Fig. 1

Bioengineering of a human osteoblast-derived microtissue and characterization. a Scaffold fabrication process using melt electrowriting and medical-grade polycaprolactone. Adapted with copyright from Farrugia et al.¹⁵ b Schematic of human primary osteoprogenitor cell seeding on a calcium phosphate-treated scaffold. The light micrograph shows scaffold fibers (arrow) and cell organization 1 day post seeding (arrow head). The culture of the cellular construct for at least 7 weeks leads to a human osteoblast-derived mineralized microtissue (hOBMT) containing live osteoblastic (hOB) and osteocytic cells (hOS), bone extracellular matrix (ECM) and hydroxyapatite (HA) mineralized nodules. c DAPI (blue) and Phalloidin (green) staining and confocal imaging (Max Proj image, 19 µm z-stack) of the hOBMT shows high cellular organization and strong directional actin filaments. d Live/dead FDA (green) and PI (red) staining and confocal imaging (Max Proj image, 100 µm z-stack) of hOBMT shows > 80% cell viability after 10 weeks in culture. e SEM imaging shows dense ECM deposition (asterisk), osteoblastic cells (arrow head), and osteocytic cells (inset). f Hematoxylin and eosin (H&E) staining on histology sections shows internal microtissue morphology, comprised of fibers (open arrows), osteocytic cells, surrounded by their lacunae (arrow head and dashed lines, respectively) and connective tissue (asterisk). g Gene expression of the hOBMT normalized to 2D cultures by RT-qPCR (N = 3, means ± SE) after 10 weeks of culture (*P < 0.05, **P < 0.01) with h heat map of ΔCq mean values. i Protein expression shows more and higher amounts of proteins (Angiogenin, thrombospondin 1 (TSP-1), vascular cell adhesion protein 1 (VCAM1), insulin-like growth factor-binding protein (IGFB-2)) expressed in the hOBMT compared to 2D hOBs (relative pixel intensity from protein microarray membranes) by heat map of means from two donors (#1, #2). j Confocal images of immunostained hOBMT for collagen-I (green) and sclerostin (green) and DAPI (blue), (Max Proj image, 50 µm z-stack)