Developmental Biology-Inspired Strategies To Engineer 3D Bioprinted Bone Construct

A major challenge in bone tissue engineering is to develop clinically conformant load-bearing bone constructs in a patient-specific manner. A paradigm shift would involve combination of developmental engineering and 3D bioprinting to optimize strategies focusing on close simulation of in vivo developmental processes using in vitro tissue engineering approaches. This study demonstrates that silk-gelatin bioink could activate the canonical Wnt/β-catenin and Indian hedgehog (IHH) pathways during osteogenic differentiation of mesenchymal stem cells (TVA-BMSC), laden in 3D bioprinted constructs. Temporal gene expression related to early and terminal osteogenic differentiation of the TVA-BMSC in 3D bioprinted constructs closely followed the in vivo processes. This was evidenced by expression of early differentiation markers (RUNX2 and COL I), mid- and mid-to-late-stage markers (ALP, ON, OPN, and OCN), and terminal osteocytic genes (PDPN, DMP1, and SOST). Furthermore, a combinatorial effect of addition of T3 and simulation of the endochondral ossification route could activate the parathyroid hormone (PTH), IHH, and Wnt/β-catenin pathways, thus improving the osteogenic differentiation potential of stem cells and improved mineralization. The endochondral ossification observed in vitro in our study shows stark similarities to in vivo endochondral ossification-based limb skeletal development, specifically (1) chondrogenic condensation and hypertrophic cartilaginous template development, (2) involvement of IHH signaling indicative of the development of bony collar by perichondral ossification, (3) involvement of Wnt/β-catenin signaling, (4) involvement of PTH signaling, and (5) synthesis and deposition of bone-specific mineral. Thus, induction of differentiation of progenitor cells to osteoblasts in 3D bioprinted constructs, while recapitulating the developmental-biology-inspired endochondral ossification route, may offer an important therapeutic proposition to develop clinically conformant bone construct.