# Aging Alters Cervical Vertebral Bone Density Distribution: A Cross-Sectional Study
## 2 Materials and Methods  
The protocol used in the current study was approved by institutional review board at The Ohio State University (Protocol no. 2011H0128). A total of 136 CBCT images was randomly selected from records on routine dental patients (63 images for male and 73 images for female) at The Ohio State University, College of Dentistry. Those 3D images were taken by a CBCT machine (iCAT, Imaging Science International, Hatfield, PA, USA) with resolutions of 0.25 mm for four images, 0.3 mm for 127 images, and 0.4 mm for five images under the same scanning energy (120 kV and 5 mA) with full field of view (FOV) at different scanning times (26.9 s, 8.9 s, and 8.9 s for images with 0.25 mm, 0.3 mm, and 0.4 mm voxel sizes, respectively). These scanning conditions are routinely used in \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Technologies**} & \multirow{2}{*}{**Voxel Size (\(\upmu\)m)**} & **Effective Radiation Dose (\(\upmu\)Sv)** & \multirow{2}{*}{**Scan Time (Second)**} \\  & & & \\ \hline DXA & 500 & 1–20 & \(\sim\)120 \\ MDCT & 156–500 & 100–8000 & \(<\)30 \\ CBCT & 130–400 & 6.3–2100 & 10–40 (Rotation) \\ Micro-CT & 0.3–100 & NA & \(>\)600 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Descriptive summary of X-ray based technologies [11]. Figure 1: (**a**) Strong positive correlations in the calibration curves of gray values for (**b**) phantoms of bone materials (hydroxyapatite) with three different densities (1000, 1250, and 1750 mg/cm\({}^{3}\)) scanned using three different resolutions (200, 300, and 400 \(\upmu\)m) of CBCT (Reprinted with unrestricted permission for non-commercial use) [11]. clinical practices. The exclusion criteria for the CBCT images were craniofacial anomalies, vertebral anomalies (fusions and major vertebral asymmetries), orthognathic patients with bone plates, and limited field of view images without second and third cervical vertebrae. Three age groups including 40-age group (20 to 49 years old, 36.61 \(\pm\) 11 years), 50-age group (50 to 59 years old, 54.55 \(\pm\) 2.94 years), and 60-age group (older than 60 years old, 64.81 \(\pm\) 2.82 years) were assigned for male (21 images for each age group) and female (29 images for 40-age group, 24 images for 50-age group, and 20 images for 60-age group). The sample size was determined using a previous clinical observation that showed the significant changes of CBCT based cervical vertebral mean gray levels with aging (1948.31 \(\pm\) 81.87 vs. 1997.26 \(\pm\) 50.03) [18]. Fifteen CBCT images turned out to provide the minimum number of samples needed to obtain significant results (_p_ < 0.05) with 80% statistical power. Therefore, the current number of CBCT images used for each age group was assumed to be sufficient to satisfy the power of statistical analysis. The 3D CBCT images were imported to image-analysis software (Image), NIH (Figure 2a), and two cervical vertebrae (C2 and C3) were digitally cropped and saved to individual image files. Non-bone voxels outside of vertebra were removed using semi-automatic heuristic algorithm [16; 18; 23]. Posterior and lateral processes were cut from 10 voxels away from the endplates of vertebral body producing an integral volume of vertebral body. A gray level of each voxel, which is proportional to BMD, was obtained (Figure 2e) and collected for its histogram of C2 and C3 (Figure 2f,g). The values of Mean, standard deviation (SD), and low and high gray levels at the 5 th and the 95 th percentiles (Low\({}_{5}\) and High\({}_{5}\)) of voxel counts in the histogram were determined (Figure 2f). Figure 2: Cont. Morphology of C2 and C3 were measured at a single mid-sagittal CBCT slide. The CBCT images were aligned at the orientation of the cervical vertebrae using imaging software (Dataviewer, Bruker, Kontich, Belgium).