Fig. 8: Evolution of constructs and potential applications of the model.

a Osteoblast constructs that were previously cultured under low-shear conditions for 7 days and subsequently seeded into fibrin matrices, are able to form micro-trabecular shaped soft structures during 3 weeks of culture and to generate nucleation points at sites far away from the initial organoid structure (red squares). This new matrix shows a mixture of protein and inorganic content and a phosphorus-led mineralisation (b). c It is possible to quantitatively assess the degree of new bone formation by measuring the ratios of elements from the initial construct (yellow shape) compared to the overall organoid (green shape) to assess the degree of change. d In these examples from osteoblastic constructs that were either cultured in a static condition (top) or simulated microgravity (bottom) for 7 days and subsequently developed in fibrin for 3 weeks, a rate of remodelling (e) could be calculated from the acquired maps by normalising the average elemental content of the organoid to that of the initial trabecular construct. Alternatively, in the example provided in f (left) from a previous organoid, regions of interest can be compared within or across samples, provided that the surface area selected is constant (f, right). As shown, the differences between Area 1, containing a visibly larger amount of new matrix compared to Area 2, can be successfully translated numerically based on the elemental content. Owing to the small diameter of organoids, constructs can be used with more automated systems to determine the effects of various agents (g). Change can be assessed by recovering the organoids and analysing them volumetrically (individually or as a mass) with technologies such as micro-CT, which facilitate a density-based selection (as exemplified here using a function selecting the highest density mineral, i.e., the initial bone). Further tests can be applied, such as illumination with polarised light, which will vary based on the orientation of crystallographic axes within the construct, linked to the degree of new bone formation. Additional colorimetric and fluorescence-based assays specific to bone tissue can be used in conjunction. Scale bars as indicated.