Name: Construction and Evaluation of a Murine Calvarial Osteolysis Model by Exposure to CoCrMo Particles in Aseptic Loosening (Video) | JoVE Journal
Uploaded: 2018-02-17T14:00:00.000+00:00
Duration: 9 min 46 s
Description: Learn to construct a murine calvarial osteolysis model using cobalt-chromium-molybdenum nanoparticles for studying aseptic loosening in arthroplasty. This method enables assessment of particle-cell interactions and bone loss mechanisms.

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5 minutes

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 CoCrMo particles into 1.5 mL of anhydrous ethanol.</li>
		<li>Resuspend CoCrMo particles in anhydrous ethanol by ultrasonic shaking at 28 kHz and 600 W for 5 min.</li>
		<li>Apply one drop (about 20 &#181;L) of the resulting suspension on the objective table of a transmission electron microscope (TEM). Capture series of TEM photos at 200 kV acceleration voltage and 0.24 nm resolution.</li>
		<li>Use the provided software to calculate the mean diameter and particle size distribution in TEM micrographs.</li>
	</ol>
	</li>
	<li>Decontaminate endotoxins
	<ol>
		<li>Autoclave 50 g of particles for 15 min at 121 &#176;C and 15 psi.</li>
		<li>Detect endotoxins by a quantitative Limulus Amebocyte Lysate (LAL) Assay (&#60;0.25% EU/mL was considered to indicate endotoxin absence)5.</li>
	</ol>
	</li>
	<li>Resuspend the particles in phosphate-buffered saline (PBS) at a concentration of 50 mg/mL as stock solution6.</li>
</ol>
2. Construction of the Calvarial Osteolysis Model
<ol>
	<li>Anesthetize 6 week old C57BL/J6 mice (six mice per group) withpentobarbital (50 mg/kg). Use the pinch test to assess the level of anesthesia. Prevent drying of eyes with normal saline.</li>
	<li>Place mice in the prone position. Remove the fur on the cranium with a shaver and disinfect the skin using medical cotton balls containing 75% ethanol.</li>
	<li>For point localization, identify two points, including the midpoints between the two eyes and ears, respectively. Then, determine the line between the two above points, and incise the skin along the above line with scissors (Figure 1A).</li>
	<li>Remove the cranial periosteum from the calvaria with a scalpel (Figure 1B)6.</li>
	<li>Suture skin at both ends with simple interrupted suture.</li>
	<li>Make a suture line through the middle of the incision without knotting. Hold the two ends of the suture line.</li>
	<li>Embed 50 &#181;L of CoCrMo particle suspension (50 mg/mL, in PBS) in the middle of the calvarias (Figure 1C)2.</li>
	<li>Knot the last stitch within simple interrupted suture (Figure 1D).</li>
	<li>Maintain mice for another 2 weeks.</li>
</ol>
3. Evaluation of Calvarial Osteolysis Model by Micro-CT Scanning
<ol>
	<li>Sacrifice the mice with carbon dioxide. Decapitate the mice in the horizontal plane. Remove the brain tissue inside, and the skin and fur outside. Harvest the calvarias for further experiments.</li>
	<li>Gently clear up all the soft tissue on the calvaria with tweezers. Fix the cleared calvarias in 4% paraformaldehyde at 4 &#176;C for 24 h. Immerse the calvarias in PBS 24 h before micro-CT scanning.</li>
	<li>Analyze the mice calvarias by high-resolution micro-CT at an isometric resolution of 18 &#181;m and X-ray energy settings of 45 kV and 550 mA.</li>
	<li>Conduct three-dimensional reconstruction of micro-CT data with the software.</li>
	<li>Qualitative and quantitative analysis.
	<ol>
		<li>First, select the square region around the midline suture as the region of interest.</li>
		<li>Secondly, measure bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation/spacing (Tb.Sp), and percentage of total porosity with the provided software for micro-CT.</li>
		<li>Thirdly, compare the three groups for various measurements by one-way ANOVA. For post hoc analysis of variance, apply the Bonferroni method2.</li>
	</ol>
	</li>
</ol>
4. Evaluation of Calvarial Osteolysis Model by H&#38;E Staining
<ol>
	<li>Decalcify calvaria samples in 15% ethylene diamine tetraacetic acid (EDTA)-PBS at 4 &#176;C. Change the decalcification solution every day for 3 weeks.</li>
	<li>Embed the decalcified samples in paraffin for a 2 cm x 1 cm x 1 cm cube, and slice them into 2 &#181;m sections in the area of particle deposition.</li>
	<li>Stain the sections with hematoxylin and eosin as previously described7.</li>
	<li>Capture micrographs of the overall pathomorphism by light microscopy.</li>
</ol>

<ol><li>Dong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antisense+oligonucleotide+targeting+TNF-alpha+can+suppress+Co-Cr-Mo+particle-induced+osteolysis%5BTitle%5D)+AND+%22J+Orthop+Res%22%5BJournal%5D)">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/pubmed?term=((SIRT1+protects+osteoblasts+against+particle-induced+inflammatory+responses+and+apoptosis+in+aseptic+prosthesis+loosening%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">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/pubmed?term=((New+animal+models+of+wear-particle+osteolysis%5BTitle%5D)+AND+%22Int+Orthop%22%5BJournal%5D)">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="https://www.researchgate.net/publication/287666498_Preparation_of_ultrafine_silver_powders_by_DC_arc_plasma">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/pubmed?term=((The+fibroblast+expression+of+RANKL+in+CoCrMo-particle-induced+osteolysis+is+mediated+by+ER+stress+and+XBP1s%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">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/pubmed?term=((Autophagy+mediated+CoCrMo+particle-induced+peri-implant+osteolysis+by+promoting+osteoblast+apoptosis%5BTitle%5D)+AND+%22Autophagy%22%5BJournal%5D)">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/pubmed?term=((Particle-induced+osteolysis+mediated+by+endoplasmic+reticulum+stress+in+prosthesis+loosening%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Adeno-associated+virus-mediated+osteoprotegerin+gene+transfer+protects+against+particulate+polyethylene-induced+osteolysis+in+a+murine+model%5BTitle%5D)+AND+%22Arthritis+Rheum%22%5BJournal%5D)">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/pubmed?term=((Autophagy+mediated+TiAl%286%29V%284%29+particle-induced+peri-implant+osteolysis+by+promoting+expression+of+TNF-alpha%5BTitle%5D)+AND+%22Biochem+Biophys+Res+Commun%22%5BJournal%5D)">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/pubmed?term=((ER+Stress+Mediates+TiAl6V4+Particle-Induced+Peri-Implant+Osteolysis+by+Promoting+RANKL+Expression+in+Fibroblasts%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((TiAl6V4+particles+promote+osteoclast+formation+via+autophagy-mediated+downregulation+of+interferon-beta+in+osteocytes%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">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/pubmed?term=((Lycorine+suppresses+RANKL-induced+osteoclastogenesis+in+vitro+and+prevents+ovariectomy-induced+osteoporosis+and+titanium+particle-induced+osteolysis+in+vivo%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">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/pubmed?term=((The+role+of+calcitonin+receptor+signalling+in+polyethylene+particle-induced+osteolysis%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/20415362">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/pubmed?term=((The+biological+response+to+nanometre-sized+polymer+particles%5BTitle%5D)+AND+%22Acta+Biomater%22%5BJournal%5D)">The biological response to nanometre-sized polymer particles.</a> Acta Biomater. 23, 38-51 (2015).</li>
</ol>

The authors have nothing to disclose.

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

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

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 ...

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.

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Research

JoVE Journal

Medicine

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

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

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

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

This protocol demonstrates step-by-step details of how to extract the mandibular first molar in the mouse. It provides an alternative method for researchers focusing on jawbone healing and regeneration.

The Establishment of a Murine Mandibular Molar Extraction Socket Healing Model

Here, we present a protocol for creating a box-cavity defect in rat femoral diaphysis tissue. This model can assess biomaterial performance under biomechanical stress and explore mechanisms of bone regeneration related to intramembranous osteogenesis.

Creating a Box-Cavity Defect Model in the Cortical Bone of Rat Femora

The murine bone marrow injury model is a useful tool for studying postnatal osteogenesis through intramembranous ossification. This simplified surgical protocol describes the bone marrow ablation procedure for downstream assessment of new bone formation and cellular responses following injury.

Improved Methodology for Studying Postnatal Osteogenesis via Intramembranous Ossification in a Murine Bone Marrow Injury Model

Learn to construct a murine calvarial osteolysis model using cobalt-chromium-molybdenum nanoparticles for studying aseptic loosening in arthroplasty. This method enables assessment of particle-cell interactions and bone loss mechanisms.

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

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