Despite bone's impressive ability to heal after traumatic injuries and fractures, a significant need still exists for developing strategies to promote healing of nonunion defects. To address this issue, we developed collagen-based hydrogels containing two-dimensional nanosilicates. Nanosilicates are ultrathin nanomaterials with a high degree of anisotropy and functionality that results in enhanced surface interactions with biological entities compared to their respective three-dimensional counterparts. The addition of nanosilicates resulted in a 4-fold increase in compressive modulus along with an increase in pore size compared to collagen-based hydrogels. In vitro evaluation indicated that the nanocomposite hydrogels are capable of promoting osteogenesis in the absence of any osteoinductive factors. A 3-fold increase in alkaline phosphatase activity and a 4-fold increase in the formation of a mineralized matrix were observed with the addition of the nanosilicates to the collagen-based hydrogels. Overall, these results demonstrate the multiple functions of nanosilicates conducive to the regeneration of bone in nonunion defects, including increased network stiffness and porosity, injectability, and enhanced mineralized matrix formation in a growth-factor-free microenvironment.

Advanced bioinks for 3D printing are rationally designed materials intended to improve the functionality of printed scaffolds outside the traditional paradigm of the "biofabrication window". While the biofabrication window paradigm necessitates compromise between suitability for fabrication and ability to accommodate encapsulated cells, recent developments in advanced bioinks have resulted in improved designs for a range of biofabrication platforms without this tradeoff. This has resulted in a new generation of bioinks with high print fidelity, shear-thinning characteristics, and crosslinked scaffolds with high mechanical strength, high cytocompatibility, and the ability to modulate cellular functions. In this review, we describe some of the promising strategies being pursued to achieve these goals, including multimaterial, interpenetrating network, nanocomposite, and supramolecular bioinks. We also provide an overview of current and emerging trends in advanced bioink synthesis and biofabrication, and evaluate the potential applications of these novel biomaterials to clinical use.

The small GTPase Rho regulates the formation of actin stress fibers in adherent cells through activation of its effector proteins Rhokinase and mDia. We found in bovine aortic endothelial cells that inhibitions of Rho, Rho-kinase, and mDia (with C3, Y27632, and F1F2⌬1, respectively) suppressed stress fiber formation, but fibers appeared after 10% cyclic uniaxial stretch (1-Hz frequency). In contrast to the predominately perpendicular alignment of stress fibers to the stretch direction in normal cells, the stress fibers in cells with Rho pathway inhibition became oriented parallel to the stretch direction. In cells with normal Rho activity, the extent of perpendicular orientation of stress fibers depended on the magnitude of stretch. Expressing active RhoV14 plasmid in these cells enhanced the stretch-induced stress fiber orientation by an extent equivalent to an additional Ϸ3% stretch. This augmentation of the stretch-induced perpendicular orientation by RhoV14 was blocked by Y27632 and by F1F2⌬1. Thus, the activity of the Rho pathway plays a critical role in determining both the direction and extent of stretch-induced stress fiber orientation in bovine aortic endothelial cells. Our results demonstrate that the stretch-induced stress fiber orientation is a function of the interplay between Rho pathway activity and the magnitude of stretching.cytoskeletal dynamics ͉ endothelial cells ͉ mechanotransduction ͉ Rho-kinase T he tension generated by contraction of adherent cells against their underlying surface results in an internal stress field that depends on the organization of the cytoskeleton and the associated adhesive contacts (see ref. 1 for review). Intracellular forces have an important role in cellular functions such as migration, proliferation, apoptosis, differentiation, and gene expression (see refs. 2-4 for reviews). Actin stress fibers, which are formed in response to cell contraction (5), consist of bundles of actin microfilaments cross-linked by ␣-actinin, myosin, myosin light-chain, tropomyosin, and other proteins arranged in a manner similar to that in muscle sarcomeres (6). Stress fibers represent the main contractile apparatus in non-muscle cells (7) and are the primary structures associated with intracellular tension. Stress fibers terminate at focal adhesions, which attach the cell to the extracellular matrix (8). Isometric contraction of a cell would result in tension development in the stress fibers, which are anchored at their ends.The activation of the small GTPase Rho leads to stress fiber assembly (9) and cell contraction by means of myosin light chain phosphorylation (5), which is regulated by Rho-kinase, a downstream effector of Rho (10). mDia, another Rho effector, is also involved in stress fiber formation downstream of Rho activation (11), possibly by regulating actin polymerization and focal adhesion turnover through its association with profilin (12, 13) and src-tyrosine-kinase (14), respectively.Cyclic uniaxial stretch induces the orientation of stress fibers in endothelial cells ...

Bioprinting is an emerging approach for fabricating cell-laden 3D scaffolds via robotic deposition of cells and biomaterials into custom shapes and patterns to replicate complex tissue architectures. Bioprinting uses hydrogel solutions called bioinks as both cell carriers and structural components, requiring bioinks to be highly printable while providing a robust and cell-friendly microenvrionment. Unfortunately, conventional hydrogel bioinks have not been able to meet these requirements and are mechanically weak due to their heterogeneously crosslinked networks and lack of energy dissipation mechanisms. Advanced bioink designs using various methods of dissipating mechanical energy are aimed at developing next-generation cellularized 3D scaffolds to mimic anatomical size, tissue architecture, and tissue specific functions. These next-generation bioinks need to have high print fidelity and should provide a biocompatible microenvironment along with improved mechanical properties. To design these advanced bioink formulations, it is important to understand the structure-property-function relationships of the hydrogel network. By specifically leveraging biophysical and biochemical characteristics of hydrogel networks, high performance bioinks can be designed to control and direct cell functions. In this review article, the authors will critically evaluate current and emerging approaches in hydrogel design and bioink reinforcement techniques. This bottom-up perspective provides a materials-centric approach to bioink design for 3D bioprinting.

Cells in the body are subjected to mechanical stresses such as tension, compression, and shear stress. These mechanical stresses play important roles in both physiological and pathological processes; however, mechanisms transducing mechanical stresses into biochemical signals remain elusive. Here, we demonstrated that equibiaxial stretch inhibited lamellipodia formation through deactivation of Rac. Nearly maximal effects on Rac activity were obtained with 10% strain. GAP-resistant, constitutively active V12Rac reversed this inhibition, supporting a critical role for Rac inhibition in the response to stretch. In contrast, activation of endogenous Rac with a constitutively active nucleotide exchange factor did not, suggesting that regulation of GAP activity most likely mediates the inhibition. Uniaxial stretch suppressed lamellipodia along the sides lengthened by stretch and increased it at the adjacent ends. A fluorescence assay for localized Rac showed comparable changes in activity along the sides versus the ends after uniaxial stretch. Blocking polarization of Rac activity by expressing V12Rac prevented subsequent alignment of actin stress fibers. Treatment with Y-27632 or ML-7 that inhibits myosin phosphorylation and contractility increased lamellipodia through Rac activation and decreased cell polarization. We hypothesize that regulation of Rac activity by tension may be important for motility, polarization, and directionality of cell movement.