Direct Mouse Trauma/Burn Model of Heterotopic Ossification

The overall goal of this procedure is to induce ectopic bone formation following a traumatic injury without the application of exogenous materials. This is accomplished by first creating a focused musculoskeletal injury. In this case an Achilles tenotomy.

In the second step, a global inflammatory condition is induced. Here, a 30%surface area partial thickness contact burn is demonstrated. Heterotopic ossification or HO is then allowed to develop in the region of the tenotomy over the next five to nine weeks.

Ultimately, the amount and location of the ectopic bone can be assessed by micro tomographic imaging. The main advantage of this technique over existing methods is that this is has a clinical correlate in this model. Mice go on to form a cartilage precursor followed by the heterotopic bone without any additional cytokines and no additional mutations in the mice.

Thus, we feel that this will allow for better testing of therapeutics and other imaging modalities, as it does mimic well the type of bone that we see in patients with heterotopic ification. The implications of this technique extend towards the therapy and the diagnosis of HO because it allows for the study of potential treatments of ectopic bone development before it OSS acidifies. We first had the idea for this method when we saw HO in patients suffering major burns that developed at various locations distant from the burn site and in other patients at locations of musculoskeletal trauma.

After administering anesthesia, confirm a lack of response to toing and administer analgesic. Apply ointment to the eyes of an eight to 10 week old C 57 BL six adult mouse. Next place the animal on a heating pad and use clippers to closely shave the hind paw from the heel to the knee.

Then beginning over the spine and extending to the left of the dorsal midline. Clear an area of at least two by three centimeters to accommodate the burn injury. When all of the hair has been removed, use three alternating povidone iodine scrubs to clean the surgical area and the hind paw, and don the proper personal protective equipment, including a face mask, hair bonnet, and a sterile gown and gloves.

Then using sterile instruments, make a 0.5 centimeter longitudinal incision along the medial aspect of the left achilles tendon. When the tendon can be visualized, insert one blade of a pair of sharp tissue, scissors in the tissue plane beneath the tendon, and dissect along the plane until the blade is at the tendon midpoint. Then close the scissor blades sharply to sever the tendon and apply pressure with a sterile gauze to achieve hemostasis.

After closing the skin incision with a five oh Vicryl stitch, eat a 35 gram two by two by three centimeter aluminum block to 60 degrees Celsius in a water bath. When the block has been heated, rested on top of the mouse for 17 seconds, taking care that the entire surface is in contact with the mouse. Then dry the dorsal partial thickness burn with gauze and apply a tegaderm dressing, resuscitate the animal with the administration of warmed lactated ringer solution by intraperitoneal and subcutaneous injection.

Then place the mouse in an individual cage for monitoring until it is fully recovered. To monitor the heterotopic ossification of the injuries by micro CT analysis at the appropriate time points post dorsal partial thickness burn, secure the anesthetized mouse onto the scanner bed in the prone position. Tape the hind limb securely to the bed to prevent a breathing motion artifact and include the air, water, and hydroxy appetite containing phantom beneath the mouse for image calibration.

Next, open the bone analysis software and define a region of interest that encompasses both of the hind limbs from the hip joint proximally to the tip of the hind paw distally. Obtain an image using the indicated parameters. Then draw a region of interest in each of the three phantom chambers and input the average density into the appropriate fields in the software to calibrate the image to hounds field units.

To achieve the clearest anatomical view, use the grabber tool in the software to reorient the image so that the tibia of the left hind limb is parallel along the Z axis. Then beginning at the knee scroll distally through the image slices. When HO is encountered, use the manual spline tool to create a region of interest around the ectopic bone on every third to fifth slice continuing distally through the paw or until the HO has been surpassed.

Then use the extrapolate tool to extend and stitch the regions of interest together into a single region of interest that contains all of the ho. Finally, create a 3D region of interest and open the analysis menu, setting the lower and upper threshold values that best show the bone window to calculate the bone volume to date. All mice that have sustained both an Achilles tenotomy and burn injury as just demonstrated, have developed a measurable amount of HO at the tenotomy site to quantify the formation of ectopic bone.

The calibrated image micro CT protocol is used at each desired time point. The addition of the burn injury in combination with the Achilles tenotomy has proven to be key in the induction of significant ho ho, indeed, with both injuries being sustained concurrently, there is a significant increase in the amount of ectopic bone that is formed at the tenotomy site compared to the Achilles tenotomy alone. Once mastered, this technique can be performed in 10 minutes or less per animal if performed properly.

While attempting this procedure, it's important to remember that all posterior tendons should be severed completely. Following this procedure, we can use imaging modalities such as RO spectroscopy, bioluminescence, or CT scan to assess how much bone is formed and the effect of certain drugs. We also could then use histology, immunohistochemistry, immunofluorescence to look at actual signaling mechanisms behind why heterotopic ossification has developed and how our drugs are working.