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In most vertebrates the embryonic cartilaginous skeleton is replaced by bone during development. During this process, cartilage cells (chondrocytes) mineralize the extracellular matrix and undergo apoptosis, giving way to bone cells (osteocytes). In contrast, sharks and rays (elasmobranchs) have cartilaginous skeletons throughout life, where only the surface mineralizes, forming a layer of tiles (tesserae). Elasmobranch chondrocytes, unlike those of other vertebrates, survive cartilage mineralization and are maintained alive in spaces (lacunae) within tesserae. However, the function(s) of the chondrocytes in the mineralized tissue remain unknown. Applying a custom analysis workflow to high-resolution synchrotron microCT scans of tesserae, we characterize the morphologies and arrangements of stingray chondrocyte lacunae, using lacunar morphology as a proxy for chondrocyte morphology. We show that the cell density is comparable in unmineralized and mineralized tissue from our study species and that cells maintain the similar volume even when they have been incorporated into tesserae. This discovery supports previous hypotheses that elasmobranch chondrocytes, unlike those of other taxa, do not proliferate, hypertrophy or undergo apoptosis during mineralization. Tessera lacunae show zonal variation in their shapes—being flatter further from and more spherical closer to the unmineralized cartilage matrix and larger in the center of tesserae— and show pronounced organization into parallel layers and strong orientation toward neighboring tesserae. Tesserae also exhibit local variation in lacunar density, with the density considerably higher near pores passing through the tesseral layer, suggesting pores and cells interact (e.g. that pores contain a nutrient source). We hypothesize that the different lacunar types reflect the stages of the tesserae formation process, while also representing local variation in tissue architecture and cell function. Lacunae are linked by small passages (canaliculi) in the matrix to form elongate series at the tesseral periphery and tight clusters in the center of tesserae, creating a rich connectivity among cells. The network arrangement and the shape variation of chondrocytes in tesserae indicate that cells may interact within and between tesserae and manage mineralization differently from chondrocytes in other vertebrates, perhaps performing analogous roles to osteocytes in bone.
Purpose/Aims of the Study: Bone’s hierarchical structure can be visualized using a variety of methods. Many techniques, such as light and electron microscopy generate two-dimensional (2D) images, while micro computed tomography (μCT) allows a direct representation of the three-dimensional (3D) structure. In addition, different methods provide complementary structural information, such as the arrangement of organic or inorganic compounds. The overall aim of the present study is to answer bone research questions by linking information of different 2D and 3D imaging techniques. A great challenge in combining different methods arises from the fact that they usually reflect different characteristics of the real structure.
Materials and Methods: We investigated bone during healing by means of μCT and a couple of 2D methods. Backscattered electron images were used to qualitatively evaluate the tissue’s calcium content and served as a position map for other experimental data. Nanoindentation and X-ray scattering experiments were performed to visualize mechanical and structural properties. Results: We present an approach for the registration of 2D data in a 3D μCT reference frame, where scanning electron microscopies serve as a methodic link. Backscattered electron images are perfectly suited for registration into μCT reference frames, since both show structures based on the same physical principles. We introduce specific registration tools that have been developed to perform the registration process in a semi-automatic way.
Conclusions: By applying this routine, we were able to exactly locate structural information (e.g. mineral particle properties) in the 3D bone volume. In bone healing studies this will help to better understand basic formation, remodeling and mineralization processes.