This method can help answer key questions in the hard and soft tissue regeneration field. The main advantage of this technique is that the use of a mouse allows for a detailed analysis of bone regeneration. Begin with a fully anesthetized adult mouse.

Carefully shave and disinfect the surgical area with 10%iodine solution. Then, administer 0.5%lidocaine hydrochloride at the right lower limb. Then, after making a 15-millimeter longitudinal skin incision at the right lower leg with a number 15 scalpel, bluntly separate the underlying muscles, taking care not to remove all periosteum.

Then, cut the fibula with scissors. Grasp the ankle with narrow, thin forceps, and use a 27-gauge needle to make a hole in the bone, about five millimeters from the heel. The needle should penetrate the skin, bone, and skin in that order.

When the needle fully penetrates, cut the tip and root with a nipper so that the needle is about 15 millimeters. Make another hole in the same manner, about two to three millimeters proximal. Next, hold around the ankle, and pass a 25-gauge needle under the knee in the same manner.

After the needle penetrates, cut the tip and root with a nipper. Make another hole in the same manner, about two to three millimeters distal. Place the custom-made distractor such that it is parallel to the extension direction.

Fix the needles and device with enough polymerization dental resin to fill the grooves of the device. Wait for the polymerization to complete, which should take approximately five minutes. Then, while being very careful not to damage the surrounding tissues, cut the middle of the diaphysis of the tibia using a very thin cutting disc while applying a saline solution.

Close the wound with a 4-0 nylon suture. Give subcutaneous injections of buprenorphine for analgesia immediately after surgery. There are various reports on the latency period and distraction rate, but here, representative protocols are shown.

After a latency period of five days, begin the distraction procedure. For extension, use the pin attached to the rapid expansion screw. Move the pin in the direction of the yellow arrow attached to the extension screw.

Hold the tail with the little finger and palm, and fix the extension device with the forefinger and thumb. Perform extension by moving the pin in the direction of the yellow arrow 0.2 millimeters for a one-quarter turn. Continue lengthening at the rate of 0.2 millimeters every 12 hours for eight days, which will result in a total gap of 3.2 millimeters.

This image shows typical radiography findings at four weeks after distraction in the model. Newly formed bone is seen at the distraction site. Hematoxylin and eosin staining results are seen here.

A newly formed bone bridge was observed, and the newly formed bone could be easily distinguished from the native bone. We presented a mouse tibial DO model developed using a custom-made distractor. The use of a mouse DO model facilitates a more detailed analysis.

It is particularly suitable for experiments using knockout mice.