Novel image registration approach for combining 2D Osterix and collagen bundles images with 3D micro-CT.

To study bone healing, different imaging techniques are typically employed. Histological and immunohistological methods allow the visualization of Osterix (OSX) expression and collagen structures, whereas μCT imaging enables the assessment of mineralized tissue. However, a clear spatial alignment between the information obtained from these 2 modalities is still lacking. In this study, we present a technological approach for registering 2D histological sections of collagen bundles and OSX signals with 3D volumetric μCT data. We applied this method to datasets from animals with rigid and semi-rigid fracture fixation to resemble fast and effective healing and delayed healing. Using our 2D-3D registration workflow, we were able to identify corresponding 2D μCT and histological slices, and we showed that the algorithm performed consistently across both fixation conditions, resembling the healing consequences. Thus, we could illustrate how such image registration techniques could be used to study the co-localization of OSX, collagen, and hydroxyapatite (HA). This framework enables the visualization and direct comparison of OSX, collagen, and HA within a single, spatially matched image, providing a tool for future studies to quantitatively explore tissue co-localization and spatial relationships during bone healing.