More than a century ago, it was proposed that mechanical forces could drive tissue formation. However, only recently with the advent of enabling biophysical and molecular technologies are we beginning to understand how individual cells transduce mechanical force into biochemical signals. In turn, this knowledge of mechanotransduction at the cellular level is beginning to clarify the role of mechanics in patterning processes during embryonic development. In this perspective, we will discuss current mechanotransduction paradigms, along with the technologies that have shaped the field of mechanobiology.

Objective. To investigate whether periosteal cells from adult humans have features of multipotent mesenchymal stem cells (MSCs) at the single-cell level.Methods. Cell populations were enzymatically released from the periosteum of the proximal tibia obtained from adult human donors and then expanded in monolayer. Single-cell-derived clonal populations were obtained by limiting dilution. Culture-expanded periosteal cell populations were tested for their growth potential and for expression of conventional markers of MSCs and were subjected to in vitro assays to investigate their multilineage potential. To assess their multipotency in vivo, periosteal cells were injected into a regenerating mouse tibialis anterior muscle for skeletal myogenesis or were either seeded into an osteoinductive matrix and implanted subcutaneously into nude mice for osteogenesis or implanted in a joint surface defect under a periosteal flap into goats for chondrogenesis. Cell phenotypes were analyzed by histochemistry and immunohistochemistry and by reverse transcriptionpolymerase chain reaction for the expression of lineagerelated marker genes.Results. Regardless of donor age, periosteal cells were clonogenic and could be expanded extensively in monolayer, maintaining linear growth curves over at least 30 population doublings. They displayed long telomeres and expressed markers of MSCs. Under specific conditions, both parental and single-cellderived clonal cell populations differentiated to the chondrocyte, osteoblast, adipocyte, and skeletal myocyte lineages in vitro and in vivo.Conclusion. Our study demonstrates that, regardless of donor age, the adult human periosteum contains cells that, upon enzymatic release and culture expansion, are multipotent MSCs at the single-cell level.

The vascular barrier that separates blood from tissues is actively regulated by the endothelium and is essential for transport, inflammation, and hemostasis1. Hemodynamic shear stress plays a critical role in maintaining endothelial barrier function2, but how this occurs remains unknown. Here, using an engineered organotypic model of perfused microvessels and confirming in mouse models, we identify that activation of the Notch1 transmembrane receptor directly regulates vascular barrier function through a non-canonical, transcription independent signaling mechanism that drives adherens junction assembly. Shear stress triggers Dll4-dependent proteolytic activation of Notch1 to reveal the Notch1 transmembrane domain – the key domain that mediates barrier establishment. Expression of the Notch1 transmembrane domain is sufficient to rescue Notch1 knockout-induced defects in barrier function, and does so by catalyzing the formation of a novel receptor complex in the plasma membrane consisting of VE-cadherin, the transmembrane protein tyrosine phosphatase LAR, and the Rac1 GEF Trio. This complex activates Rac1 to drive adherens junction assembly and establish barrier function. Canonical Notch transcriptional signaling is highly conserved throughout metazoans and is required for many processes in vascular development, including arterial-venous differentiation3, angiogenesis4, and remodeling5; here, we establish the existence of a previously unappreciated non-canonical cortical signaling pathway for Notch1 that regulates vascular barrier function, and thus provide a mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodeling.

Tissue vascularization and integration with host circulation remains a key barrier to the translation of engineered tissues into clinically relevant therapies. Here, we used a microtissue molding approach to demonstrate that constructs containing highly aligned "cords" of endothelial cells triggered the formation of new capillaries along the length of the patterned cords. These vessels became perfused with host blood as early as 3 d post implantation and became progressively more mature through 28 d. Immunohistochemical analysis showed that the neovessels were composed of human and mouse endothelial cells and exhibited a mature phenotype, as indicated by the presence of alpha-smooth muscle actin-positive pericytes. Implantation of cords with a prescribed geometry demonstrated that they provided a template that defined the neovascular architecture in vivo. To explore the utility of this geometric control, we implanted primary rat and human hepatocyte constructs containing randomly organized endothelial networks vs. ordered cords. We found substantially enhanced hepatic survival and function in the constructs containing ordered cords following transplantation in mice. These findings demonstrate the importance of multicellular architecture in tissue integration and function, and our approach provides a unique strategy to engineer vascular architecture.tissue engineering | regenerative medicine | angiogenesis | vascular biology | liver

Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is classically thought to be mediated by different cytokines such as the bone morphogenetic proteins (BMPs). Here, we report that cell adhesion to extracellular matrix (ECM), and its effects on cell shape and cytoskeletal mechanics, regulates BMP-induced signaling and osteogenic differentiation of hMSCs. Using micropatterned substrates to progressively restrict cell spreading and flattening against ECM, we demonstrated that BMP-induced osteogenesis is progressively antagonized with decreased cell spreading. BMP triggered rapid and sustained RhoA/Rho-associated protein kinase (ROCK) activity and contractile tension only in spread cells, and this signaling was required for BMPinduced osteogenesis. Exploring the molecular basis for this effect, we found that restricting cell spreading, reducing ROCK signaling, or inhibiting cytoskeletal tension prevented BMP-induced SMA/mothers against decapentaplegic (SMAD)1 c-terminal phosphorylation, SMAD1 dimerization with SMAD4, and SMAD1 translocation into the nucleus. Together, these findings demonstrate the direct involvement of cell spreading and RhoA/ROCK-mediated cytoskeletal tension generation in BMP-induced signaling and early stages of in vitro osteogenesis, and highlight the essential interplay between biochemical and mechanical cues in stem cell differentiation.