Centimeter-sized biomimetic bone constructs fabricated via CBD-BMP2-collagen microcarriers and BMSC-gelatin microspheres.

To advance the "bottom-up" approaches for bone tissue engineering, we developed rat bone mesenchymal stem cell (BMSC)-gelatin microspheres (BGMs) as cell-culture modules and collagen binding domain-bone morphogenetic protein2 (CBD-BMP2)-collagen microcarriers (CCMs) as differentiation-control modules. BMSCs were efficiently seeded onto gelatin microspheres in a spinner flask to form BGMs with a high cell density. CBD-BMP2 was recombined and bound with collagen microfibers to form CCMs, in which BMP2 was controllably released. BGM and CCM were assembled as building blocks to fabricate macroscopic bone-like constructs in vitro and in vivo, where BMSCs were directly induced to differentiate into osteocytes. The osteogenic differentiation of BMSCs was confirmed with increasing mineral deposition (alkaline phosphatase stain, alizarin red stain, calcium content and micro-CT), gene expression of osteogenic markers (alkaline phosphatase, bone sialoprotein, osteocalcin and collagen type I) as well as alkaline phosphatase activity. The in vivo formed bone-like tissues had the compression modulus of 2.62 ± 1.41 MPa, similar to that in the in vivo bone tissues, much higher than those in BGM samples of 0.03 ± 0.012 MPa. Our results suggesting these proof-of-concept strategies, assembling cell-culture modules and function-control modules to modularly engineer clinical-relevant large tissue replacements, have great potential applications in tissue engineering.