HIGH-RESOLUTION 3D IMAGING OF BOVINE TAIL INTERVERTEBRAL DISC DEGENERATION USING IODINE-ENHANCED X-RAY MICROSCOPY

Abstract

INTRODUCTION

The vertebral endplates of the intervertebral disc (IVD) consist of two structurally distinct layers: the cartilaginous endplate (CEP) and the bony endplate (BEP). While most research on IVD degeneration has focused on the biochemical or biomechanical failures of the annulus fibrosus (AF) and nucleus pulposus (NP), the physiology and microstructure of the CEP have often been overlooked. To address this gap, we employed iodine-enhanced X-ray microscopy (XRM) in a bovine tail IVD degeneration model. This approach enabled the simultaneous visualization of soft and hard tissues, with a specific focus on the CEP.

OBJECTIVE

1) To simultaneously visualize soft and hard tissues in IVDs, with a specific focus on detecting changes in the CEP using iodine-enhanced XRM. 2) To validate the observed structural changes through histological analysis.

METHODS

34 IVDs with intact vertebral endplates were harvested from six fresh bovine tails. Samples were cultured in Dulbecco’s Modified Eagle Medium (DMEM) for 11 days under unloaded conditions. On day one, approximately 70-100 μL of chondroitinase ABC (chABC, 0.5 U/mL), a pro-inflammatory cytokine cocktail containing interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) (each at 100 ng/mL), or a sham control solution of phosphate-buffered saline (PBS) with 0.1% bovine serum albumin (BSA) was injected into the NP using a 21G needle. Additional control samples received no injection. On day 11, IVDs were fixed in 4% formaldehyde and dehydrated. Samples were immersed in 1% (w/v) iodine (I₂) in 100% ethanol and stained for a minimum of two weeks. Following staining, samples were washed, embedded in 1% agarose, and imaged with an XRM (Zeiss Xradia Versa 610; source voltage: 60kV; exposure: 4-6 sec; voxel size: 9.9-15.6 µm). Post-imaging, iodine was removed, and samples were decalcified and paraffin-embedded. Thin sections (7-10 µm) were prepared and stained with hematoxylin and eosin (H&E) and safranin-O and fast green. Reconstituted XRM image stacks were processed using built-in noise filtering software (Zeiss). Dragonfly 3D world (Comet) software was used for visualization and segmentation. XRM images were qualitatively compared with histological sections to assess changes in soft and hard tissues (Figures 1 and 2).

RESULTS

The interface between mineralized and non-mineralized cartilage (tidemark) was visualized using XRM, enabling the identification of calcified cartilage and CEP (Figure 1). Iodine-based contrast provided sufficient resolution to detect structural malalignments among the BEP, CEP, and NP (Figure 2). Notably, even sham injections with PBS induced degenerative changes in the disc.

CONCLUSION

Non-destructive iodine-enhanced XRM enables clear visualization of the CEP, providing sufficient contrast to simultaneously assess structural changes in both soft and hard tissues. This approach offers a powerful tool for evaluating IVD degeneration on ex vivo models.