Fig. 6: Encapsulation of trabecular organoids into biochemical microenvironments that can generate bone physiological or pathological prototypes.

a and b present two examples each of constructs that were encapsulated into a physiological (human fibrin) and tumorigenic environment (Matrigel^®). Constructs that were encapsulated in fibrin (day 19) create extensive projections (orange arrows) into the surrounding environment (a-top), which stretch from multiple points on the surface (middle). These constructs are heavily populated with cells (bottom). Constructs embedded in Matrigel^® (day 16) also display highly proliferative cells, however, the pattern and morphology of these cells is more representative of a tumouroid, as they are densely packed into atypical phenotypes (pink arrows). These lobular proliferative regions have been delimited with a dotted white line for easier visualisation. c The chemical composition of fibrin-embedded constructs and nature of projections was probed using X-ray fluorescence mapping to allow for spatial detection of new features, with different elements coding for distinct anatomical structures. The projections emerging from the construct surface (grey arrows and black arrows) contain a large amount of Phosphorus (P). Calcium (Ca) is also present in the newly forming structures. Potassium (K), an intracellular ion present in cells is seen within these dendritic structures (pink arrows) and co-localised with the Phosphorus deposits, however, discrete deposits of P are found on their own surrounding the K structures (green arrows). Areas rich in Sulphur (S) are also seen localised to the regions where projections and matrix are forming (white arrows). Organoids were scanned with Micro-CT as a fused mass (d) to detect compositional gradients based on density. Owing to the dense nature of the trabecula, organoids behave like bone tissue when subjected to X-Ray radiation and as such, display selective attenuation at the surface (the beam hardening effect). Therefore, beam artefacts were removed and a function was applied to remove the lower density ranges of data (e). White arrows show discrete pockets of a denser (blue) material in different constructs in the near vicinity or emerging from the very dense (red) surface, and trapped within the lowest density (green), surrounding fibrin material. Scale bars as indicated.