Gostaríamos de agradecer ao Dr. George Commons e Dr. Dean Vistnes por seu apoio e colaboração da nossa pesquisa. Este trabalho é apoiado pelo Instituto Nacional de Bolsas de pesquisa Dental e Craniofacial um R21 DE019274-01, R01EB009689 e RC2 DE020771-02, da Fundação Oak e Hagey Laboratório de Medicina Regenerativa de Pediatria para MTL Dr. Hyun é apoiado pela Saint Joseph Mercy Hospital GME .

1. Isolamento de células e Expansão <ol><li> Todos consentimento do paciente e os protocolos experimentais foram analisados ​​e aprovados pela Universidade de Stanford Conselho de Revisão Institucional (Protocolo n º 2188 e n º 9999). </li><li> Obtenção de tecido adiposo humano subcutânea de procedimentos lipoaspiração eletivas sob local / anestesia geral. </li><li> Haverá duas camadas na lipoaspirado (Figura 1A). O sobrenadante contém a grande maioria do material processado celular. A camada inferior é geralmente injectado salino. Derivadas de tecido adiposo células estromais podem ser colhidas a partir de qualquer camada, mas o rendimento é muito maior do sobrenadante. </li><li> Para isolar a fracção do estroma vascular (SVF), lava-se a lipoaspirado extensivamente com volumes iguais de 1X tampão de fosfato salino (PBS) contendo 2,5% de betadine (x2), seguido de igual volume de PBS 1X (x1) sem betadine. Permitir que a solução de lavagem a precipitar. </li><li> Tenha o cuidado de manter te estérilchnique ao longo do processo como a contaminação com o tecido humano processado pode acontecer. </li><li> Aspirar a camada inferior e descartar depois de cada lavagem. </li><li> Alíquotas de 15 ml de tecido adiposo em tubos de 50 ml, BD Falcon cónicos. </li><li> Digerir o tecido adiposo, com um volume igual (15 ml) de filtrado de 0,075% de colagenase do Tipo II em Solução Salina Equilibrada de Hank (HBSS) a 37 ° C em banho-maria durante 60 min, sob agitação constante (cerca de 180 agitações / min) - ventilar cada tubo a cada 15 min. </li><li> Neutralizar a actividade da enzima com 15 ml de PBS contendo 10% (soro fetal bovino) de FBS, e centrifuga-se a 1000 rpm durante 5 min a 4 ° C para se obter uma alta densidade de SVF pellet. O sedimento será uma mistura de derivadas de tecido adiposo células estromais e eritrócitos. </li><li> Desprezar o sobrenadante sem perturbar o sedimento. </li><li> Ressuspender o sedimento em 10 ml de meio de crescimento tradicional (Dulbecco Modified Eagle Médium [DMEM] / FBS a 10% / 1% de solução de penicilina-estreptomicina). </li><li> Filtro de suspensão<html> Sion através de um coador de células 100 uM de nylon para remover os restos celulares. </li><li> Combinar 3 tubos em um limpo de 50 ml. Centrifugar a 1000 rpm durante 5 min a 4 ° C. </li><li> Desprezar o sobrenadante sem perturbar o sedimento. </li><li> Ressuspender o sedimento em 5 ml de meio de crescimento tradicional </li><li> Combinar duas cônicas 50 ml por uma placa de 10 cm ou cinco cônicas 50 ml para uma placa de 15 cm. </li><li> Estabelecer culturas primárias durante a noite a 37 ° C/21% de O 2, 5% de CO 2. </li><li> Após incubação durante a noite, lava-se as placas extensivamente com PBS para remover residuais não aderentes glóbulos vermelhos. As populações de células resultantes são derivadas de tecido adiposo células estromais. </li><li> Manter as células em níveis sub-confluentes para prevenir a diferenciação espontânea a 37 ° C/21% de O 2, 5% de CO 2 em meio de crescimento. As células geralmente precisam ser divididas a cada 3-4 dias em meio de crescimento regular quando plaqueadas a 50% de confluência. </li></ol>ve_title &quot;&gt; 2. Preparação Andaime <ol><li> Os andaimes foram feitas pelo Dr. Min Lee na Universidade da Califórnia, Los Angeles - Faculdade de Odontologia. </li><li> Andaimes PLGA foram fabricados a partir de 85/15 poli (ácido láctico-co-glicólico) (viscosidade inerente = 0,61 dl / g, Birmingham Polymers) por evaporação de solvente e de um processo de lixiviação de partículas. </li><li> PLGA / clorofórmio soluções foram misturadas com sacarose 200-300 um de diâmetro para se obter 92% de porosidade (percentagem, em volume), e comprimida em folhas finas de um molde de Teflon. </li><li> Após liofilização durante a noite, os andaimes foram imersos em três mudas de bidestilada (dd) H 2 O para dissolver a sacarose, e suavemente removida da placa de Teflon com uma espátula de ponta fina. </li><li> Após a lixiviação das partículas, todos os suportes foram desinfectados por imersão em 50%, 60% e etanol a 70% durante 30 min cada, seguido de três lavagens de DDH 2 O. </li><li> Os andaimes foram secos sob uma câmara de fluxo laminar. </li><li> Após a fabricação de andaime, scaffolds foram revestidos com apatite. <ol class="ualpha"><li> SBF solução (fluido corporal simulado) foi preparada com concentrações de iões que eram 5 vezes aquela do plasma de sangue humano. Todas as soluções foram esterilizadas por filtração através de uma membrana de 0,22 ^ M PES (Nalgene). Imediatamente antes do processo de revestimento, secaram-se os andaimes de PLGA foram submetidos a descarga luminescente, de árgon-plasma etching (Harrick Scientific) para melhorar a molhagem e a uniformidade do revestimento. </li><li> Andaimes gravadas PLGA foram então incubadas em SBF a 37 ° C dentro de uma incubadora com camisa de água durante 12 horas, seguido de Mg 2 + e HCO 3 - SBF livre 2 por um outro 12 hr a 37 ° C sob agitação suave. </li><li> Andaimes revestidos PLGA foram lavadas suavemente com estéril DDH 2 O, para lavar o excesso de solução de cloreto de sódio, secou-se numa câmara de fluxo laminar e desinfectada com álcool a 70%. </li><li> Integridade do revestimento apatita foi analisada com um microscópio eletrônico de varredura (MEV). Apatita revestido scaffolds foram montadas em bases de SEM (Ted Pella) e revestidas com carbono para melhorar a condutividade. O modo de elétron secundário foi aplicado durante SEM observação (FEI / Phillips XL-30). Espectro de dispersão de energia de raios X foram obtidos a confirmar a composição elementar das estruturas de apatite. </li></ol></li></ol> 3. Sementeira celular <ol><li> Ao atingir os níveis de sub-confluência, lavar as células com PBS (x2) e Tripsinizar. </li><li> Contar as células de quantificar para propagação </li><li> Semente em pré-cortadas 4,0 andaimes com 1,5 mm de diâmetro x 10 5 células. <ol class="ualpha"><li> Lugar individual andaime em um poço de uma placa de 96 poços </li><li> Re-suspender as células em meio de crescimento regular com uma concentração de cerca de 1,5 x 10 5 células por 20 uL de meios </li><li> Pipetar 20 uL diretamente sobre o andaime e coloque em cultura de células de incubadora para 30 min. </li><li> Após 30 min, adiciona-se 200 ul de meio e permitir que as células a incubarno andaime durante a noite antes da cirurgia </li><li> Não é necessário para assegurar a adesão das células sobre o andaime como semeando um número suficiente de células irá garantir que a maioria vai anexar para o andaime. Este foi validado em nosso laboratório in vivo com a bioluminescência e análise histológica. </li></ol></li></ol> 4. Criação de defeitos de calvárias e na implantação Vivo <ol><li> Anesthetize adulto (60 dias de idade) CD-1 ratinhos nus com cocktail anestésico. (Cetamina / xilazina / acepromazina) na concentração de 50% (diluição 1:1 com NaCl 0,9%), ou por recomendada regime anestésico por protocolos instituição. Estes ratos são então as hASCs atímicos não irá causar uma reacção imune. <ol class="lalpha"><li> Dosagem: cloridrato de cetamina (80 mg / kg), xilazina (2,5 mg / kg) de Acepromazina (2,5 mg / kg) </li><li> Duração do efeito: aproximadamente 30 min </li></ol></li><li> Cirurgicamente armar o animal e esterilizar o cirúrgicolocal com betadine e álcool (x3) e cobrir os olhos do rato com óleo antes de fazer uma incisão na linha média sagital no escalpo do animal para expor o osso parietal direito. Remover o pericrânio do osso parietal direito com raspagem romba (Figura 1B). </li><li> Criar um unilateral de 4 mm de espessura total defeito no osso parietal direito não sutura associada com um estéril diamantada broca trefina. Cuidado extremo deve ser tomado para não perturbar a dura-máter subjacente (Figura 1B) que a espessura do mouse calvária é &lt;0,3 milímetros. </li><li> Antes de implantação, lavar andaimes com PBS estéril para evitar a transferência de factores de crescimento derivados das médias. </li><li> Coloque o andaime no defeito. </li><li> Finalmente, suturar a pele fechada e monitorar o animal por protocolos estabelecidos no pós-operatório. </li><li> Use analgesia estabelecido como dirigido por seus protocolos de animais cuidados necessários no pós-operatório, por exemplo. buprenorfina 0,1 mg / kg. </li></ol> 5. Quantificação de osteóide <ol><li> Após MicroCT foi realizada no rato, a imagem tridimensional foi reconstruída utilizando software MicroView como previamente descrito 1. </li><li> Utilizando o Adobe Photoshop, as imagens foram dimensionados para uma altura padrão. </li><li> Usando o recurso Magic Wand, os pixels foram medidos do defeito ósseo craniano. </li><li> Percentual de cura do defeito foi determinada por exames posteriores CT medir a área do defeito ósseo craniano e determinar o número de pixels e dividindo-o pelo número de pixels do defeito original </li><li> Consequentemente, se o MicroCT não estiver disponível, é uma alternativa utilizando Adobe Photoshop e colheita da calvária em pontos de tempo determinados para histologia. </li><li> Usando manchas histológico como anilina azul e Pentachrome que denotam a formação de osteóide, várias seções ao longo do período do defeito deve ser manchado </li><li> Utilizando o Adobe Photoshop, recortar todas as áreas que não sãono defeito ósseo craniano </li><li> Use o recurso de varinha mágica e determinar o número de pixels de formação de osso novo na área do defeito e comparar com controles ou outras variáveis. </li></ol> 6. Resultados representativos O tecido adiposo não tem o potencial de servir um papel vital na geração de células progenitoras para aplicação clínica. O tecido adiposo não tem uma vantagem única na medida em que existe um fornecimento facilmente disponível que pode ser colhida de um procedimento relativamente simples que envolve mínima morbidade e mortalidade. Uma vez que o tecido é colhido e recolhido, o nosso protocolo é descrito na Figura 2. Derivadas de tecido adiposo células estromais são isoladas através de uma série de passos de lavagem, a digestão com colagenase, e centrifugação. Uma vez que as células são plaqueadas em placas de cultura, que pode ser expandido, colocado em vários protocolos de diferenciação in vitro, ou colocado directamente in vivo. O creation do defeito crítico 4-mm de tamanho calvária proporciona um fácil acesso e reprodutível modelo in vivo para testar a capacidade de diferenciação osteogénica nossos derivadas de tecido adiposo células estromais. Através da utilização de MicroCT, que são capazes de seguir a formação do tecido ósseo in vivo e controlar o progresso das intervenções (Figura 3). O defeito de tamanho crítico calvária não cicatrizam dentro do tempo de vida do animal, e podemos ver cerca de 90% do defeito permanecer patente cerca de 8 semanas. O andaime em si (ver discussão) tem propriedades osteogénicas indutivas e tem demonstrado a capacidade de induzir alguma regeneração óssea. Os resultados típicos de colocação de andaime sem células mostra que cerca de um terço do defeito terá de formação de osso novo às 8 semanas. Com o aumento das derivadas de tecido adiposo células estromais, inteiramente de dois terços ou mais do defeito irá mostrar regeneração óssea em cerca de 8 semanas, embora haja variability entre cada animal e cirurgia. A formação de tecido esquelético pode ser quantificado através de exame histológico e os resultados típicos mostram o aumento na formação osteóide em amostras com ASC por meio de coloração com azul de anilina e Pentachrome (Figura 3C). Além disso, mostra-se que as células implantadas humanos contribuem para a formação de base de novo óssea através da utilização de hASCs GFP-etiquetados que mostram a coloração in vivo de 2 semanas, perto da zona de formação de osso novo no defeito ósseo craniano (Figura 3D) . Além disso, podemos usar imuno-histoquímica com o anticorpo humano específico para mostrar a sobrevivência de células humanas e de contribuição na área do defeito (Figura 3E). <img alt="Figura 1" src="/files/ftp_upload/4221/4221fig1.jpg" /> Figura 1 A -. Lipoaspirado A tem duas camadas. A camada superior contém os adipócitos e que a maioria do processes material celular, enquanto a camada inferior contém a solução salina utilizada durante o procedimento de lipoaspiração. B - a criação do defeito ósseo craniano através de uma incisão na linha média ao longo do pericrânio para isolar o osso parietal direito, subsequente criação de um defeito de 4 milímetros de tamanho crítico calvária sem rompimento da dura-máter subjacente. <img alt="Figura 2" src="/files/ftp_upload/4221/4221fig2.jpg" /> Figura 2. Resumo da colheita e da aplicação de lipoaspirado a partir do isolamento de células derivadas de tecido adiposo do estroma para a sua expansão, diferenciação e utilização in vitro e in vivo. <img alt="Figura 3" src="/files/ftp_upload/4221/4221fig3.jpg" /> Figura 3. MicroCT-A mostra na cura vivo do defeito de tamanho crítico calvária com a aplicação de ASC por meio de um hydroxyapatitie andaime (linha de fundo). Os controles incluem nenhum andaime e defeito (linha superior) e defeito com a colocação do andaime sem células (linha do meio) B - Quantificação de cicatrização óssea do MicroCT mostrando cura aumentou significativamente no grupo ASC. C - Histologia mostrando aumento da formação de osteóide do grupo ASC (linha de fundo) através de azul de anilina e coloração Pentachrome. Para Anilina Azul, as manchas de osteóide azul escuro e para Pentachrome, as manchas de osteóide amarelo. D - hASCS marcadas com GFP foram semeadas sobre um andaime e colocados em um defeito calvária e sacrificados às 2 semanas. A coloração foi feita com um anticorpo marcado GFP para mostrar as células humanas que contribuem para a regeneração na área do defeito. E -. Imunohistoquímica antigénio nuclear humano que mostra a prevalência de células humanas na área do defeito de 2 semanas <a href="http://www.jove.com/files/ftp_upload/4221/4221fig3large.jpg" target="_blank">para ver figura maior</a> .

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Uma vez que o isolamento do derivado de tecido adiposo a partir de células do estroma 2 lipoaspirado, estas células foram diferenciadas em uma grande variedade de linhagens celulares. O tecido adiposo é de origem mesodérmica e, portanto, multipotentes derivadas de tecido adiposo células estromais serão provavelmente mais eficaz com a aplicação para uma linhagem mesodérmica. A capacidade de gerar tecido ósseo é especialmente crítico dada a escassez de área doadora de um autoenxerto e as limitaçõ...

<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></td><td></td><td></td><td> Lipoaspirado Colheita </td></tr><tr><td> PBS </td><td> Gibco </td><td> 10010-023 </td><td></td></tr><tr><td> Solução Salina Equilibrada de Hank </td><td> Cellgro </td><td> 21-023 CV- </td><td></td></tr><tr><td> Colagenase </td><td> Sigma </td><td> C6885-500mg </td><td></td></tr><tr><td> Célula de 100 um filtro </td><td> BD Falcon </td><td> 352360 </td><td></td></tr><tr><td> Steri-top 500 ml 0,22 mM filtro </td><td> Millipore </td><td> SCGPT05RE </td><td></td></tr><tr><td></td><td></td><td></td><td> Defeito calvária </td></tr><tr><td> Z500 Brushless MicromotorsUM50C </td><td> NSK </td><td> NSKZ500 </td><td></td></tr><tr><td> Circular Faca 4,0 milímetros </td><td> Xemax Cirúrgica </td><td> CK40 </td><td></td></tr></tbody></table>

repair of a critical-sized calvarial defect model using adipose-derived stromal cells harvested from lipoaspirate

Reparação e regeneração do esqueleto craniofacial oferece a promessa de formação de tecido de novo através de uma abordagem com base em células, utilizando células estaminais. Derivadas de tecido adiposo células do estroma (ASC) têm provado ser uma fonte abundante de células estaminais multipotentes capazes de sofrer diferenciação osteogénica condrogénica, adipogênica e miogénica. Muitos estudos têm explorado o potencial osteogénico destas células in vivo com o uso de biomateriais de andaimes diferentes para entrega celular. Tem sido demonstrado que, utilizando um osteocondutor, hidroxiapatite revestido de poli (ácido láctico-co-glicólico) (PLGA-HA) scaffold semeado com ASCs, um defeito de tamanho crítico calvária, um defeito que é definido pela sua incapacidade de sofrer espontânea cura durante o tempo de vida do animal, podem ser eficazmente mostrar regeneração óssea robusta. Este modelo in vivo demonstra a base de abordagens de translação com vista a regenerar o tecido ósseo - o celularcomponente e matriz biológica. Este método serve como um modelo para a aplicação final clínico de uma célula progenitora para a reparação de um defeito do tecido específico.

Watch this Scientific Journal Video about Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate at JoVE.com

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Este protocolo descreve o isolamento de derivadas de tecido adiposo a partir de células do estroma lipoaspirado e a criação de um defeito de 4 milímetros de tamanho crítico calvária em avaliar a regeneração óssea.

Reparação de um modelo de defeito crítico porte calvária Usando derivadas de tecido adiposo células do estroma colhidas lipoaspirado

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

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15.8K visualizações.  Stanford University. Este protocolo descreve o isolamento de derivadas de tecido adiposo a partir de células do estroma lipoaspirado e a criação de um defeito de 4 milímetros de tamanho crítico calvária em avaliar a regeneração óssea.

Vídeo: Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Geração Experimental de Carcinoma Associado fibroblastos (FAC) de Fibroblastos Humanos mamária

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Medicina

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

Um Modelo contundente de lesão da medula espinhal cervical unilateral Usando o Impactor horizonte infinito

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

Criando Fraturas rigidamente estabilizadas para a Avaliação ossificação intramembranosa, Distração Osteogênica, ou Cura de defeitos críticos Sized

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Um modelo de pele Rato Fetal de reparação de feridas Scarless

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

Um Defeito modelo simples Critical porte Femoral em Ratos

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Criação e transplante de uma folha de células-tronco adiposo-derivado (ASC) em um modelo de cicatrização de feridas diabética

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

Isolamento de células regenerativas derivadas de adipose para o tratamento da disfunção erétil após a prostatectomia radical

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Um modelo de rato BALB/c neonatal de Enterocolite Necrotizante

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

<meta charset="utf8"></head><body>Fração vascular estromal mecânica em hidrogel de fibrina para medicina regenerativa

The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the past, the extraction of these cells from adipose tissue required a multistep enzyme-based process, resulting in a heterogenous cell mix consisting of ACSs and other cells, which are jointly termed the stromal vascular fraction (SVF). More recently introduced mechanical SVF (mSVF) isolation protocols are less time-consuming and bypass regulatory concerns. We recently proposed a protocol that generates mSVF rich in stromal cells based on a combination of emulsification and centrifugation. One current issue in mSVF application for wound therapy application is the lack of a scaffold providing protection from mechanical manipulation and desiccation. Fibrin hydrogels have been shown to be a useful adjunct in cell transfer for wound healing purposes in the past. In the work herein, we delineate the preparation steps of an mSVF-fibrin hydrogel construct as a novel approach for translational research and clinical application.

<meta charset="utf8"></head><body>Autor em destaque: Explorando o potencial da fração vascular estromal derivada de gordura para a cicatrização de feridas

Mechanically isolated stromal vascular fraction (SVF) in combination with a fibrin hydrogel offers an easy and efficient carrier for viable adipose-derived stromal cells for various indications, including tissue engineering and or wound healing purposes. Here, we present the preparation of a mechanical SVF (mSVF)-fibrin hydrogel construct for translational research and clinical application.

A combinação de fração vascular estromal isolada mecanicamente e hidrogel de fibrina: um protocolo de processamento

The regenerative potential of adipose-derived stromal cells (ASCs) has gained significant attention in regenerative and translational research. In the ...