Mineral tessellation in mouse enthesis fibrocartilage, Achilles tendon, and Hyp calcifying enthesopathy: A shared 3D mineralization pattern

juin 22, 2023

Daniel J. Buss (1), Katya Rechav (2), Natalie Reznikov (1) (3) (4), Marc D. McKee (1) (4)
Bone. Volume 174 (June 2023). DOI: https://doi.org/10.1016/j.bone.2023.116818


Keywords

Enthesis, mineralization, X-linked hypophosphatemia, Hyp mice, mineral tessellation, fibrocartilage, tendon, PHEX, osteopontin


Abstract

The hallmark of enthesis architecture is the 3D compositional and structural gradient encompassing four tissue zones – tendon/ligament, uncalcified fibrocartilage, calcified fibrocartilage and bone. This functional gradient accommodates the large stiffness differential between calcified bone and uncalcified tendon/ligament. Here we analyze in 3D the organization of the mouse Achilles enthesis and mineralizing Achilles tendon in comparison to lamellar bone. We use correlative, multiscale high-resolution volume imaging methods including μCT with submicrometer resolution and FIB-SEM tomography (both with deep learning-based image segmentation), and TEM and SEM imaging, to describe ultrastructural features of physiologic, age-related and aberrant mineral patterning. We applied these approaches to murine wildtype (WT) Achilles enthesis tissues to describe in normal calcifying fibrocartilage a crossfibrillar mineral tessellation pattern similar to that observed in lamellar bone, but with greater variance in mineral tesselle morphology and size. We also examined Achilles enthesis structure in Hyp mice, a murine model for the inherited osteomalacic disease X-linked hypophosphatemia (XLH) with calcifying enthesopathy. In Achilles enthesis fibrocartilage of Hyp mice, we show defective crossfibrillar mineral tessellation similar to that which occurs in Hyp lamellar bone. At the cellular level in fibrocartilage, unlike in bone where enlarged osteocyte mineral lacunae are found as peri-osteocytic lesions, mineral lacunar volumes for fibrochondrocytes did not differ between WT and Hyp mice. While both WT and Hyp aged mice demonstrate Achilles tendon midsubstance ectopic mineralization, a consistently defective mineralization pattern was observed in Hyp mice. Strong immunostaining for osteopontin was observed at all mineralization sites examined in both WT and Hyp mice. Taken together, this new 3D ultrastructural information describes details of common mineralization trajectories for enthesis, tendon and bone, which in Hyp/XLH are defective.


How Our Software Was Used

Mineralized regions and structures were segmented using the deep-learning engine in Dragonfly.


Author Affiliation

(1) Department of Anatomy and Cell Biology, School of Biomedical Sciences, Faculty of Medicine and Health Sciences, McGill University, Montreal, Quebec, Canada
(2) Electron Microscopy Unit, Weizmann Institute of Science, Rehovot, Israel
(3) Department of Bioengineering, Faculty of Engineering, McGill University, Montreal, Quebec, Canada
(4) Faculty of Dental Medicine and Oral Health Sciences, McGill University, Montreal, Quebec, Canada