A 3-D Visualization Technique for Bone Remodeling in a Suture Expansion Mouse Model

The interest in craniofacial sutures has been driven by the clinical needs to understand the mechanism of orthodontic and the orthopedic treatment. The scope of our study is to establish a standardized suture expansion mice model, and the 3D visualization method to study the mechanobiology code changes of suture and bone remodeling upon expansion. PEGASOS tissue clearing technique has been combined with virus labeling methods to obtain versatile information of suture and bone remodeling in space perspective.

This model is suitable for various strengths of transgenic mice of different ages. It is convenient to adjust the magnitude of tensile stress and withdraw force at any time. With the PEGASOS technique, we can observe the three dimensional spatial temporal distribution of cranial suture stem cells during bone remodeling.

With the PEGASOS technique, we will further explore the relationship between such stem cells and mechanical stress as well as its specific mechanisms. Begin by preparing two retention holders. Use light wire pliers and a 0.014 inch Australian wire to make a helical loop.

Then, prepare customized stainless steel springs and cut the spring into an appropriate length. Also, prepare one straight Australian wire or straight steel wire of length seven millimeters, and cut two pieces of paper with diameters of two millimeters to use as barriers. Next, place an anesthetized mouse on the operating table and carefully remove the fur on top of the head with hair removal cream or a shaver.

Then, disinfect the scalp of the mice using alternating rounds of iodophor and 75%alcohol. Position the mouse with its head on the top and use surgical tapes to fix the limbs on the operating table. Now, use surgical scissors to perform an arcuate flap along the scalp near the neck of the mouse, fully exposing the sagittal suture and surrounding skull.

Then, fix the scalp flap with a 6-0 suture on the operating table. Dry the skull and etch it with 37%phosphoric acid for 20 seconds. Then, use normal saline to clean up the acid etching.

Place the two holders on both sides of the parietal bones, three millimeters from the sagittal sutures, and glue them with a light-cured adhesive. Then, put back the scalp flap, label the position of the holders, and cut two small holes in the scalp at the corresponding positions. Reset the flapped scalp by simultaneously passing the small loops through the holes to expose the skin's surface.

To install the spring and guide wire, cut the spring one millimeter longer than the distance between the two holders. Compress the pressure spring and place it between the small coils on both sides. Pass the stainless steel wire through the small coils and the spring, and release the spring to obtain a starting thrust of about 30G.

Next, place two scraps of paper between the spring and the holders and bond them with a light-cured adhesive to set up barriers at both ends of the spring. After expansion, Micro CT was used to visualize the changes in the cranial suture. In comparison to controls, the cranial suture gradually and significantly expanded after applying force for one day, and fluffy bony protrusions appeared on the bone edge after day five.

Also, visualization of mineralization using double labeling method showed that force loading significantly activated osteogenesis. Newly mineralized bone was observed after seven days, indicating the bone remodeling process after suture expansion. To begin, fix the limbs of an anesthetized mouse with adhesive tape, hold the skin of the abdomen upward, and carefully cut along the circumference of the thorax to fully expose it.

Make a small cut in the right atrium with ophthalmic scissors, and immediately insert a perfusion 22 gauge needle at the apex of the left ventricle. Then, push 30 to 50 milliliters of cardiac perfusion fluid through the syringe. After the outflow fluid from the right atrium becomes completely clear, infuse an equal volume of 4%PFA solution.

Dissect the tissues and organs and fix them overnight with 4%PFA at four degrees Celsius. To induce tissue decalcification, place the fixed hard tissue in 10 milliliters of EDTA solution on a shaking table at 37 degrees Celsius. Incubate for about two days and change the fluid daily.

Place the fixed calvarial bones in 20 milliliters of 25%Quadral solution for tissue decolorization. Incubate on a shaking table at 37 degrees Celsius for one day and change the fluid once. Next, place the tissue sequentially in 30%tertiary butanol or TB solution, then 50%TB solution, and then 70%TB solution to perform gradient degreasing.

Subsequently, dehydrate the samples and TB PEG solution for six hours on a shaking table at 37 degrees Celsius. Finally, place the completely dehydrated tissue in BB PEG solution on a shaking table at 37 degrees Celsius. After two to four hours of incubation, the tissue will turn transparent.

The PEGASOS tissue clearing method resulted in transparent tissue. Cleared tissue with whole mount EDU staining showed that the labeling was efficient and well-preserved. In the control group, several EDU-positive cells were diffusely distributed throughout the sutures.

After one day of suture expansion, proliferating cells were observed in the middle and edges of the suture. As the suture widened, the number of proliferating cells decreased over time. The green labeled cells were small round cells in the bone marrow, which differed from the EDU-positive suture cells.