Name: construction and evaluation of a murine calvarial osteolysis model by exposure to cocrmo particles in aseptic loosening
Uploaded: 2018-02-17T14:00:00.000+00:00
Duration: 9 min 46 s
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 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/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>

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 bone tissue. The calvarial osteolysis model has several advantages over the air-pouch counterpart. First, wear particles are directly exposed to the calvaria, making it possible to assess interactions between wear particles and bone homeostasis, including bone resorption, and osteoblast, osteoclast and macrophage activities9,10. Secondly, quantitative measurements of bone loss are available, allowing the assessment of various potential genetic approaches and biological agents in bone loss prevention11. Thirdly, it is possible to assess the relationship between wear particles and bone loss in various genetic backgrounds, including transgenic and gene knockout mice12,13. Fourthly, it can be used to screen a large number of compounds at various doses. However, the success rate of the traditionalcalvarial osteolysis model is relatively low, and using bone histomorphometry to measure osteolysis makes the results less objective1.
To improve the success rate of the model and render results more objective, several modifications have been made. First, nanoscale particles were used to enhance the interactions between the calvaria and wear particles. Indeed, the interactions between nanoscale particles and the calvaria is enhanced compared with commercially alloy particles, with a mean diameter of 1.5 &#181;m14,15. Secondly, a 1.0 cm2 area on the calvaria was delineated to achieve adequate exposure of the calvaria. Thirdly, micro-CT and three-dimensional reconstruction were used to quantify bone loss.
There are several limitations in the present model. First, micro-CT equipment for mice is not widely available for researchers, and technicians are required for scanning and three-dimensional reconstruction. Secondly, the CoCrMo nanoparticles used in the present model are not commercially available, and their production relies on support from materials science technicians. Thirdly, this model does not represent chronic effects of particles on bone mass, and lacks non-biological factors related to osteolysis, such as oscillatory fluid pressure or mechanical forces. The efficacy of the model could be significantly increased with the help of nanoscale particles and enough exposure of the calvaria. Bone loss could be quantified, and more objective data obtained with micro-CT.
A murine calvarial osteolysis model established by exposure to CoCrMo particles is an ideal tool to assess the interactions between CoCrMo particles and different cells such as macrophages, fibroblasts, osteoblasts and osteoclasts in aseptic loosening. In addition, a series of medications can be tested of their effects on aseptic loosening using this model.
There are many critical steps of this procedure. The first is the application of CoCrMo nanoparticles with a mean diameter of 50 nm. Secondly, adequate exposure of the calvaria was achieved. A 1.0 cm2 area on the calvaria was enough in this model, and the periosteum on the calvaria should be dissected carefully and completely. A clear dissection of the periosteum intensifies the interactions between the particles and calvaria. Thirdly, a quantitative measurement of bone loss by micro-CT is possible. Quantitative measurements of bone loss from three dimensional reconstruction, such as BMD, BV/TV, Tb.N, Tb.Th, Tb.Sp and percentage of total porosity, make it easier to distinguish bone loss differences in various treatments, and provide solid evidence of osteolysis compared with the traditional bone histomorphometry.

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

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 harvested. Then, all the soft tissue was gently cleared up, and micro-CT was used to quantify bone loss. From both three-dimensional reconstruction images and representative coronal photographs at the cross-section, significant bone loss was observed in mice treated with CoCrMo particles (Figure 3). Bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were significantly reduced, while total porosity and trabecular separation/spacing (Tb.Sp) were significantly increased in the CoCrMo group compared with the control and sham operation groups (Figure 4). Student t test was used to assess differences between groups, and p&#60;0.05 was considered statistically significant. Furthermore, H&#38;E staining of calvaria sections confirmed bone loss in mice treated with CoCrMo particles (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56276/56276fig1.jpg" /> 
Figure 1: Schematic of the particle-induced osteolysis (PIO) mouse model. The left side shows the mouse position in modeling. (A) Point localization. Determine the midpoints between the two eyes and ears, respectively, and incise the skin along the line between them. (B) Expose and remove the cranial periosteum from the calvaria. (C) Embed CoCrMo particle suspension in the middle of the calvaria. (D) Suture skin in simple interrupted suture. <a href="/files/ftp_upload/56276/56276fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56276/56276fig2.jpg" /> 
Figure 2: Transmission electron microscopy scanning of CoCrMo particles. (A) Representative Transmission electron microscopy images of CoCrMo particles. (B) Particle size distribution of CoCrMo particles was quantified with the software. Each bar represents frequency normalized to the total number of particles. <a href="/files/ftp_upload/56276/56276fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56276/56276fig3.jpg" /> 
Figure 3: Micro-CT analysis with 3-dimensional reconstruction of samples from control mice and those treated with PBS (sham operation) and CoCrMo particles. The white horizontal line indicates the location of the cross-section image. The white arrow indicates bone loss in the CoCrMo implantation group. <a href="/files/ftp_upload/56276/56276fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56276/56276fig4.jpg" /> 
Figure 4: Quantitative analysis of micro-CT images after three-dimensional reconstruction. Quantification of bone mineral density (BMD) (A), bone volume/total volume (BV/TV) (B), percentage of total porosity (C), trabecular number (Tb.N) (D), trabecular thickness (Tb.Th) (E), and trabecular separation/spacing (Tb.Sp) (F), in mean&#177;standard error. Student t test was used to assess differences between the groups, with p&#60;0.05 considered statistically significant. **, P&#60;0.01; ***, P&#60;0.001. n = 6 mice per group. <a href="/files/ftp_upload/56276/56276fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56276/56276fig5.jpg" /> 
Figure 5: Representative images of H&#38;E staining of calvaria samples (10&#215;) from control mice and those treated with PBS (sham operation) and CoCrMo particle. Red arrow indicates osteolysis. <a href="/files/ftp_upload/56276/56276fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

7.9K Views.  Nanjing University. Dit manuscript beschrijft een model lymfkliertest calvarial osteolyse door blootstelling aan CoCrMo deeltjes, die een ideale diermodel vormt voor de beoordeling van de interacties tussen deeltjes van slijtage en verschillende cellen in aseptische losraken.

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

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

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

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.

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Cancer Research

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

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

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.

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Developmental Biology

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

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

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

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.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Bioengineering

A Simple Critical-sized Femoral Defect Model in Mice

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

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

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

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

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

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

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

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

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

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.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

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.

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

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

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.

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

Establishment and Evaluation of a Rat Femoral Box: Cavity Defect Model

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

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which involves the application of cells, scaffolds, and cytokines, is considered a promising solution for bone regeneration. Consequently, various animal models that simulate bone defects play a crucial role in exploring the therapeutic potential of tissue engineering for bone healing. In this study, we established a box-shaped cortical bone defect model in the mid-femur of rats, which could serve as an ideal model for assessing the function of biomaterials in promoting bone healing. This box-shaped cortical bone defect was drilled using an oral low-speed handpiece and shaped by a lathe needle. Post-operative micro-CT analysis was immediately conducted to confirm the successful establishment of the box-cavity cortical bone defect. The femurs on the operated side of the rats were then harvested at multiple time points post-surgery (0 days, 2 weeks, 4 weeks, and 6 weeks). The healing process of each sample&#39;s defect area was evaluated using micro-CT, hematoxylin and eosin (H&#38;E) staining, and Masson trichrome staining. These results demonstrated a healing pattern consistent with intramembranous ossification, with healing essentially complete by 6 weeks. The categorization of this animal model&#39;s healing process provides an effective in vivo method for investigating novel biomaterials and drugs that target intramembranous ossification during bone tissue defect healing.

Author Spotlight: The Box-Cavity Cortical Approach for Enhanced Evaluation of Biomaterials and Bone Regeneration

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.

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which ...

The Establishment of a Murine Mandibular Molar Extraction Socket Healing Model

This study introduces the development of a molar extraction model in the murine mandible to provide a practicable model for studying alveolar bone regeneration and intramembranous ossification. C57/J6 mice were used to extract the mandibular first molar to establish this model. They were euthanized, and the bilateral mandibles harvested, at 1 week and 4 weeks post-surgery, respectively. Subsequent serial stereoscopic harvest, histological assessment, and immunofluorescence staining were performed to demonstrate successful surgery. Immediately after surgery, the stereoscopic images displayed an empty extraction socket. The hematoxylin and eosin (H&#38;E) at 1 week and Masson staining at 4 weeks post-surgery showed that the area of the original root was partially and fully filled with bone trabeculae, respectively. The immunofluorescence staining showed that, compared with the homeostasis side, the Sp7 expression increased at 1 week post-surgery, suggesting vigorous osteogenesis in the alveolar fossa. All these results demonstrated a practicable murine tooth extraction socket healing model. Upcoming studies revealing the mechanisms of jawbone defect healing or socket healing could adopt this method.

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.

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

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