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

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.