I wanna develop a better treatment for children with broken bones. Specifically, I wanna prevent bone formation in the fractured cartilage growth plate because, otherwise. the broken limb won't lengthen normally.
Step one, we had to develop a more accurate mouse model to test new therapies and determine mechanism of action. Conventional treatments involve resecting the bony bridge after injury, and inserting interpositional materials like fat tissue. These methods do not regenerate growth plate cartilage, and often require additional surgeries.
However, emerging tissue engineering approaches we are now testing, include localized molecules to boost cartilage regeneration while preventing bony bridge formation. Animal models are essential for testing tissue engineering approaches to injured growth plate cartilage regeneration. There are trade-offs between precision and cost.
Larger animals like sheep offer higher precision, and consistency in injury modeling, but are much more expensive than smaller animals like mice. The goal of our effort here is to understand the cellular basis for this tissue response that occurred after the injury to the growth plate. Using our three-color fluorescent mice, we are able to map the cells that are emanating these colors, along with other fluorescent stains, and somatic mineral from antibodies, and map them back to a familiar chromogenic stain.
This allows us to appreciate the dynamism of the repair, and to identify the cells that are responsible for this tissue response. The native fluorescence of the hypertrophic chondrocytes in these young transgenic mice allows the researcher under live imaging to precisely create a clinically relevant growth plate injury that mimics a child's injury. Also, the articular cartilage remains uninjured, and the extensive reagents available for mechanistic studies in mice can be utilized.
To begin, position three anesthetized mice at a time in parallel on their stomachs in the X-ray cabinet. Splay the legs of the mice so the tibial bones are not obscured under them. For recording the initial limb length position a radiopaque scale near the mice, and perform X-ray imaging with 26 kilovolts, and 800 milliamperes to capture their tibial images.
In the animal surgery room, assemble the electronic high-speed dental drilling system. Connect the electronic foot controller, and the handpiece to the control unit, and cover the handpiece cord with a disinfected surface barrier tube socks. Set the controller at a drive ratio of one-to-one with a maximum of 30, 000 revolutions per minute.
Next, place the flexible isoflurane machine hose covered with a disinfected tube socks on the fluorescent stereo microscope stage. To begin, determine the initial limb length of the mouse with X-ray imaging. Attach the sterile 0.5 millimeter round dental bur to the handpiece, and select the other surgical instruments needed.
After anesthetizing the animal, immediately administer half the prescribed dose of buprenorphine subcutaneously. Apply ocular lubricant to protect the mouse's eyes from drying, and transfer the mouse in a supine position on the stereo microscope stage. Disinfect the right hind limb, pelvic region, anterior aspect of the left hind limb and tail sequentially with povidone iodine, followed by 70%ethanol.
Under bright light illumination employing a number 15 scalpel, create a five millimeter skin incision just below the knee joint to reveal the proximal end of the right tibia. Keep the left contralateral tibia uninjured so that it serves as an internal uninjured control. Next, using the backside of the number 15 scalpel blade, perform a vertical blunt dissection through the overlying muscle at the proximal tibia, removing the soft tissue for clear exposure of the tibial head.
After turning off the operating room lights, select the correct fluorescence channel to illuminate the desired region of the growth plate. Next, adjust the mouse's skin opening slightly more proximal, and then distal, to ensure the hypertrophic growth plate area of the tibial growth plate is in view, and not the femur growth plate. To create a Salter-Harris Type II-like lesion, position the 0.5 millimeter dental drill bur in the middle of the hypertrophic growth plate zone.
Keep the bur and limb parallel to the work surface, so that the bur entry pathway will not angle into the epiphysis, nor go through the entire soft growth plate. Apply pressure on the drill pedal to initiate bur rotation, and gently depress the bur into the growth plate, stopping before the defect goes any deeper than the bur diameter. Irrigate the lesion site with a drop of sterile PBS to remove any debris.
Using a periodontal probe, confirm that the defects depth is 0.5 millimeters. Then, carefully realign the skin edges. Then, employ an interrupted suturing technique with 5-0 polyglycolic acid sutures to seal the skin incision.
For tissue dissection, isolate both intact hind limbs from the euthanized animal, and remove the skin and the muscle from the bone and knee capsular area. Then, using micro dissecting scissors, excise the patella carefully. Employ a 29 gauge insulin syringe to thoroughly distribute cold 10%buffered formalin within all areas of the knee cavity.
Finally, sever the diaphyseal region of the femur and tibia to improve fixative access to the marrow space. Tie the joint tissue with gauze to a thin dowel. Put the hind limbs into the fixative, and place the tissue at four degrees Celsius to maintain it in a fully extended position for 24 to 36 hours.
Injured tibias displayed a reduced growth in length compared to uninjured controls at three weeks post-surgery in 2D cross-sections of the bone. Injured growth plates showed disruption of the hypertrophic zone, and provisionally calcified layer with only slight disturbance in the proliferative zone. Bony bridge formation within the injured growth plates was consistently observed in all six mice, and the majority formed near the middle of the growth plate, despite the lateral approach.
The multicolor fluorescence approach enabled a detailed examination of chondrocyte differentiation within the bony bridge area.
