A pesquisa reportada nesta publicação foi apoiada pelo Instituto Nacional de artrite e osteomuscular e doenças de pele do institutos nacionais da saúde sob concessão número F30AR071201 e R01AR066028.

<ol>
	<li>Corso, P., Finkelstein, E., Miller, T., Fiebelkorn, I., Zaloshnja, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+and+lifetime+costs+of+injuries+in+the+United+States.">Incidence and lifetime costs of injuries in the United States.</a> Injury Prevention. 12 (4), 212-218 (2006).</li><li>Nguyen, N. D., Ahlborg, H. G., Center, J. R., Eisman, J. A., Nguyen, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+lifetime+risk+of+fractures+in+women+and+men.">Residual lifetime risk of fractures in women and men.</a> Journal of Bone and Mineral Research: The Official Journal of the American Society for Bone and Mineral Research. 22 (6), 781-788 (2007).</li><li>Thompson, Z., Miclau, T., Hu, D., 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=A+model+for+intramembranous+ossification+during+fracture+healing.">A model for intramembranous ossification during fracture healing.</a> Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society. 20 (5), 1091-1098 (2002).</li><li>Cheung, K. M. C., Kaluarachi, K., Andrew, G., Lu, W., Chan, D., Cheah, K. S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+externally+fixed+femoral+fracture+model+for+mice.">An externally fixed femoral fracture model for mice.</a> Journal of Orthopaedic Research. 21 (4), 685-690 (2003).</li><li>Connolly, C. K., 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+reliable+externally+fixated+murine+femoral+fracture+model+that+accounts+for+variation+in+movement+between+animals.">A reliable externally fixated murine femoral fracture model that accounts for variation in movement between animals.</a> Journal of Orthopaedic Research. 21 (5), 843-849 (2003).</li><li>Histing, T., 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=An+internal+locking+plate+to+study+intramembranous+bone+healing+in+a+mouse+femur+fracture+model.">An internal locking plate to study intramembranous bone healing in a mouse femur fracture model.</a> Journal of Orthopaedic Research. 28 (3), 397-402 (2010).</li><li>Gr&#246;ngr&#246;ft, I., 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=Fixation+compliance+in+a+mouse+osteotomy+model+induces+two+different+processes+of+bone+healing+but+does+not+lead+to+delayed+union.">Fixation compliance in a mouse osteotomy model induces two different processes of bone healing but does not lead to delayed union.</a> Journal of Biomechanics. 42 (13), 2089-2096 (2009).</li><li>Bonnarens, F., Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+standard+closed+fracture+in+laboratory+animal+bone.">Production of a standard closed fracture in laboratory animal bone.</a> Journal of Orthopaedic Research. 2 (1), 97-101 (1984).</li><li>Huang, C., 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=The+spatiotemporal+role+of+COX-2+in+osteogenic+and+chondrogenic+differentiation+of+periosteum-derived+mesenchymal+progenitors+in+fracture+repair.">The spatiotemporal role of COX-2 in osteogenic and chondrogenic differentiation of periosteum-derived mesenchymal progenitors in fracture repair.</a> PloS One. 9 (7), 100079 (2014).</li><li>Waki, T., 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=Profiling+microRNA+expression+during+fracture+healing.">Profiling microRNA expression during fracture healing.</a> BMC Musculoskeletal Disorders. 17, 83 (2016).</li><li>Yee, C. S., 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=Sclerostin+antibody+treatment+improves+fracture+outcomes+in+a+Type+I+diabetic+mouse.">Sclerostin antibody treatment improves fracture outcomes in a Type I diabetic mouse.</a> Bone. 82, 122-134 (2016).</li><li>Wong, E., 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+low-molecular-weight+compound+enhances+ectopic+bone+formation+and+fracture+repair.">A novel low-molecular-weight compound enhances ectopic bone formation and fracture repair.</a> The Journal of Bone and Joint Surgery. American Volume. 95 (5), 454-461 (2013).</li><li>Prodinger, P. M., 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=Does+Anticoagulant+Medication+Alter+Fracture-Healing?+A+Morphological+and+Biomechanical+Evaluation+of+the+Possible+Effects+of+Rivaroxaban+and+Enoxaparin+Using+a+Rat+Closed+Fracture+Model.">Does Anticoagulant Medication Alter Fracture-Healing? A Morphological and Biomechanical Evaluation of the Possible Effects of Rivaroxaban and Enoxaparin Using a Rat Closed Fracture Model.</a> PloS One. 11 (7), 0159669 (2016).</li><li>Menzdorf, L., 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=Local+pamidronate+influences+fracture+healing+in+a+rodent+femur+fracture+model:+an+experimental+study.">Local pamidronate influences fracture healing in a rodent femur fracture model: an experimental study.</a> BMC Musculoskeletal Disorders. 17, 255 (2016).</li><li>Hagiwara, Y., 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=Fixation+stability+dictates+the+differentiation+pathway+of+periosteal+progenitor+cells+in+fracture+repair.">Fixation stability dictates the differentiation pathway of periosteal progenitor cells in fracture repair.</a> Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society. 33 (7), 948-956 (2015).</li><li>Gardner, M. 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=Differential+fracture+healing+resulting+from+fixation+stiffness+variability:+a+mouse+model.">Differential fracture healing resulting from fixation stiffness variability: a mouse model.</a> Journal of Orthopaedic Science: Official Journal of the Japanese Orthopaedic Association. 16 (3), 298-303 (2011).</li><li>Catma, M. 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=Remote+ischemic+preconditioning+enhances+fracture+healing.">Remote ischemic preconditioning enhances fracture healing.</a> Journal of Orthopaedics. 12 (4), 168-173 (2015).</li><li>Lichte, P., 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=Impaired+Fracture+Healing+after+Hemorrhagic+Shock.">Impaired Fracture Healing after Hemorrhagic Shock.</a> Mediators of Inflammation. 2015, 132451 (2015).</li><li>Lopas, L. A., 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=Fractures+in+geriatric+mice+show+decreased+callus+expansion+and+bone+volume.">Fractures in geriatric mice show decreased callus expansion and bone volume.</a> Clinical Orthopaedics and Related Research. 472 (11), 3523-3532 (2014).</li><li>Bonnarens, F., Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+standard+closed+fracture+in+laboratory+animal+bone.">Production of a standard closed fracture in laboratory animal bone.</a> Journal of orthopaedic research. 2 (1), 97-101 (1984).</li><li>Marturano, J. E., 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=An+improved+murine+femur+fracture+device+for+bone+healing+studies.">An improved murine femur fracture device for bone healing studies.</a> Journal of Biomechanics. 41 (6), 1222-1228 (2008).</li><li>Jackson, R. W., Reed, C. A., Israel, J. A., Abou-Keer, F. K., Garside, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+standard+experimental+fracture.">Production of a standard experimental fracture.</a> Canadian Journal of Surgery. Journal Canadien De Chirurgie. 13 (4), 415-420 (1970).</li><li>Byrne, M., Cleveland, B., Marturano, J., Wixted, J., Billiar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+reproducible+murine+femoral+fracture+device.+Conference:+Bioengineering+Conference,+2007.">Design of a reproducible murine femoral fracture device. Conference: Bioengineering Conference, 2007.</a> NEBC '07. IEEE 33rd Annual Northeast. , (2007).</li><li>Carter, D. R., Hayes, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compact+bone+fatigue+damage-I.+Residual+strength+and+stiffness.">Compact bone fatigue damage-I. Residual strength and stiffness.</a> Journal of Biomechanics. 10 (5), 325-337 (1977).</li><li>McGee, A., Qureshi, A., Porter, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+biomechanics+and+patterns+of+limb+fractures.">Review of the biomechanics and patterns of limb fractures.</a> Trauma. 6 (1), 29-40 (2004).</li></ol>

Os autores não têm nada para divulgar.

Este protocolo de otimização e geração de fratura fornece a pesquisadores com um método eficiente para derivar parâmetros de fratura e realizar um procedimento minimamente invasivo, que produz fraturas transversais, precisas e reproduzíveis. Além disso, o presente protocolo estabelece um conjunto de parâmetros de geração de fratura, que promove a consistência do método entre os investigadores. Esses parâmetros permitirá a criação de um banco de dados comum da fratura para estabelecer padrões de fratura ...

Pesquisa sobre a consolidação da fratura é necessária para resolver um grande problema clínico e económico. Cada ano mais 12 milhões de fraturas são tratadas no Estados Unidos1, custando US $ 80 bilhões por ano2. A probabilidade de um macho ou fêmea a sofrer uma fratura ao longo da vida é de 25% e 44%, respectivamente3. Problemas associados com a consolidação da fratura são esperados para aumentar com comorbidades mais que a população envelhece. Para estudar e resolver este problema, modelos sólidos de geração da fratura e estabilização são necessários. Modelos de roedores são ideais para essa finalidade. Eles fornecem a relevância clínica e podem ser modificados para as condições específicas do endereço(ou seja, múltiplas lesões, fracturas abertas, fechadas, isquêmicas e infectadas). Além de replicar cenários clínicos, modelos animais de fratura são importantes para a compreensão da biologia óssea e em desenvolvimento de terapêuticas e dispositivos. No entanto, as tentativas para estudar as diferenças entre as intervenções podem ser complicadas pela variabilidade introduzida pela geração de fratura inconsistente. Assim, gerar fraturas reproduzíveis e consistentemente fechadas em modelos animais é essencial para o campo de pesquisa músculo-esquelética.
Apesar de controlar corretamente para potenciais heterogeneidade do assunto, garantindo o fundo genético apropriado, sexo, idade e condições ambientais, a produção de lesões ósseas consistentes clinicamente relevante é uma variável significativa que afectam reprodutibilidade que deve ser controlada. Comparações estatísticas usando inconsistentes fraturas são atormentadas com ruído experimental e uma alta variabilidade4; Além disso, a variabilidade de fratura pode resultar em morte desnecessária de animais devido à necessidade de aumentar o tamanho da amostra ou a necessidade de eutanásia em animais com fraturas cominutivas ou malpositioned. O objetivo do método aqui descrito é otimizar os parâmetros de geração de fratura que são específicos para o tipo de amostra e produzir uma fratura consistente localização e padrão.
Modelos atuais de geração de fratura caem em duas categorias amplas, cada um com suas próprias forças e fraquezas. Modelos de aberto-fratura (osteotomia) se submeter à cirurgia para expor o osso, após o qual uma fratura é induzida pela corte do osso ou enfraquecimento isso e depois quebrá-lo manualmente5,6,7,8. Os benefícios deste método são a visualização directa do local da fratura e um local de fratura mais consistente e padrão. No entanto, a relevância clínica e fisiológica da abordagem e mecanismo de lesão são limitadas. Além disso, o open métodos de geração de fratura requerem uma abordagem cirúrgica e encerramento com períodos prolongados, durante o qual os roedores estão expostos a um risco aumentado de contaminação.
Técnicas fechadas abordar muitas das limitações da técnica aberta. Fechado técnicas produzem fraturas usando um aplicados externamente um traumatismo que provoca lesão no osso e tecidos circundantes, mais semelhantes aos observados em lesões clínicas humanas. O método mais comum foi descrito por Bonnarens e Einhorn em 19849. Eles descreveram uma guilhotina ponderada, sendo usada para transmitir um trauma para quebrar o osso, sem causar quaisquer feridas de pele externa. Este método tem sido amplamente adotado para estudar o efeito da genética10,11, terapia farmacológica12,13,14,15, mecânica16, 17e fisiologia18,19,20 na consolidação óssea em camundongos e ratos. Enquanto o benefício de métodos fechados é fisiologicamente relevantes fraturas, rigor e reprodutibilidade experimental são limitados pela heterogeneidade de fratura. A geração de fratura inconsistente resulta em uma diferenciação entre grupos limitada, espécimes perdidos e um aumento em animais necessários para alcançar significância estatística.
Controlar a variabilidade na geração de fratura e estabilização é essencial para produzir resultados significativos. A fim de estudar adequadamente a biologia do reparo de fratura, é necessário um modelo de fratura simples, porém robusta. O modelo deve ser traduzível para roedores, tipos de osso (fêmur ou tíbias, por exemplo) e através de origens genéticas do mouse variável e induzidas mutações. Além disso, o procedimento ideal deve ser tecnicamente simples e produzir resultados consistentes. A heterogeneidade de fratura de endereço, o método descrito neste documento é a construção de um dispositivo de fratura bem controlados que pode ser usado para otimizar parâmetros e gerar fraturas consistentemente fechadas, independentemente da idade, sexo ou genótipo.

<table border="0" cellpadding="0" cellspacing="0" style="width:911px;" width="910">
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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">Support Subassembly</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td style="width:315px;">Supplementary Figure 1</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Jaw Section&#160;</td>
			<td style="width:209px;">80/20</td>
			<td>1003 x 9.00</td>
			<td>w/ #7042 at A, C, in Left End</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Horizontal Section</td>
			<td style="width:209px;">80/20</td>
			<td>1002 x 14.00</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Vertical 1</td>
			<td style="width:209px;">80/20</td>
			<td>1050 x 10.50&#160;</td>
			<td>w/ #7042 at A in Left End and at A in Right End</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Vertical 2</td>
			<td style="width:209px;">80/20</td>
			<td>1010 x 10.50&#160;</td>
			<td>w/ #7042 at D, B in Left End and at A in Right End</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Plate Mount</td>
			<td style="width:209px;">80/20</td>
			<td>1030 x 8.00&#160;</td>
			<td>w/ #7036 at Left End</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Magnet</td>
			<td style="width:209px;">80/20</td>
			<td>1010 x 13.50&#160;</td>
			<td>w/ #7042 at A, C, in Right End</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Anchors (3)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">3392</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Double Anchor (3)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">3091</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Bolt Assembly (6)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">3386</td>
			<td>1/4-20 x 3/8&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Button Head Socket Cap Screw (6)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">3604</td>
			<td>1/4-20 x 3/4&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ram Subassembly</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 2</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Block, Stop</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 3</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Block, Guide</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 3</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Rod, Ram</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 4</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Alignment Screw</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 5</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Plate, Mounting</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 6</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Linear Sleeve Bearing (2)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">8649T2</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Hex Nut (3)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>92673A125</td>
			<td>3/8-16 UNC</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Socket Cap Screw (8)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">92196A108</td>
			<td>4/40 x 3/8&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Socket Cap Screw (6)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">92196A032</td>
			<td>4/40 x 1 1/8&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Socket Cap Screw (1)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">92196A267&#160;</td>
			<td>10/32 3/8&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Magnet Subassembly</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 7</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Mount, Magnet</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 8</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Power Supply</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>70235K23</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Foot Switch</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>7376k2</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Electromagnet</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>5698k111</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Wire - 10 feet</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">9936k12</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Rod, Magnet</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>95412A566</td>
			<td>1/4&#34; Threaded Rod x 7&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Corner Bracket (6)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">4108</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Socket Cap Screw (1)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">92196A705</td>
			<td>10/32 1 1/4&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Hex Nut (4)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>92673A113</td>
			<td>1/4-20 UNC</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Complete Assembly</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 9</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Bracket, Leg Jaw (2)</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 10</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Platform, Fracture</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 11</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Jig, Positioning-Fracture</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 12</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Other</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Pin Cutter</td>
			<td style="width:209px;">Medical Supplies and Equipment</td>
			<td style="width:147px;">150S</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Needles</td>
			<td style="width:209px;">Sigma</td>
			<td style="width:147px;">Z192430, Z192376&#160;</td>
			<td>23g x 1.5&#34; - mouse femur, 27g x 1.25&#34; - mouse tibia</td>
		</tr>
	</tbody>
</table>

fracture apparatus design and protocol optimization for closed-stabilized fractures in rodents

A geração confiável de fraturas estabilizadas consistentes em modelos animais é essencial para compreender a biologia de regeneração óssea e desenvolvimento de terapêuticas e dispositivos. No entanto, modelos de lesão disponíveis são atormentados por inconsistência, resultando em desperdício de animais e recursos e dados imperfeitos. Para resolver este problema da heterogeneidade de fratura, o objetivo do método aqui descrito é otimizar a geração de fratura parâmetros específicos para cada animal e produzir uma fratura consistente localização e padrão. Este protocolo é responsável por variações no tamanho do osso e morfologia que possam existir entre estirpes de rato e pode ser adaptado para gerar fraturas consistentes em outras espécies, como o rato. Além disso, um aparelho de fratura cost-effective, ajustável é descrito. Em comparação com as técnicas atuais de fratura estabilizada, o protocolo de otimização e novos aparelhos de fratura demonstram maior consistência nos padrões de fratura estabilizada e locais. Usando otimizado parâmetros específicos para o tipo de amostra, o protocolo descrito aumenta a precisão dos traumas induzidos, minimizando a heterogeneidade de fratura normalmente observada nos processos de geração de fratura fechada.

A guilhotina usada anteriormente em nosso laboratório foi desenvolvida em 2004 e foi baseada em modelos publicados por Einhorn21. O design não permitia ajustes para explicar adequadamente quaisquer diferenças na morfologia do osso e não permitiam um posicionamento reprodutível do membro. Além disso, o aparelho anterior necessárias duas pessoas para operá-lo. Portanto, nós concebido, projetado e construído um novo aparelho de fratura. O objetivo principal ...

Watch this Scientific Journal Video about Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents at JoVE.com

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

O objetivo do protocolo é otimizar os parâmetros de geração de fratura para produzir fraturas consistentes. Este protocolo é responsável para as variações no tamanho do osso e morfologia que possam existir entre os animais. Além disso, um aparelho de fratura cost-effective, ajustável é descrito.

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 ...

fracture-apparatus-design-protocol-optimization-for-closed-stabilized

Research

JoVE Journal

Medicine

13.3K visualizações.  Michigan State University. O objetivo do protocolo é otimizar os parâmetros de geração de fratura para produzir fraturas consistentes. Este protocolo é responsável para as variações no tamanho do osso e morfologia que possam existir entre os animais. Além disso, um aparelho de fratura cost-effective, ajustável é descrito.

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

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

O Uso de cistometria em pequenos roedores: Um Estudo de Chemosensation Bexiga

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Medicina

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

Protocolo de Coleta para Pâncreas Humano

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Parabiosis in Mice: A Detailed Protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 18641, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival2. In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

Parabiose em Ratos: Um protocolo detalhado

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

Baseado em celular Protocolo Ensaio para a predição prognóstica de escoliose idiopática Usando celular dielétrica Espectroscopia

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Seqüente<em&gt; In vivo</em&gt; Imagem de osteogênicas células-tronco / progenitoras durante o reparo de fratura

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

Cateterismo da artéria carótida e jugular para executar as medidas hemodinâmicas, infusões e Sangue de amostragem em um Rat Consciente Modelo

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

-Nervo poupadores Mid-obstrução uretral (Nemo) em fêmeas pequenos roedores

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Na Vivo Avaliação do desenvolvimento de calo de fratura durante a consolidação óssea em ratos usando um dispositivo de osteossíntese compatível com MRI para o fêmur de Mouse

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Um modelo minimamente invasivo para analisar a fratura endocondral cura em ratos sob condições biomecânicas padronizadas

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

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 ...