Abstract
Introduction
Histology is still considered the gold standard method for the evaluation of soft tissues in the musculoskeletal field, thanks to the possibility of studying structures using different staining and high magnification microscopy. To overcome the intrinsic limits of this method, contrast enhanced microtomographic (CE- microCT) protocols are constantly evolving to allow 3D study of soft tissues. However, no standardized approaches are available, and many concerns exist about the alterations induced to the samples.
Method
microCT/histology protocols were explored on human tendons and menisci. To enhance contrast tissues for microCT scanning 1) examethyldisilazane drying 2) 2% phosphotungstic acid (PTA) in alcoholic solution exposition and 3) 2% PTA in aqueous solution exposition were performed; to observe PTA contrast progression, three exposition and scanning times were selected. microCT images were compared to histological slices obtained from the same samples, after rehydration protocols, or from adjacent tissues portion, stained with Picrosirius red to highlight the peculiar collagenic structures.
Result
Exposition times influence PTA diffusion and tissue contrast; its specificity for collagenic structure allow a clearer contrast of the tissues. Histological processing on the same samples is possible: PTA removal requires careful washing in basic solution to reduce the hardening of the sample, while drying can be reverted applying inverse protocol. Comparison with microCT images is really accurate if histology is performed on the same sample, although all protocols induce tissue shrinkage with relative packing of collagen fibers.
Conclusion
The contrast approaches tested proved effective in highlighting the structures of both tendons and menisci, but the structural effects induced by tissue shrinkage do not allow a completely real microCT visualization of native tissue. Histology can be the reference method to monitor the efficacy of the contrast methods and the alterations induced to define the possibility of improvement of the technique.
Acknowledgement
PR23-PAS-P4 “ADJOINT 2”- INAIL
