Name: construction and evaluation of a murine calvarial osteolysis model by exposure to cocrmo particles in aseptic loosening
Uploaded: 2018-02-17T14:00:00
Description: Watch this Scientific Journal Video about Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening at JoVE.com

This study was supported by the National Natural Science Foundation of China (81572111), the Clinical Science and Technology Project Foundation of Jiangsu Province (BL2012002), the Scientific Research Project of Nanjing (201402007), the Natural Science Foundation of Jiangsu Province (BK20161385), and the Special Foundation of Chinese Medical Doctor Association (2015COS0810).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing University.
1. CoCrMo Particle Preparation
<ol>
	<li>Obtain CoCrMo particles by using a fabricated high-vacuum three-electrode direct current4. Place CoCrMo alloy in the instrument under 10-3 Pa vacuum, 0.04 MPa argon and hydrogen 3:2 (v/v), and 650 A cathode current.</li>
	<li>Measure the diameters of CoCrMo particles.
	<ol>
		<li>Add 1 mg of...

<ol>
	<li>Dong, 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=Antisense+oligonucleotide+targeting+TNF-alpha+can+suppress+Co-Cr-Mo+particle-induced+osteolysis.">Antisense oligonucleotide targeting TNF-alpha can suppress Co-Cr-Mo particle-induced osteolysis.</a> J Orthop Res. 26 (8), 1114-1120 (2008).</li><li>Deng, Z., 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=SIRT1+protects+osteoblasts+against+particle-induced+inflammatory+responses+and+apoptosis+in+aseptic+prosthesis+loosening.">SIRT1 protects osteoblasts against particle-induced inflammatory responses and apoptosis in aseptic prosthesis loosening.</a> Acta Biomater. 49, 541-554 (2017).</li><li>Langlois, J., Hamadouche, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+animal+models+of+wear-particle+osteolysis.">New animal models of wear-particle osteolysis.</a> Int Orthop. 35 (2), 245-251 (2011).</li><li>Wang, P., Zhao, F. X., Zhang, Z. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+ultrafine+zinc+powders+by+DC+arc+plasma+evaporation+method.">Preparation of ultrafine zinc powders by DC arc plasma evaporation method.</a> Chinese Journal of Nonferrous Metals. 21 (9), 2236-2241 (2011).</li><li>Wang, Z., 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+fibroblast+expression+of+RANKL+in+CoCrMo-particle-induced+osteolysis+is+mediated+by+ER+stress+and+XBP1s.">The fibroblast expression of RANKL in CoCrMo-particle-induced osteolysis is mediated by ER stress and XBP1s.</a> Acta Biomater. 24, 352-360 (2015).</li><li>Wang, Z., 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=Autophagy+mediated+CoCrMo+particle-induced+peri-implant+osteolysis+by+promoting+osteoblast+apoptosis.">Autophagy mediated CoCrMo particle-induced peri-implant osteolysis by promoting osteoblast apoptosis.</a> Autophagy. 11 (12), 2358-2369 (2015).</li><li>Wang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle-induced+osteolysis+mediated+by+endoplasmic+reticulum+stress+in+prosthesis+loosening.">Particle-induced osteolysis mediated by endoplasmic reticulum stress in prosthesis loosening.</a> Biomaterials. 34 (11), 2611-2623 (2013).</li><li>Yang, S. 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=Adeno-associated+virus-mediated+osteoprotegerin+gene+transfer+protects+against+particulate+polyethylene-induced+osteolysis+in+a+murine+model.">Adeno-associated virus-mediated osteoprotegerin gene transfer protects against particulate polyethylene-induced osteolysis in a murine model.</a> Arthritis Rheum. 46 (9), 2514-2523 (2002).</li><li>Liu, N., 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=Autophagy+mediated+TiAl(6)V(4)+particle-induced+peri-implant+osteolysis+by+promoting+expression+of+TNF-alpha.">Autophagy mediated TiAl(6)V(4) particle-induced peri-implant osteolysis by promoting expression of TNF-alpha.</a> Biochem Biophys Res Commun. 473 (1), 133-139 (2016).</li><li>Wang, Z., 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=ER+Stress+Mediates+TiAl6V4+Particle-Induced+Peri-Implant+Osteolysis+by+Promoting+RANKL+Expression+in+Fibroblasts.">ER Stress Mediates TiAl6V4 Particle-Induced Peri-Implant Osteolysis by Promoting RANKL Expression in Fibroblasts.</a> PLoS One. 10 (9), e0137774 (2015).</li><li>Wang, Z., 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=TiAl6V4+particles+promote+osteoclast+formation+via+autophagy-mediated+downregulation+of+interferon-beta+in+osteocytes.">TiAl6V4 particles promote osteoclast formation via autophagy-mediated downregulation of interferon-beta in osteocytes.</a> Acta Biomater. 48, 489-498 (2017).</li><li>Chen, 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=Lycorine+suppresses+RANKL-induced+osteoclastogenesis+in+vitro+and+prevents+ovariectomy-induced+osteoporosis+and+titanium+particle-induced+osteolysis+in+vivo.">Lycorine suppresses RANKL-induced osteoclastogenesis in vitro and prevents ovariectomy-induced osteoporosis and titanium particle-induced osteolysis in vivo.</a> Sci Rep. 5, 12853 (2015).</li><li>Neuerburg, 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+role+of+calcitonin+receptor+signalling+in+polyethylene+particle-induced+osteolysis.">The role of calcitonin receptor signalling in polyethylene particle-induced osteolysis.</a> Acta Biomater. 14, 125-132 (2015).</li><li>Catelas, I., Jacobs, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologic+activity+of+wear+particles.">Biologic activity of wear particles.</a> Instr Course Lect. 59, 3-16 (2010).</li><li>Liu, 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=The+biological+response+to+nanometre-sized+polymer+particles.">The biological response to nanometre-sized polymer particles.</a> Acta Biomater. 23, 38-51 (2015).</li></ol>

There are two main methods for wear particle-induced osteolysis in mice: the air-pouch model and the calvarial osteolysis model. In the air-pouch model, a subcutaneously generated air-pouch is first established, followed by wear particle introduction and implantation into the bone tissue8. The pouch wall mimics the periosteum in aseptic loosening. However, bone implantation is nonvascular with no biological activity, which makes it difficult to assess direct interactions between particles and the ...

Aseptic loosening is the most common cause of total hip arthroplasty (THA) and total knee arthroplasty (TKA) failure, which requires revision surgery1. However, the underlying mechanism remains unclear2. A long follow-up is required to detect particle-induced osteolysis, whose occurrence is rare; therefore, it is challenging to explore its pathogenesis in clinical cases. Hence, further studies focusing on complex cellular and tissue mechanisms require both in vivo experiments in wear particle-induced osteolysis models and in vitro assays in cells related to bone homeostasis3. A valid animal model is important in revealing the effects of wear particles on bone loss, providing evidence for further cellular assays. 
A murine calvarial osteolysis model constructed by exposure to CoCrMo particles is an effective and valid method for assessing the interactions between particles and various cells in aseptic loosening. In this model, CoCrMo particles cause calvarial osteolysis by inducing inflammatory cytokines in macrophages, activating osteoclasts, inhibiting osteoblast proliferation, and promoting osteoblast apoptosis.
It only takes two weeks to establish this model. Osteolysis can be visualized and quantified by hematoxylin and eosin (H&amp;E) staining and micro computed tomography (micro-CT)2. In addition, this model has a relatively low cost, and transgenic and knockout mouse models can be used to screen a large number of compounds at various doses3.
The procedure to establish and evaluate this model is simple. First, CoCrMo particles were obtained by high-vacuum three-electrode direct current and resuspended in phosphate-buffered saline (PBS) at a concentration of 50 mg/mL. Then, 50 &#181;L of the resulting suspension was applied to the middle of the murine calvaria after separation of the cranial periosteum by sharp dissection. The mice were sacrificed after two weeks, and calvaria samples were harvested; qualitative and quantitative analyses were performed by H&amp;E staining andmicro-CT. 
A murine calvarial osteolysis model constructed by exposure to CoCrMo particles is an ideal tool for assessing the interactions between CoCrMo particles and various cells, such as macrophages, fibroblasts, osteoblasts, and osteoclasts, in aseptic loosening.

<table border="0" cellpadding="0" cellspacing="0" width="684">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">CoCrMo alloy from prosthesis</td>
			<td style="width:183px;">Waldemar Link GmbH &#38; Co</td>
			<td style="width:119px;">GEMINI MK II</td>
			<td style="width:167px;">Raw material to obtain CoCrMo nanoparticles</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fabricated high-vacuum three-electrode direct current</td>
			<td style="width:183px;">College of Materials Science &#38; Engineering , Nanjing University of Technology</td>
			<td style="width:119px;"></td>
			<td>Self designed machine</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">6 week old male C57BL/6J mice</td>
			<td style="width:183px;">Model animal research center of Nanjing University</td>
			<td style="width:119px;">N000013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100% Ethanol</td>
			<td style="width:183px;">Nanjing Reagent</td>
			<td>C0691514023</td>
			<td>Solvent of CoCrMo nanoparticles for transmission electron microscope scanning</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1.5 ml Microcentrifuge tubes</td>
			<td style="width:183px;">Taizhou Weierkang Medical Supplies co., LTD</td>
			<td>W603</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Microanalytical balance</td>
			<td style="width:183px;">Shenzhen Qun long Instrument Equipment Co,. LTD</td>
			<td>EX125DZH</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultrasonic shaker</td>
			<td style="width:183px;">Shanghai Yuhao scientific instrument co., LTD</td>
			<td style="width:119px;">YH-200DH</td>
			<td>To suspend CoCrMo nanoparticles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transmission Electron Microscope</td>
			<td style="width:183px;">FEI</td>
			<td style="width:119px;">Tecnai G20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SimplePCI software</td>
			<td style="width:183px;">Compix Inc.</td>
			<td style="width:119px;">6.6 version</td>
			<td>To calculate the mean diameter and particle size distribution.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High-handed sterilization pan</td>
			<td style="width:183px;">QIULONGYIQI</td>
			<td style="width:119px;">KYQL-100DS</td>
			<td>To decontaminate endotoxin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Limulus Amebocyte Lysate (LAL) Assay</td>
			<td style="width:183px;">Charles River</td>
			<td style="width:119px;">R13025</td>
			<td>To detect endotoxin&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml Microcentrifuge tubes</td>
			<td style="width:183px;">Taizhou Suyi Medical</td>
			<td style="width:119px;">B122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate-buffered saline</td>
			<td style="width:183px;">Boster Biological Technology</td>
			<td style="width:119px;">AR0030</td>
			<td>Solvent of CoCrMo nanoparticles stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pentobarbital Sodium</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:119px;">P3761</td>
			<td>To anesthetize mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normal saline</td>
			<td style="width:183px;">SACKLER</td>
			<td style="width:119px;">SR8572EP-15</td>
			<td>To prevent drying of mice eyes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">75% Ethanol</td>
			<td style="width:183px;">Nanjing Reagent</td>
			<td style="width:119px;">C0691560275</td>
			<td>Disinfection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Medical cotton ball</td>
			<td style="width:183px;">Shuitao</td>
			<td style="width:119px;">1278298933</td>
			<td>Disinfection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shaver</td>
			<td style="width:183px;">Kemei</td>
			<td style="width:119px;">KM-3018</td>
			<td>To shave the fur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissor</td>
			<td style="width:183px;">RWD LIFE SCIENCE</td>
			<td style="width:119px;">S12005-10</td>
			<td>To incise skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Suture</td>
			<td style="width:183px;">RWD LIFE SCIENCE</td>
			<td style="width:119px;">F34001-01</td>
			<td>To suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle holder</td>
			<td style="width:183px;">RWD LIFE SCIENCE</td>
			<td style="width:119px;">F33001-01</td>
			<td>To suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle</td>
			<td style="width:183px;">RWD LIFE SCIENCE</td>
			<td style="width:119px;">R14003-12</td>
			<td>To suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vessel forceps</td>
			<td style="width:183px;">RWD LIFE SCIENCE</td>
			<td style="width:119px;">F22003-09</td>
			<td>To suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel</td>
			<td style="width:183px;">RWD LIFE SCIENCE</td>
			<td style="width:119px;">S31010-01</td>
			<td>To harvest calvaria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tweezers</td>
			<td style="width:183px;">RWD LIFE SCIENCE</td>
			<td style="width:119px;">F12006-10</td>
			<td>To harvest calvaria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 &#181;L pipettes</td>
			<td style="width:183px;">Eppendorf</td>
			<td style="width:119px;">3120000240</td>
			<td>To embed particles suspension in the calvatias</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 &#181;L pipette tips</td>
			<td style="width:183px;">AXYGEN</td>
			<td style="width:119px;">T-200-Y</td>
			<td>To embed particles suspension in the calvatias</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5 ml Microtubes</td>
			<td style="width:183px;">Taizhou Weierkang Medical Supplies co., LTD</td>
			<td>W621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4% Paraformaldehyde</td>
			<td style="width:183px;">Servicebio</td>
			<td style="width:119px;">G1101</td>
			<td>Fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro Computed Tomography&#160;</td>
			<td style="width:183px;">SkyScan</td>
			<td style="width:119px;">SkyScan1176</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethylene Diamine Tetraacetic Acid</td>
			<td style="width:183px;">Servicebio</td>
			<td style="width:119px;">G1105</td>
			<td>Decalcification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraffin</td>
			<td style="width:183px;">Servicebio</td>
			<td style="width:119px;">#0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraffin slicing machine</td>
			<td style="width:183px;">Leica</td>
			<td style="width:119px;">RM2125RTS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass slide</td>
			<td style="width:183px;">Servicebio</td>
			<td style="width:119px;">G6004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cover glass</td>
			<td style="width:183px;">Servicebio</td>
			<td style="width:119px;">200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HE staining kit</td>
			<td style="width:183px;">Servicebio</td>
			<td style="width:119px;">#1-5</td>
			<td>HE staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Light microscope</td>
			<td style="width:183px;">Nikon</td>
			<td style="width:119px;">E200</td>
			<td></td>
		</tr>
	</tbody>
</table>

construction and evaluation of a murine calvarial osteolysis model by exposure to cocrmo particles in aseptic loosening

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long follow-ups necessary for detection and sporadic occurrence, it is challenging to assess the pathogenesis ofparticle-induced osteolysis in clinical cases. Hence, optimal animal models are required for further studies. The murine model of calvarial osteolysis established by exposure to CoCrMo particles is an effective and valid tool for assessing the interactions between particles and various cells in aseptic loosening. In this model, CoCrMo particles were first obtained by high-vacuum three-electrode direct current and resuspended in phosphate-buffered saline at a concentration of 50 mg/mL. Then, 50 &#181;L of the resulting suspension was applied to the middle of the murine calvaria after separation of the cranial periosteum by sharp dissection. After two weeks, the mice were sacrificed, and calvaria specimens were harvested; qualitative and quantitative evaluations were performed by hematoxylin and eosin staining and micro computed tomography. The strengths of this model include procedure simplicity, quantitative evaluation of bone loss, rapidity of osteolysis development, potential use transgenic or knockout models, and a relatively low cost. However, this model cannot to be used to assess the mechanical force and chronic effects of particles in aseptic loosening. Murine calvarial osteolysis model generated by exposure to CoCrMo particles is an ideal tool for assessing the interactions between wear particles and various cells, e.g., macrophages, fibroblasts, osteoblasts and osteoclasts, in aseptic loosening.

The in-house produced nanoscale CoCrMo particles were around 50 nm (standard error of 3.56) in diameter, as quantified by TEM (Figure 2). After exposure of mouse calvarias to CoCrMo particles, the animals (n=6 per group) were maintained for another two weeks. Within the two weeks, the calvarial incision was completely healed, and the suture may fall. Any local infection or nonunion may affect bone loss assessment. After mouse sacrifice, calvaria samples were ...

Watch this Scientific Journal Video about Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening at JoVE.com

Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening

This manuscript describes a murine calvarial osteolysis model by exposure to CoCrMo particles, which constitutes an ideal animal model for assessing the interactions between wear particles and various cells in aseptic loosening.

Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanism remains unclear. Due to long ...

The overall goal of this procedure is to construct and evaluate the Murine Calvarial Osteolysis Model by exposure to cobalt-chromium-molybdenum nanoparticles. Wear particle-induced osteolysis is a major cause of aseptic loosening in arthroplasty failure, but the underlying mechanics of this are unclear. Due to the long follow-up time and the sporadic occurrence, it is hard to explore the parthogenesis of aseptic loosening in clinical cases.Hence, a wear animal model is necessary for further study. In our lab, a murial calvarial osteolysis model constructed by cobalt alloy particle is an effective model to study the parthogenesis of aseptic loosening. This is accomplished by first generating nanoscale cobalt chromium molybdenum particles from cobalt alloy prosthesis and re suspending them in phosphate buffer saline at a concentration of 15 milligram per millilitre.The next step is to identify the incision position at a line between the two midpoints between two eyes and ears, respectively. Then, remove the cranial periosteum from the calvarium by shock dissection. Next, take 50 microlitres of cobalt alloy nanoparticles stock and embed them in the middle suture of the calvaria.After two weeks of maintenance, sacrifice some mice and collect the calvaria for osteolysis evaluation. Osteolysis is obvious in cobalt alloy nanoparticle treated mice, both in micro-CT and H and E staining images. Obtain cobalt chromium molybdenum alloy from cobalt alloy prosthesis, obtaining cobalt chromium molybdenum particles from cobalt chromium molybdenum alloy by fabricated high-vaccuum three electrode direct current.In order to quantify the mean diameter of the cobalt chromium molybdenum particles, take one milligram of particles into 1.5 milliliter anhydrous ethanol and suspend them by ultrasonic shaking. Apply one drop, about 20 microlitres, of the suspension on the objective table of the transmission electron microscopy and acquire a series of images. Use the provided software to calculate the mean diameter and particle size distribution in TEM images.Here are representative TEM images on the left panel the right one shows the distribution of nanoparticle size of cobalt chromium molybdenum alloy calculated by our commercial software. The mean diameter is around 50 nanometers. Autoclave the particles for 15 minutes to remove endotoxins.Then, re suspend them in phosphate-buffered saline at a concentration of 15 milligram per milliliter as stock solution. Next is the construction of the calvarial osteolysis model. Select and anesthetize six week old mice:six mice per group.Place the animals in the prime position. Remove the fur and disinfect skin on the cranium with 75 percent ethanol. For incise, identify the two midpoints between the two eyes and ears, respectively, and incise skin along the line joining them.Carefully and completely remove the cranial periosteum from the calvarium by shock dissection. A clear dissection of the periosteum intensifies the interactions between particles in the calvaria. The suture is performed carefully at both ends of the incision in simple interrupted suture.Usually, three stitches are needed for a complete suture. For the last stage, pass the suture line through the incision without knotting, holding both ends of the suture line. Take 15 microlitres cobalt alloy stock solution nanoparticles and embed them in the middle suture of the calvaria.Knot the last stitch in simple interrupted suture. Maintain the mice for another two weeks. Two weeks later, sacrifice the animal and harvest calvaria.Gently clear up all the soft tissue on calvaria samples with tweezers, and affix the tissue in four percent paraformaldehyde at four degrees Celsius. For osteolysis evaluation in the cobalt chromium molybdenum nanoparticle induced model, perform high resolution micro-CT 24 hours before micro-CT scanning. Transfer calvaria samples in phosphate-buffered saline from paraformaldehyde.On the day of micro-CT scanning, collect calvaria specimens from phosphate-buffered saline, and fix them on a holder associated with an instrument. Scan calvaria on micro-CT instrument. Conduct three dimensional reconstruction with assorted software.H and E staining is another tool for osteolysis evaluation in a nanoparticle-induced model. De-calcify calvaria samples in the phosphate-buffered saline, containing 15 percent of ehylenediaminetetraacetic for three weeks at four degrees Celsius. Embed the decalcified calvaria in paraffin and slice them into two micrometer sections in the area of particle deposition.Stain the sections with hematoxylin and eosin. Observe the overall pass through morphism and by light microscopy and acquire micrographs. Here are representative micro-CT images.Three dimensional reconstruction images of micro-CT data are shown in the left panel. Cross-section images from control, sham operation, and nanoparticle treated mice are shown in the right panel. Osteolysis is obvious in the nanoparticle treatment group.Here is a quantitative analysis of micro-CT photographs by the instruments associated software. Measurements include bone mineral density, bone volume, or total volume ratio, percentage of total porosity, trabecular number, trabecular thickness, and trabecular separation or spacing. Mice treated with cobalt alloy nanoparticles show significantly lower bone mineral density and bone volume, with higher percentage of total porosity compared with control animals.Here are representative H and E staining images for calvaria samples from control, sham operation and nanoparticle treated mice. Osteolysis is also obvious in the nanoparticle treatment group. Murine calvarial osteolysis model constructed by cobalt alloy nanoparticles have high success rate in modeling wear particle-induced osteolysis.Following are several key factors. First, the cobalt alloy nanoparticles enhance the interaction between wear particles and calvaria. Second, adequate exposure of calvaria and clear dissection of periosteum makes the interaction between particles and calvaria more intensive.Thirdly, quantitative measurements of bone loss by micro-CT makes it easier to access differences in bone loss among various treatments, providing us solid evidence for osteolysis compared with the traditional bone histomorphometry. Wear calvaria osteolysis model constructed by cobalt alloy particle is an ideal model to investigate the interaction between wear particle and different cells in aseptic loosening. Such as microphage, fibroblasts, osteoclasts, and osteoblasts.We hope our model will be helpful for the study on aseptic loosening in the future.

construction-evaluation-murine-calvarial-osteolysis-model-exposure-to

Research

JoVE Journal

Medicine

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...