Multifactorial bottom-up bioengineering approaches for the development of living tissue substitutes

Bottom-up bioengineering utilizes the inherent capacity of cells to build highly sophisticated structures with high levels of biomimicry. Despite the significant advancements in the field, monodomain approaches require prolonged culture time to develop an implantable device, usually associated with cell phenotypic drift in culture. Herein, we assessed the simultaneous effect of macromolecular crowding (MMC) and mechanical loading in enhancing extracellular matrix (ECM) deposition while maintaining tenocyte (TC) phenotype and differentiating bone marrow stem cells (BMSCs) or transdifferentiating neonatal and adult dermal fibroblasts toward tenogenic lineage. At d 7, all cell types presented cytoskeleton alignment perpendicular to the applied load independently of the use of MMC. MMC enhanced ECM deposition in all cell types. Gene expression analysis indicated that MMC and mechanical loading maintained TC phenotype, whereas tenogenic differentiation of BMSCs or transdifferentiation of dermal fibroblasts was not achieved. Our data suggest that multifactorial bottom-up bioengineering approaches significantly accelerate the development of biomimetic tissue equivalents.