A Simple Critical-sized Femoral Defect Model in Mice

The overall goal of this procedure is to generate a simple, experimental, critical size defect in a mouse femur. This is accomplished by first making a medullary pin from surgical steel tubing. In the second step, a critical size defect is generated in the femur of an anesthetized mouse.

The pin is then implanted to stabilize the bone. Ultimately, radiography and histology can be used to assess the degree of bone healing. While attempting this procedure, it's important to remember to secure the bone only with the current forceps and to ensure that the collar is flushed with the edge of the cut bone.

Begin by placing 10 microliters of cyanoacrylate adhesive onto the middle of the shaft of a nine millimeter piece of 22 gauge stainless hypodermic tubing. Pass a three millimeter collar over the shaft until it reaches the center, and then twist it to evenly distribute the glue between the collar and shaft as illustrated here. Various pin configurations can be prototyped for the specific purposes of the study and the recipient.

Next, measure the dimensions of the pin with a pair of digital calipers. Each section should be about three millimeters long. Allow the collar to set for at least 15 hours, and then use a two 20 grit Emory impregnated disc to remove any burrs or excessive glue.

Next, use a rotary tool to polish the pin with a felt polishing disc, and then rinse the pin with deionized water and dry it with compressed air. Now, test the integrity by placing a 19 gauge hypodermic blunt needle over the shaft of the pin and push the needle against the collar to confirm that the collar can resist approximately 25 grams of weight. Then rinse the pin in sterile filtered PBS and test the fit by slipping the pin into the femoral medullary cavity of a bone specimen, ensuring that the shaft fits snugly with the collar flush against the edges of the cut bone.

Make sure that the end of the pin penetrates into the trabecula bone of the diaphysis as well, so as to maximize the fixation and improve the interference fit. Punch out a three millimeter cylinder of gel foam with a skin biopsy knife and pierce the center with a 21 gauge needle. Then pass the gelatin foam cylinder and align it with the collar.

Finally, autoclave the pin on a dry cycle at 120 degrees Celsius and 15 PSI. The foam will dry and darken in color After confirming sedation by the appropriate institutionally approved method. Begin the surgical procedure by placing an anesthetized with the hind limbs facing upwards on a sterile drape.

Apply eye ointment to the animal, and then after removing the fur, cover the animal with a clean fenestrated drape and locate the proximal and distal lens of the femur. Incise the skin for five to 10 millimeters along the longitudinal axis. Then use a number 15 scalpel to separate the skin layer from the fascia, exposing a lateral approach to the femur via the biceps for MAs and vestus lateralis.

Locate where the septa of both muscles. Meet a line of white tissue against the pink coloration of the muscle where the scalpel carefully dissect along this intermuscular boundary until the femur is visible. Next, use a blunt periosteal elevator to develop the incision so as to expose the entire diaphysis.

Then further expose the central two thirds of the femur taking care to preserve the posterior neurovascular bundle on the medial side. Use a scalpel to gently scrape the soft tissue off of the bone and then dry the bone with a sterile cotton tipped applicator. Now use a sterile ruler or calipers to locate the center of the femur and mark the bone 1.5 millimeters approximately and distally from the center.

Then gently grasp the femur with a pair of fine nosed forceps previously fashioned in the current style, and use a fine drill fitted with a fine diamond grit coated cutting wheel to make the first cut with the elevator in place to protect tissue.Below. Raising the cut femur to 45 degrees while firmly holding the extremity of the diaphysis. Make the second cut removing a three millimeter segment with the bone immobilized by forceps.

Carefully ream the medullary cavities of each end with a blunt 23 gauge hypodermic needle. Then using a pre-made depth gauge made from a length of 22 gauge tubing placed within a 19 gauge tube, confirmed that the depth of the cavity is three millimeters. When the bone is ready, insert the prepared pin into the proximal, then distal medullary cavities to bring the femur back to its original length, and to establish a stable three millimeter gap.

Confirm that the pin fits snugly into both medullary cavities and that the edges of the cut bone are flushed with the collar. Then reposition the muscle and peripheral tissue over the pin and close it with a continuous absorbable five zero suture. Close the skin with five seven square knot nylon, five zero sutures, and seal the incision with surgical adhesive animals.

Were euthanized according to A VMA guidelines and also under an iacuc approved protocol. After two to five weeks, expose the proximal femur and the pelvis from the medial side of the animal to carefully dissect away the hind limb. Then gently press the joint inwards from the lateral side of the limb while using scalpel to detach the femoral head from the acetabulum.

Use a sharp scalpel to cut away the remaining muscle and skin releasing the entire limb from the pelvis. Then cut away excess tissue and the lower hind limb approximately five millimeters below the knee joint and remove the skin, leaving the muscle in place. Next, fix the tissue in 10%Buffered formin supplemented with 10 millimolar calcium chloride fixative for one week, followed by storage and PBS supplemented with 10 millimolar calcium chloride for up to one month prior to imaging.

To analyze the specimens, wrap the tissues in a thin layer of plastic ceiling. Film and position them vertically, one at a time. In the micro CT imaging chamber, set the scan to the following parameters.

Voltage to 29 kilovolts current to 661 milliamps power to 19 watts. Image pixel size to 21 microns. 360 degree rotation to yes frame averaging to on rotation, step to one degree and random movement to on.

As each image is acquired, save them as jpeg files in a single destination folder per scan. Then using the axial images and analytical software for each file, select the sections that encompass the collar only to set the proximal and distal edges of the original defect, and to define the region of interest. Finally, draw an exclusion zone with a 100 micron margin around the collar to exclude it from the calculations, and then transfer the zone to each of the sections in the region of interest.

Without therapeutic intervention, the edges of the defect typically extend 0.5 millimeters during the first 21 days post-surgery. After 14 to 21 days of healing, the volume of the de novo bone can be readily determined from the axial images generated by the micro CT scanning. The volume of the new bone generated increases with time, but does not generally exceed a total of one cubic millimeter.

In the absence of therapeutic intervention after 21 days, the polar moment of inertia of the de novo bone 0.25 millimeters from the lesion edges, ranges from between 0.05 to 0.35 millimeters fourth compared to 0.02 to 0.08 millimeters fourth at the center of the lesion. Further confirming the presence of the non-union in untreated cases as shown in this representative image, Mason's TRI chro, staining of decalcified longitudinal sections permits the visualization of the endochondral bone outgrowth with the leading edge of cartilage followed by cancellous bone. While some damage will inevitably occur during the dissection, if the pin is removed carefully, the histological structure of the bone and connective tissue will remain clear.

Alternatively, methylmethacrylate embedding and sectioning of non demineralized sections may be performed with the pain in place Once mastered, this technique can be done in 30 minutes. Following this procedure of methods like CAR TPCR Microarray or immuno blotting can be performed on the recovered tissue to answer additional questions like, how do certain genotype cells, proteins, or even compounds affect bone repair? In our studies, we use human cells and human proteins in immune compromised mice.