Image analysis pipeline for segmentation of a biological porosity network, the lacuno-canalicular system in stingray tesserae.

A prerequisite for many analysis tasks in modern comparative biology is the segmentation of 3-dimensional (3D) images of the specimens being investigated (e.g. from microCT data). Depending on the specific imaging technique that was used to acquire the images and on the image resolution, different segmentation tools are required. While some standard tools exist that can often be applied for specific subtasks, building whole processing pipelines solely from standard tools is often difficult. Some tasks may even necessitate the implementation of manual interaction tools to achieve a quality that is sufficient for subsequent analysis. In this work, we present a pipeline of segmentation tools that can be used for the semiautomatic segmentation and quantitative analysis of voids in tissue (i.e. internal structural porosity). We use this pipeline to analyze lacuno-canalicular networks in stingray tesserae from 3D images acquired with synchrotron microCT.•The first step of this pipeline, the segmentation of the tesserae, was performed using standard marker-based watershed segmentation.•The efficient processing of the next two steps, that is, the segmentation of all lacunae spaces belonging to a specific tessera and the separation of these spaces into individual lacunae required recently developed, novel tools.•For error correction, we developed an interactive method that allowed us to quickly split lacunae that were accidentally merged, and to merge lacunae that were wrongly split.•Finally, the tesserae and their corresponding lacunae were subdivided into structural wedges (i.e. specific anatomical regions) using a semi-manual approach. With this processing pipeline, analysis of a variety of interconnected structural networks (e.g. vascular or lacuno-canalicular networks) can be achieved in a comparatively high-throughput fashion. In our study system, we were able to efficiently segment more than 12,000 lacunae in high-resolution scans of nine tesserae, providing a robust data set for statistical analysis.

Co-aligned chondrocytes: Zonal morphological variation and structured arrangement of cell lacunae in tessellated cartilage.

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 functions 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 and that cells maintain similar volume even when they have been incorporated into tesserae. Our findings support 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. The lacunae 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, and that pores may contain a nutrient source. We propose 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 elongated 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.