Micro-CT

Bone samples from 4 sites, including the mandible, spine (third lumbar vertebra), femur, and tibia, were harvested and analyzed by micro-CT and a histology test. All samples were fixed in 4% concentration of paraformaldehyde and scanned using a micro-CT system (micro-CT 50 scanner, Scanco Medical, Bassersdorf, Switzerland), and they were reconstructed with a voxel size of 20 μm. The scanning system was set to 70 kV, 114 μA, and 700 ms of integration time. After scanning, we used a 3-dimensional Gaussian filter (sigma = 1.2, support = 2) to eliminate noise in the volumes. The region of interest (ROI) only included the trabecular bones, without cortical bones, because measuring trabecular bones might be more appropriate to show the bone mass changes in T1DM patients.[17]

Four skeletal sites were chosen as the representative areas, including the mandibular bone, proximal tibia, femur greater trochanter, and spine. All 4 sites were commonly used in many bone research studies, and they had a good site-specific response in past studies. According to previous literature in OVX rats, osteoporosis affected jaw bones much less than long bones.[11] Thus, we suppose that DOP may affect areas with dense cancellous bone much less than general areas. Therefore, we chose the femur and greater trochanter as another position because these bones are comprised of dense cancellous bones.

All of the ROI diagrams were shown in Fig 1

Mandible: The trabecular area between the first molar root was selected as the ROI of the mandible, as a past study reported[18].

Spine: A 2-mm thickness of the trabecular bone area in the middle of the third lumber vertebral body was selected.[11]

Femur: A 1-mm thickness(50 slices) of the greater trochanter in the femur from the slice with the largest diameter in the axial plane was selected.

Tibia: A 2-mm thickness of the trabecular bone of the tibia in the axial plane (from 1 mm below the growth plate) was selected to avoid the effect of new bone growth near the growth plate[19].

For all 4 bone sites, the following indexes of the trabecular bone were chosen to evaluate the bone density and microstructure: BMD, percent bone volume ratio (BV/TV, %), trabecular number (Tb.N, /mm), trabecular separation (Tb.Sp, mm), and trabecular thickness (Tb.Th, mm), where higher values indicate reduced connectivity [20]. All these parameters can indicate the destruction of bones.