Fig. 7. From bone loss to bone remodelling and fracture repair.

Mixed-cell organoids were investigated following 23 days of total culture, including 5 days inside a simulated microgravity vessel under two rotating conditions (simulated Micro G and Normal G Dynamic). a When imaged using brightfield microscopy, areas of significant bone loss (red squares) can be observed at various locations adjacent to the organoid surface in both conditions. Large tubular projections (black arrows) can be observed emerging in both conditions from the original organoid and into the surrounding fibrin matrix. Some dense mineral-tubular structures (T) are seen forming from the surface (top panel, white dotted lines). These projections carry mineral (red arrows) to sites away from the main structure. These structures can reach ~1 millimetre, as observed in this simulated microgravity-cultured construct (b). These large projections were chemically mapped at high resolution using Micro-X-Ray Fluorescence and were shown to contain a mixture of protein and mineral content (c, d), where the elements Sulphur, Phosphorus and Calcium were co-localised in both conditions along the length of these tubule formations (d). e Whole maps of constructs revealed intricate networks of mineralised matrix projections also containing cells in both conditions (coloured arrows) and differences could be assessed morphologically. In these particular examples selected, a construct from the normal gravity condition (top) contains a higher number of projections compared to the simulated microgravity construct (bottom), which contains a main tubular structure forming into the matrix, but which displays many potentially emerging structures, based on the distribution of potassium (K), in networks surrounding the initial structure. Molecularly, a non-quantitative protein assessment of constructs revealed that the Sclerostin and RANKL presence was pronounced within each condition compared to that of the endocrine Parathyroid Hormone Receptor 1 and to the shear-responsive Connexin 43 channels (f). The presence of PTHR1 in the modelled microgravity group was not clearly detected, however, it was present in the normal gravity condition, which may indicate differences in the regulatory pathway controlling the bone remodelling process between the two contexts.