados com enxertos de pele de doadores ou de terceiros pode ser realizada em modelos de transplante mais complexos como um método alternativo e simples para avaliar a tolerância específica do dador. As complicações são baixos e são, em geral, limitado a uma overdose de anestesia ou angústia respiratória após o procedimento. falha do enxerto, por outro lado, ocorre geralmente como um resultado da má preparação da Grpopa, posicionamento incorreto no leito do enxerto, ou posicionamento inadequado de o curativo. Neste artigo, apresentamos um protocolo para o transplante de pele de espessura total em ratos e descrever os passos importantes necessários para o sucesso do procedimento.

A colocação do curativo sobre o mouse destinatário é um passo importante do processo. O enxerto de pele é posicionada no tronco recipiente, entre o ombro, anca e espinha (Figura 1). A ligadura é feita com gaze dobrada e a combinação de duas ataduras adesivas de plástico. O rato receptor é colocado com o enxerto para baixo sobre a gaze sobre o centro da ligadura. Usando dois micro pinças curvas, a extremidade inferior da ligadura é primeiro removido, e em segu...

Watch this Scientific Journal Video about Murine Full-thickness Skin Transplantation at JoVE.com

Murine Full-thickness Skin Transplantation

transplante de pele de espessura total murino é um modelo bem estabelecido para estudar a rejeição em um ambiente alloimmune. Aqui, nós fornecemos um tutorial de cada etapa envolvida na realização de um / c BALB -&gt; C57BL / 6 transplante de pele de espessura total.

Murino Transplante de pele de espessura total

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

murine-full-thickness-skin-transplantation

Research

JoVE Journal

Immunology and Infection

20.0K visualizações.  Johns Hopkins University School of Medicine.  transplante de pele de espessura total murino é um modelo bem estabelecido para estudar a rejeição em um ambiente alloimmune. Aqui, nós fornecemos um tutorial de cada etapa envolvida na realização de um / c BALB -> C57BL / 6 transplante de pele de espessura total. 

Vídeo: Murine Full-thickness Skin Transplantation

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

A Pele Tridimensional Humanos Reconstruir Modelo: uma Ferramenta para o Estudo da Pele Normal e Progressão Melanoma

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Bioengenharia

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

Transplante de pele murinos

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Biologia

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

A pele completa Defeito Modelo Estudar a vascularização de Biomateriais<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Modelo Murino de Cicatrização

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Medicina

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 ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

O transplante de pele da cauda para estudar as respostas das células T CD4 alogênico em camundongos

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Imunologia e Infecção

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

Geração de organoides pele 3D do cordão hemoderivados induzida por células-tronco pluripotentes

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Biologia do Desenvolvimento

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

Modelo de pele de xenoenxerto para manipular respostas imunes humanas in vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Um modelo murino de uma ferida de queimadura reconstruída com um enxerto de pele aogenética

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

A Technique for Human Skin Grafting in a Mouse Model

Source: Moss, M. I. et al., <a href="https://app.jove.com/v/64040">Xenograft Skin Model to Manipulate Human Immune Responses&#160;In Vivo.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates a method for grafting human skin onto an immunodeficient mouse with a suppressed neutrophil response. The method involves applying human split-thickness skin to the mouse&#39;s graft bed, sealing it, and covering it for recovery. Eventually, the human skin adheres to the mouse&#39;s skin, establishing vascularization and circulation, resulting in a successful skin xenograft.

Uma técnica para enxerto de pele humana em um modelo de camundongo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...