Vertebral bone microarchitecture and osteocyte characteristics of three toothed whale species with varying diving behaviour

Abstract

Although vertebral bone microarchitecture has been studied in various tetrapods, limited quantitative data are available on the structural and compositional changes of vertebrae in marine mammals. Whales exhibit exceptional swimming and diving behaviour, and they may not be immune to diving-associated bone pathologies. Lumbar vertebral bodies were analysed in three toothed whale species: the sperm whale (Physeter macrocephalus), orca (Orcinus orca) and harbour porpoise (Phocoena phocoena). The bone volume fraction (BV/TV) did not scale with body size, although the trabeculae were thicker, fewer in number and further apart in larger whale species than in the other two species. These parameters had a negative allometric scaling relationship with body length. In sperm whales and orcas, the analyses revealed a central ossification zone ("bone-within-bone") with an increased BV/TV and trabecular thickness. Furthermore, a large number of empty osteocyte lacunae was observed in the sperm whales. Quantitative backscattered electron imaging showed that the lacunae were significantly smaller and less densely packed. Our results indicate that whales have a unique vertebral bone morphology with an inside-out appearance and that deep diving may result in a small number of viable osteocytes because of diving depth-related osteocyte death.

Figure 1

Bone morphometry and scaling relationship of L1. (a) Three-dimensional reconstruction of the lumbar vertebral body L1 of the harbour porpoise (p), orca (o) and sperm whale (s). (b) Overview of the bone microstructure of the three whale species. Images obtained by backscattered electron microscopy (BSEM). (c) Comparative histomorphometric analyses revealed significant differences between the three whale species, with the sperm whales showing the thickest, fewest and most separated trabeculae and the orca showing the highest bone volume fraction. *P < 0.05. (d) Trabeculae scale with body size and show a negative allometric relationship for Tb.Th, Tb.N and Tb.Sp but not BV/TV. (e) Anterior vertebral body height and bone structure parameters present the same negative allometric scaling relationships.

Figure 2

Central bone densification (bone-within-bone) in orca and sperm whale. (a) Orcas and sperm whales show a central ossification zone with contact radiography (midsagittal slice of 0.8 cm). Note the dimensions of the vertebral bodies. (b) Punch biopsies from endplate to endplate were taken. In the orca (o) and sperm whales (s), further quantification of the different regions of interest (red boxes) using HR-pQCT revealed an increased bone volume fraction (BV/TV) with thicker trabeculae (Tb.Th) in the central parts, and it corresponded to the “bone-within-bone” structure seen in radiographies. (c) Representative images of the scanned subregions in HR-pQCT indicate that the trabecular network was denser in the central parts.

Figure 3

Histology of the vertebral bodies. (a) In peripheral zones, the trabeculae were thinner and more separated. (b) In the centre of the vertebral body of orcas and sperm whales, the trabeculae converged to a dense structure with a compact appearance that included osteonal structures (Masson Goldner staining, 50x magnification). Harbour porpoise vertebral bone did not show this phenomenon.

Figure 4

Quantitative backscattered electron imaging (qBEI) of the trabecular bone. (a) Pseudo-coloured images of the trabecular bone. (b–f) CaMean, CaPeak, CaLow and CaHigh did not differ between the groups, indicating a similar mineralisation pattern, whereas CaWidth was increased significantly in sperm whales, which indicates higher mineralisation heterogeneity. (g) Histogram of the bone mineral density distribution (BMDD).

Figure 5

Osteocyte characteristics. (a) Representative images obtained using BSEM show the differences in lacunar number and size. (b) The number of osteocyte lacunae per bone area (N.Ot.Lc/B.Ar) was significantly lower in the sperm whales than that in the other whale species. (c) Osteocyte lacunar area (Lc.Ar) was smaller in the sperm whales. *P < 0.05. (d) Toluidine blue-stained sections of undecalcified bone specimens revealed that the harbour porpoise had several osteocytes with 2 cell nuclei, the orca had typically shaped osteocytes and the sperm whale showed only small and empty lacunae; 400x magnification. (e) Percentage of occupied (viable) lacunae was significantly decreased in the sperm whales. **P < 0.001. (f) Images of the osteocyte lacunar connections performed via acid etching of the acrylate embedded specimens followed by scanning electron microscopy (SEM); the images show a well-developed lacunocanalicular system in the orca (left image), whereas poor connections via canaliculi are observed among osteocytes in the sperm whale bone (right image).