Optimal Contrast Agent Staining of Ligaments and Tendons for X-Ray Computed Tomography

Abstract

X-ray computed tomography has become an important tool for studying the microstructures of biological soft tissues, such as ligaments and tendons. Due to the low X-ray attenuation of such tissues, chemical contrast agents are often necessary to enhance contrast during scanning. In this article, the effects of using three different contrast agents—iodine potassium iodide solution, phosphotungstic acid and phosphomolybdic acid—are evaluated and compared. Porcine anterior cruciate ligaments, patellar tendons, medial collateral ligaments and lateral collateral ligaments were used as the basis of the study. Three samples of each of the four ligament/tendon types were each assigned a different contrast agent (giving a total of twelve samples), and the progression of that agent through the tissue was monitored by performing a scan every day for a total period of five days (giving a total of sixty scans). Since the samples were unstained on day one, they had been stained for a total of four days by the time of the final scans. The relative contrast enhancement and tissue deformation were measured. It was observed that the iodine potassium iodide solution penetrated the samples fastest and caused the least sample shrinkage on average (although significant deformation was observed by the time of the final scans), whereas the phosphomolybdic acid caused the greatest sample shrinkage. Equations describing the observed behaviour of the contrast agents, which can be used to predict optimal staining times for ligament and tendon X-ray computed tomography, are presented.

Phosphotungstic acid‐enhanced microCT: Optimized protocols for embryonic and early postnatal mice

“…Our protocol improves upon existing procedures by addressing a number of potentially problematic issues with contrast‐enhanced microCT. Numerous studies have reported PTA to be a less than ideal staining option, particularly as compared with iodine, due to a slower diffusion time through tissues, requiring a longer staining time . As a larger molecule, PTA will necessarily require longer staining protocols to permit full penetration into deeper tissue structures.…”

“…One recent study suggests that the high degree of tissue shrinkage may be related to a delay between specimen collection and staining, noting that large Alligator and Gallus specimens that were immediately fixed and stained following euthanasia did not show evidence of soft tissue shrinkage in the cranium . Balint et al reported generalized soft tissue shrinkage with morphology altering deformation in iodine‐stained porcine ligaments, occurring to a greater extent relative to ligaments stained with either PTA or PMA . Even more recent protocols that involve a tissue‐stabilization step prior to iodine staining still note observable tissue shrinkage, which may affect morphological analyses …”

“…The concentration of the PTA staining solution likely plays a role in the degree of tissue shrinkage. Using a 10% w/v PTA solution in water to examine porcine tendon resulted in tissue shrinkage of up to 20%, exceeding shrinkage rates for aqueous iodine, although the authors acknowledged that some degree of shrinkage may have resulted from drying that occurred during scanning . The unusually high concentration of PTA may also have played a role, as the concentration of iodine has been found to contribute to tissue shrinkage.…”

Three-dimensional visualisation of soft biological structures by X-ray computed micro-tomography

“…1. Recently, there have been substantial advances in microCT technology, both in terms of the types of scanner available, which now allow sub-micron resolution, capture of phase-contrast information and faster scan acquisition, and in the development of imaging protocols (Withers, 2007;Stock, 2008;Bravin et al, 2013;Maire and Withers, 2014;Shearer et al, 2014;Balint et al, 2016). Collectively, these advances can be exploited to image soft (non-calcified) tissues.…”

Three-dimensional imaging of internal tooth structures: Applications in dental education

“…Prior to µCT scanning at 17 µm isotropic voxel resolution, the entire organism of about 6 cm in length was immersed for four weeks in an ethanol solution with added phosphotungstic acid [83]. It would be of great interest to further explore the usefulness of this µCT-based approach for the 3D visualization of the pulp cavity and all soft tissues surrounding the human tooth, in particular since new protocols for the staining of specific tissues are now increasingly becoming available [84,85].…”