Este trabalho foi financiado pelo R01-AR053645 de NIAMS.

Todos os procedimentos foram aprovados pelo Animal Care UCSF institucional e do uso Comissão e em conformidade com as diretrizes nacionais. 1. Preparação de Fixadores Antes de Cirurgia <ol><li> Antes de criar fratura estabilizada, é preciso montar o design personalizado aparelho de fixação externa. Os quadros personalizado concebido externos de fixação consistia de dois anéis de alumínio estabilizados por três aço inoxidável-# 0/56 hastes roscadas, 8 # 2/56 porcas hexagonais, e 17 correspondentes parafusos (Small Inc., Miami Lakes, FL 3). </li><li> Dois anéis de alumínio são necessárias por fratura. Para montar um anel 4 # 2/56 porcas hexagonais (pequena parte Inc., Miami Lakes, FL) e parafusos nos furos correspondentes adequados, estes devem permanecer solto. Este será o anel proximal. Para o anel distal montar 3 porcas e parafusos, mais uma vez não aperte totalmente. </li></ol> 2. Anestesia Criação fratura, fixador e Aplicação <ol><li> Os ratinhos (idade, sexo, e do corpopeso são determinados pelo utilizador) são anestesiados por injecção intraperitoneal de 50mg/ml cetamina, 0,5 mg / ml Medetomidina (0,03 ml rato /). </li><li> Aplique lubrificante ocular para os olhos dos camundongos e coloque um pedaço de fita em toda a sua cabeça para proteger os olhos durante o procedimento. </li><li> O animal é colocado sobre uma almofada de aquecimento para o resto do procedimento. </li><li> Transfixar das metáfises proximal e distal da tíbia esquerda com quatro pinos de 0,25 mm de insetos estéreis (Anticorro: Ferramenta de Ciência Fine, Foster City, CA), anexando os pinos de uma ferramenta Dremel e perfurando-los através do osso. </li><li> Pinos são orientadas perpendicularmente ao eixo longo da tíbia, e 90 ° entre si. Pelo menos 10 mm entre os pinos proximais e distais. </li><li> Posicione o primeiro anel acima dos pinos proximais com as porcas voltado para dentro, em direção aos pinos. Proteger os pinos ao anel mantendo a tíbia posicionado centralmente dentro do anel de alumínio. </li><li> Posicione o segundo anel abaixo do dipinos Stal e fixar os pinos ao anel usando as porcas hexagonais, como anteriormente. Adicionar a combinação porca e parafuso quarto para o fixador distal. </li><li> Criar a fratura transversal por flexão de três pontos com um dispositivo apropriado. Nós utilizamos o sistema de gravidade da Bonnarens e Einhorn 4. Descanse a superfície póstero-lateral da tíbia entre os blocos, levante o peso até uma altura pré-determinada ao longo do braço de queda e deixar cair o peso de modo que entra em contato com o aspecto ântero-lateral da tíbia para produzir a fratura fechado. A altura do peso é retirado da necessidades de ser determinado para cada estirpe de rato para garantir uma fractura simples exclusivamente da tíbia. </li><li> Fracture é confirmado por raio-X. </li><li> Após a confirmação da fractura, três aço inoxidável # 0/56 hastes roscadas estão ligados aos anéis. A partir da extremidade distai inserir a haste e enfiar uma porca no anel distai para fixar a haste. Em seguida, coloque outra porca na haste, o anel proximal vai sentar-se no topo desta nut. Certifique-se que os dois anéis será realizada a distâncias iguais e não sobrecarregar os pinos. Fixar o anel proximal para as hastes com uma porca terceiro. </li><li> As injecções subcutâneas de buprenorfina (1,0 mg / kg) para a analgesia são dadas imediatamente após a cirurgia e os animais são monitorizados para a dor e buprenorfina dada como necessário. </li><li> A anestesia é revertida pela administração Atipamezole, e os animais são monitorados até que eles são sensíveis e deambulando. </li></ol> 3. Distração Osteogênica (ver também: 12,13) Para modificar este procedimento para acomodar distração osteogênica é simples. Os anéis são mantidas em posição por hastes roscadas, e ligando as porcas que prendem as hastes no lugar dos anéis pode ser movido de intervalo. <ol><li> Latência: Para padrão distração osteogênica usando este modelo, os fixadores são colocados na tíbia e deixados sozinhos por um período de latência de 5 dias. </li><li> Distração: osteog Distraçãoenesis pode ser realizada ligando as porcas sobre as hastes de estabilização ¼ de volta (0,25 mm) a cada dia, 7 dias. </li><li> Maturação: Bony ponte é observado por 10 dias e consolidação completa é observado por 27 dias após a conclusão de distracção. </li></ol> 4. Criação de um defeito crítico Sized Para criar um defeito de tamanho crítico os fixadores externos são aplicados como anteriormente descrito com as seguintes modificações. <ol><li> Antes de iniciar o procedimento da pele é limpo com etanol a 70%. </li><li> Os fixadores são aplicados completamente antes de criar o defeito. </li><li> O rato é depois colocar sob o microscópio de dissecação para visualizar a tíbia medial, e uma incisão (5-7 mm) é feita com uma lâmina de # 10 em um bisturi. </li><li> O músculo é suavemente seccionado e separado para expor o meio-diáfise da tíbia. </li><li> Um segmento de 3 mm do osso é então removido das tesouras meados de diáfise usando. Utilizando tele ponta da tesoura, o osso é lentamente snipped afastado e fragmentos de osso são removidos com uma pinça, se necessário. Deve ser tomado cuidado com as pontas dos tesoura para evitar o deslocamento da tíbia com uma grande força mecânica. </li><li> Examinar o local cirúrgico para assegurar que não existem fragmentos de osso, os quais podem promover a cicatrização. </li><li> Substituir o músculo em torno das extremidades embotadas da tíbia. </li><li> Feche a incisão na pele com suturas 2-3 de 6-0 monofilamento Polyamine. </li><li> A anestesia é revertida e os animais são monitorizados para a dor e dado analgésicos, como descrito anteriormente. </li></ol> 5. Os resultados representativos Quando adequadamente aplicada, os fixadores externos proporcionar mais estabilidade rígida da fractura fechada tibial com excelente redução (Figs. 1, 2). No entanto, em alguns casos, a redução inadequada (uma diferença óbvia e grande entre as extremidades do osso ou fracturas múltiplas ocorrer (Fig. 3 </strong&gt;), E estes ratinhos são excluídos da análise. Fracturas estabilizado usando este método curar principalmente através ossificação intramembranosa (Fig. 4). Em contraste, se a fractura não é estabilizada um calo cartilagem grande é formada na abertura fractura (Fig. 5), e este é substituído por osso através do processo de ossificação intramembranosa. <img alt="A Figura 1" src="/files/ftp_upload/3552/3552fig1.jpg" /> Figura 1. Radiografias ilustrando um dispositivo de fixação externa utilizado para estabilizar a fratura da tíbia. Radiografia tirada após fratura mostrando alguns segmentos ósseos bem alinhados (seta). <img alt="A Figura 2" src="/files/ftp_upload/3552/3552fig2.jpg" /> Figura 2. Imagem de um rato após o fixador foi aplicado. <img alt="A Figura 3" src="/files/ftp_upload/3552/3552fig3.jpg" /> Figura 3. Radiografia depois de fracture mostra segmentos ósseos desalinhadas e fragmentadas (seta). <img alt="A Figura 4" src="/files/ftp_upload/3552/3552fig4.jpg" /> Figura 4. Fratura estabilizada cura através de ossificação intramembranosa. Coloração Tricrômico de fratura estabilizada mostra algum osso novo (b) no local da fratura. Barra de escala = 500 mm. <img alt="A Figura 5" src="/files/ftp_upload/3552/3552fig5.jpg" /> Figura 5. Fratura não estabilizada cura através de ossificação endocondral. A coloração tricromo de fractura não-estabilizada mostra cartilagem (c) e osso (b) no local da fractura. Barra de escala = 500 mm.

<ol>
	<li>Ilizarov, G. A., Lediaev, V. I., Shitin, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+course+of+compact+bone+reparative+regeneration+in+distraction+osteosynthesis+under+different+conditions+of+bone+fragment+fixation+(experimental+study).">The course of compact bone reparative regeneration in distraction osteosynthesis under different conditions of bone fragment fixation (experimental study).</a> Eksperimentalnaia Khirurgiia i Anesteziologiia. 14, 3-12 (1969).</li><li>Ilizarov, G. A., Deviatov, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+elongation+of+the+leg.">Surgical elongation of the leg.</a> Ortopediia Travmatologiia i Protezirovanie. 32, (1971).</li><li>Thompson, Z., Miclau, T., Hu, D., Helms, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+fracture+repair+in+the+absence+of+MMP9.">Altered fracture repair in the absence of MMP9.</a> Development. 130, 4123-4133 (2003).</li><li>Lange, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+IL-1beta+during+fracture+healing.">Action of IL-1beta during fracture healing.</a> J. Orthop. Res. 28, 778-784 (2010).</li><li>Xing, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+roles+for+CCR2+during+fracture+healing.">Multiple roles for CCR2 during fracture healing.</a> Dis. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia+leads+to+delayed+union+during+fracture+healing:+a+mouse+model.">Ischemia leads to delayed union during fracture healing: a mouse model.</a> J. Orthop. Res. 25, 51-61 (2007).</li><li>Miclau, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+delayed+stabilization+on+fracture+healing.">Effects of delayed stabilization on fracture healing.</a> J. Orthop. Res. 25, 1552-1558 (2007).</li><li>Tay, B. K., Le, A. X., Gould, S. E., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+and+molecular+analyses+of+distraction+osteogenesis+in+a+mouse+model.">Histochemical and molecular analyses of distraction osteogenesis in a mouse model.</a> Journal of Orthopaedic Research. 16, 636-642 (1998).</li><li>Choi, P., Ogilvie, C., Thompson, Z., Miclau, T., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+characterization+of+a+murine+non-union+model.">Cellular and molecular characterization of a murine non-union model.</a> J. Orthop. Res. 22, 1100-1107 (2004).</li><li>Buckwalter, J. A., Marsh, E. T., J, L., Heckman, J. D., Bucholz, R. 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Não temos nada a divulgar.

Ossos curar por duas modalidades diferentes, dependendo da estabilidade mecânica (revisto em: 14). Quando deixou instáveis ​​se forma um molde grandes de cartilagem no intervalo fractura que é substituído por osso para preencher as duas extremidades do osso partido. Proximal e distal à quebra, o osso faz diretamente por ossificação intramembranosa dentro do periósteo e endósteo. Em contraste, na cicatrização de fracturas estável ocorre exclusivamente através de ossificação intramembranosa <...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Pin inseto 0,25 milímetros </td><td> Ferramenta de Ciência multa </td><td> 26000-25 </td><td> Aço Anodizado enegrecido, 0,25 milímetro diâmetro da haste, comprimento 4 centímetros </td></tr><tr><td> Porca sextavada de aço inoxidável </td><td> Peças pequenas </td><td> # 2-56 </td><td> 1/8 &quot;comprimento, 56 fios por polegada </td></tr><tr><td> Porca sextavada de aço inoxidável </td><td> Peças pequenas </td><td> # 0-80 </td><td> 1/8 &quot;comprimento, 80 fios por polegada </td></tr><tr><td> Parafuso de aço inoxidável </td><td> Peças pequenas </td><td> # 0-80 </td><td> 1/8 &quot;comprimento, 80 fios por polegada </td></tr><tr><td> Máquina de Corte em Aço Inoxidável haste roscada </td><td> Peças pequenas </td><td> # 0-80 </td><td> 6 comprimento &quot;, 80 fios por polegada </td></tr><tr><td> 18-8 Stainless Steel Machine Head Screw </td><td> McMaster-Carr </td><td></td><td> Tópicos 2-56, 3/6 comprimento &quot; </td></tr><tr><td> Aparelho de fixação externa </td><td> Oficina mecânica </td><td> Design personalizado </td><td></td></tr></tbody></table>

creating rigidly stabilized fractures for assessing intramembranous ossification, distraction osteogenesis, or healing of critical sized defects

Avaliando os modos de reparação do esqueleto é essencial para o desenvolvimento de terapias para ser usados ​​clinicamente para tratar fracturas. A estabilidade mecânica desempenha um papel importante na cicatrização de lesões ósseas. No pior cenário de instabilidade mecânica pode resultar em seres humanos atrasados ​​ou não-união em. No entanto, o movimento também pode estimular o processo de cicatrização. Nas fraturas que têm formas de cartilagem de movimento para estabilizar as extremidades ósseas da fratura, e essa cartilagem é gradualmente substituída por osso através de recapitulação do processo de desenvolvimento de ossificação endocondral. Em contraste, se uma fractura óssea está rigidamente estabilizado osso forma directamente através de ossificação intramembranosa. Clinicamente, tanto ossificação endocondral e intramembranosa ocorrem simultaneamente. Para efectivamente replicar este investigadores processo de inserção de um pino dentro do canal medular do osso fracturado, como descrito por Bonnarens 4. Este método experimental fornece uma excelente estabilidade lateral, permitindo simultaneamente rotational instabilidade persistir. No entanto, a nossa compreensão dos mecanismos que regulam esses dois processos distintos também pode ser reforçada por experimentalmente isolando cada um desses processos. Nós desenvolvemos um protocolo de estabilização que fornece a estabilização de rotação e lateral. Neste modelo, a ossificação intramembranosa é o único modo de cura que é observado, e os parâmetros de cura podem ser comparados entre as diferentes estirpes de ratos geneticamente modificados 5-7, após a aplicação de moléculas bioactivas 8,9, depois de alterar os parâmetros fisiológicos de cura 10, depois de modificar a quantidade ou o tempo de estabilização 11, depois de distracção osteogénese 12, após a criação de um não-união 13, ou depois da criação de um defeito de tamanho crítico. Aqui, nós ilustrar como aplicar os fixadores Ilizarov modificados para estudar a consolidação da fratura da tíbia e distração osteogênica em camundongos.

Watch this Scientific Journal Video about Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects at JoVE.com

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

Este artigo descreve um método para a estabilização de fracturas de ossos longos que é baseado na aplicação de fixadores externos modificados Ilizarov<sup&gt; 1-3</sup&gt;. Após a aplicação dos fixadores e criação da lesão óssea, a cura pode ser avaliada, distração osteogênica pode ser realizada, ou não-união ou defeito crítico porte pode ser criado e utilizado para estudar as intervenções terapêuticas.

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 ...

creating-rigidly-stabilized-fractures-for-assessing-intramembranous

Research

JoVE Journal

Medicine

17.3K visualizações.  University of California, San Francisco. Este artigo descreve um método para a estabilização de fracturas de ossos longos que é baseado na aplicação de fixadores externos modificados Ilizarov 1-3. Após a aplicação dos fixadores e criação da lesão óssea, a cura pode ser avaliada, distração osteogênica pode ser realizada, ou não-união ou defeito crítico porte pode ser criado e utilizado para estudar as intervenções terapêuticas.

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

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

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

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. ...

Medicina

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. ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

Avaliar as alterações na volátil Geral Anestésica Sensibilidade de Ratos após intervenção farmacológica local ou sistêmica

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

Minimally Invasive Thumb-sized Pterional Craniotomy for Surgical Clip Ligation of Unruptured Anterior Circulation Aneurysms

Less invasive surgical approaches for intracranial aneurysm clipping may reduce length of hospital stay, surgical morbidity, treatment cost, and improve patient outcomes. We present our experience with a minimally invasive pterional approach for anterior circulation aneurysms performed in a major tertiary cerebrovascular center and compare the results with an aged matched dataset from the Nationwide Inpatient Sample (NIS). From August 2008 to December 2012, 22 elective aneurysm clippings on patients &#8804;55 years of age were performed by the same dual fellowship-trained cerebrovascular/endovascular neurosurgeon. One patient (4.5%) experienced transient post-operative complications. 18 of 22 patients returned for follow-up imaging and there were no recurrences through an average duration of 22 months. A search in the NIS database from 2008 to 2010, also for patients aged &#8804;55 years of age, yielded 1,341 hospitalizations for surgical clip ligation of unruptured cerebral aneurysms. Inpatient length of stay and hospital charges at our institution using the minimally invasive thumb-sized pterional technique were nearly half that of NIS (length of stay: 3.2 vs 5.7 days; hospital charges: $52,779 vs. $101,882). The minimally invasive thumb-sized pterional craniotomy allows good exposure of unruptured small and medium-sized supraclinoid anterior circulation aneurysms. Cerebrospinal fluid drainage from key subarachnoid cisterns and constant bimanual microsurgical techniques avoid the need for retractors which can cause contusions, localized venous infarctions, and post-operative cerebral edema at the retractor sites. Utilizing this set of techniques has afforded our patients with a shorter hospital stay at a lower cost compared to the national average.

Minimally invasive thumb-sized pterional craniotomy for aneurysm clipping has afforded our patients with a shorter hospital stay at a lower cost compared to the national average.

Minimamente Invasiva Thumb porte pterional craniotomia para pinça de aperto cirúrgica de Unruptured Anterior Aneurismas de circulação

Less invasive surgical approaches for intracranial aneurysm clipping may reduce length of hospital stay, surgical morbidity, treatment cost, and improve ...

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 ...

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

Direto Rato Trauma / Burn Modelo de ossificação heterotópica

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

Estabelecimento de um Defeito Modelo segmentar Femoral Critical-size em Ratos estabilizada por osteossíntese Placa

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal abnormalities. In addition, pathological myopia is a common irreversible cause of visual impairment and blindness1-3. Our study demonstrates the effect of scleral crosslinking using riboflavin and ultraviolet-A radiation on the development of axial myopia in a rabbit model. The axial length of the eyeball was measured by A-scan ultrasound in New Zealand white rabbits aged 13 days (male and female). The eye then underwent 360&#176; conjunctival peritomy with scleral crosslinking, followed by tarsorrhaphy. Axial elongation was induced in 13 day-old New Zealand rabbits by suturing their right eye eyelids (tarsorrhaphy). The eyes were divided into quadrants, and every quadrant had two scleral irradiation zones, each with an area of 0.2 cm&#178; and a radius of 4 mm. Crosslinking was performed by dropping 0.1% dextran-free riboflavin-5-phosphate onto the irradiation zones 20 sec before ultraviolet-A irradiation and every 20 sec during the 200 sec irradiation time. UVA radiation (370 nm) was applied perpendicular to the sclera at 57 mW/cm&#178; (total UVA light dose, 57 J/cm&#178;). Tarsorrhaphies were removed on day 55, followed by repeated axial length measurements. This study demonstrates that scleral crosslinking with riboflavin and ultraviolet-A radiation effectively prevents occlusion-induced axial elongation in a rabbit model.

We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy).

Escleral reticulação Usando ultravioleta e riboflavina-A radiação de Prevenção de Axial Miopia em um modelo de coelho

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

A geração de fraturas fechadas de fêmur em ratos: um modelo para estudar a consolidação óssea

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing therapeutics and devices. However, available injury models are plagued by inconsistency resulting in wasted animals and resources and imperfect data. To address this problem of fracture heterogeneity, the purpose of the method described herein is to optimize fracture generation parameters specific to each animal and yield a consistent fracture location and pattern. This protocol accounts for variations in bone size and morphology that may exist between mouse strains and can be adapted to generate consistent fractures in other species, such as rat. Additionally, a cost-effective, adjustable fracture apparatus is described. Compared to current stabilized fracture techniques, the optimization protocol and new fracture apparatus demonstrate increased consistency in stabilized fracture patterns and locations. Using optimized parameters specific to the sample type, the described protocol increases the precision of induced traumas, minimizing the fracture heterogeneity typically observed in closed-fracture generation procedures.

The goal of the protocol is to optimize the fracture generation parameters to yield consistent fractures. This protocol accounts for the variations in bone size and morphology that may exist between animals. Additionally, a cost-effective, adjustable fracture apparatus is described.

Fratura aparelhos Design e otimização de protocolo para fraturas fechadas-estabilizado em roedores

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing ...

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

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