3D characterization of morphological changes in the intervertebral disc and endplate during aging: A propagation phase contrast synchrotron micro-tomography study

Abstract

A better understanding of functional changes in the intervertebral disc (IVD) and interaction with endplate is essential to elucidate the pathogenesis of IVD degeneration disease (IDDD). To date, the simultaneous depiction of 3D micro-architectural changes of endplate with aging and interaction with IVD remains a technical challenge. We aim to characterize the 3D morphology changes of endplate and IVD during aging using PPCST. The lumbar vertebral level 4/5 IVDs harvested from 15-day-, 4- and 24-month-old mice were initially evaluated by PPCST with histological sections subsequently analyzed to confirm the imaging efficiency. Quantitative assessments of age-related trends after aging, including mean diameter, volume fraction and connectivity of the canals, and endplate porosity and thickness, reached a peak at 4 months and significantly decreased at 24 months. The IVD volume consistently exhibited same trend of variation with the endplate after aging. In this study, PPCST simultaneously provided comprehensive details of 3D morphological changes of the IVD and canal network in the endplate and the interaction after aging. The results suggest that PPCST has the potential to provide a new platform for attaining a deeper insight into the pathogenesis of IDDD, providing potential therapeutic targets.

Figure 1. Corresponding sagittal and coronal sections acquired using conventional micro-CT (C-μCT) and propagation phase contrast synchrotron micro-tomography (PPCST).

(A) The PPCST reconstructed images show a markedly superior soft tissue contrast of the intervertebral disc (purple) as well as the delineation of the endplate (green) compared to the C-μCT images. The canals in the endplate could be detected in both C-μCT and PPCST images. (B) A representative intensity profile across the dashed line drawing in the images in (A). Scale bar = 1 mm. VB = vertebral body, EP = endplate, GP = growth plate, IVD = intervertebral disc.

Figure 2. Comparison of the surface rendering images of the sagittal and coronal sections acquired with PPCST with images of histological samples.

(A and B) Virtual sagittal and coronal sections acquired using PPCST. Canals located in the endplate and disc were clearly visualized. (C and D) Corresponding section images with Saf-O staining. Scale bar = 1 mm. VB = vertebral body, EP = endplate, GP = growth plate, IVD = intervertebral disc.

Figure 3. Representative 3D images of the canals in the endplate and IVD and corresponding pseudo-color images obtained by PPCST.

(A) Intact 3D morphology of the lumbar functional unit. (B) 3D images of the canals, endplate, canals located in endplate and IVD. (C) Original and pseudo-colored images of the 3D canal network in the cranial and caudal endplates. (D) Original and pseudo-colored images of the upper and lower surfaces of the IVD. The pseudo-color bar in the lower-right corner indicates the diameter ranges of the canals in the endplate or the IVD thickness distribution.

Figure 4. Virtual micro-endoscopy of the canals in the endplate.

(A) Lateral view of the 3D canal network extracted from the endplate. (B) The inner surface of the 3D canal network is clearly present. (C) Virtual navigation within the multiple levels of the internal surface of the canal. (A) and (B) scale bar = 1 mm, (C) scale bar = 500 μm.

Figure 5. 3D digitalized map quantitative information of the canal network for various ages.

(A) Lateral view of 3D image of the canal network in the endplate after aging obtained using PPCST with the cranial and caudal endplates separately displayed. The endplate was divided into three different regions, anterior, center and posterior. (B) Representative image of the corresponding histology sections of the cranial and caudal endplates for various ages. (C) Quantitative analysis of the morphological parameters in the cranial and caudal endplates in three regions after aging. Scale bar = 1 mm; A = anterior; C = center; P = posterior. Data are the means ± S.D., n = 8 mice in each group. In panel (B), a indicates a significant difference between the posterior region and the center region, and b indicates a significant difference between the posterior region and the anterior region (p < 0.05, determined by two-way repeated measures ANOVA with Bonferroni’s post hoc test).

Figure 6. 3D characterization of the endplate at different ages.

(A) Vertical view of the 3D pseudo-color image of the endplate among mice of different ages. (B) Quantification of the endplate thickness and porosity after aging. A = anterior; C = center; P = posterior. The pseudo-color bar in the lower-right corner indicates the diameter ranges of the endplate thickness. Data are the means ± S.D., n = 8 mice in each group. In panel (B), a indicates a significant difference between the posterior region and the center region, and b indicates a significant difference between the posterior region and the anterior region (p < 0.05, determined by two-way repeated measures ANOVA with Bonferroni’s post hoc test).

Figure 7. 3D characterization of IVD at different ages.

(A) Vertical view of the 3D images of the IVD from mice of different ages. (B) Corresponding pseudo-color image coding with the IVD thickness; the upper and lower surfaces of the IVD are separately displayed. The pseudo-color bar in the lower right corner indicates the diameter ranges of IVD thickness. The blue color represents the largest diameter, whereas the red indicates the smallest diameter. (C) Quantitative analysis of changes in volume and thickness distribution in anterior, center and posterior regions and of IVD for different ages. Scale bar = 1 mm, A = anterior; C = center; P = posterior. In panel (B), a indicates a significant difference between the anterior region and the posterior region, and b indicates a significant difference between the anterior region and the posterior region (p < 0.05, determined by two-way repeated measures ANOVA with Bonferroni’s post hoc test).

Figure 8. Representative PPCST image of a canal network in the endplate and IVD in a plane view.

The vertical view was then further divided into anterior, central, and posterior areas. Five regions of interest were randomly selected to determine the average canal diameter, volume fraction and connectivity, and endplate porosity and thickness in each of these regions. The IVD thickness distribution was also calculated for these three regions. The pseudo-color bar in the lower-right corner indicates the diameter ranges of the canals in the endplate or the IVD thickness distribution.