Directional bone matrix mineralization in the CAM assay is governed by vascular integration and matrix remodeling

Bone matrix mineralization plays an important role in maintaining bone health but is a highly dynamic process for which few physiologically relevant model systems exist. The chorioallantoic membrane (CAM) assay is one such assay and has been used to study biomineralization; however, the mechanisms controlling mineral deposition in the CAM assay remain poorly understood. Here, we implanted decellularized, organic bone matrices onto the CAM and investigated their mineralization using a combination of micro-computed tomography, Raman spectroscopy, scanning electron microscopy, and histochemistry. These studies revealed increased mineralization and microvessel density on the shell-facing interface of the implanted matrices, while their embryo-facing counterpart lacked mineralization but was densely infiltrated by cells. Seeding organic bone matrices with mesenchymal stromal cells (MSCs) prior to CAM-implantation prevented mineralization by altering matrix micro- and nanostructure and limiting vessel integration. Collectively, our results suggest that mineral precursors from the eggshell and vasculature combine to mineralize organic bone matrix in the CAM assay, while proteolytic remodeling by invaded or implanted stromal cells inhibits that process. Our findings provide critical new insights into the interplay between acellular and cellular drivers of bone matrix mineralization, and will inform future studies of biomineralization using the CAM assay.