Scaffold-free human mesenchymal stem cell construct geometry regulates long bone regeneration

Scaffold-based bone tissue engineering approaches frequently induce repair processes dissimilar to normal developmental programs. In contrast, biomimetic strategies aim to recapitulate aspects of development through cellular self-organization, morphogenetic pathway activation, and mechanical cues. This may improve regenerative outcome in large long bone defects that cannot heal on their own; however, no study to date has investigated the role of scaffold-free construct geometry, in this case tubes mimicking long bone diaphyses, on bone regeneration. We hypothesized that microparticle-mediated in situ presentation of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) to engineered human mesenchymal stem cell (hMSC) tubes induces the endochondral cascade, and that TGF-β1 + BMP-2-presenting hMSC tubes facilitate enhanced endochondral healing of critical-sized femoral segmental defects under delayed in vivo mechanical loading conditions compared to loosely-packed hMSC sheets. Here, localized morphogen presentation imparted early chondrogenic lineage priming, and stimulated robust endochondral differentiation of hMSC tubes in vitro. In an ectopic environment, hMSC tubes formed a cartilage template that was actively remodeled into trabecular bone through endochondral ossification without lengthy predifferentiation. Similarly, hMSC tubes stimulated in vivo cartilage and bone formation and more robust healing in femoral defects compared to hMSC sheets. New bone was formed through endochondral ossification in both groups; however, only hMSC tubes induced regenerate tissue partially resembling normal growth plate architecture. Together, this study demonstrates the interaction between mesenchymal cell condensation geometry, bioavailability of multiple morphogens, and defined in vivo mechanical environment to recapitulate developmental programs for biomimetic bone tissue engineering.Significance StatementEngineered bone constructs must be capable of withstanding and adapting to harsh conditions in a defect site upon implantation, and can be designed to facilitate repair processes that resemble normal developmental programs. Self-assembled tubular human mesenchymal stem cell constructs were engineered to resemble the geometry of long bone diaphyses. By mimicking the cellular, biochemical, and mechanical environment of the endochondral ossification process during embryonic development, successful healing of large femoral segmental defects upon implantation was achieved and the extent was construct geometry dependent. Importantly, results were obtained without a supporting scaffold or lengthy predifferentiation of the tubular constructs. This indicates that adult stem/progenitor cells retain features of embryonic mesenchyme, and supports the concept of developmental engineering for bone regeneration approaches.