Microfluidic Formulation of Topological Hydrogels for Microtissue Engineering.

Microfluidics has recently emerged as a powerful tool in generation of submillimeter-sized cell aggregates capable of performing tissue-specific functions, so-called microtissues, for applications in drug testing, regenerative medicine, and cell therapies. In this work, we review the most recent advances in the field, with particular focus on the formulation of cell-encapsulating microgels of small "dimensionalities": "0D" (particles), "1D" (fibers), "2D" (sheets), etc., and with nontrivial internal topologies, typically consisting of multiple compartments loaded with different types of cells and/or biopolymers. Such structures, which we refer to as topological hydrogels or topological microgels (examples including core-shell or Janus microbeads and microfibers, hollow or porous microstructures, or granular hydrogels) can be precisely tailored with high reproducibility and throughput by using microfluidics and used to provide controlled "initial conditions" for cell proliferation and maturation into functional tissue-like microstructures. Microfluidic methods of formulation of topological biomaterials have enabled significant progress in engineering of miniature tissues and organs, such as pancreas, liver, muscle, bone, heart, neural tissue, or vasculature, as well as in fabrication of tailored microenvironments for stem-cell expansion and differentiation, or in cancer modeling, including generation of vascularized tumors for personalized drug testing. We review the available microfluidic fabrication methods by exploiting various cross-linking mechanisms and various routes toward compartmentalization and critically discuss the available tissue-specific applications. Finally, we list the remaining challenges such as simplification of the microfluidic workflow for its widespread use in biomedical research, bench-to-bedside transition including production upscaling, further in vivo validation, generation of more precise organ-like models, as well as incorporation of induced pluripotent stem cells as a step toward clinical applications.

Tendon Tissue Engineering: Effects of Mechanical and Biochemical Stimulation on Stem Cell Alignment on Cell-Laden Hydrogel Yarns.

Fiber-based approaches hold great promise for tendon tissue engineering enabling the possibility of manufacturing aligned hydrogel filaments that can guide collagen fiber orientation, thereby providing a biomimetic micro-environment for cell attachment, orientation, migration, and proliferation. In this study, a 3D system composed of cell-laden, highly aligned hydrogel yarns is designed and obtained via wet spinning in order to reproduce the morphology and structure of tendon fascicles. A bioink composed of alginate and gelatin methacryloyl (GelMA) is optimized for spinning and loaded with human bone morrow mesenchymal stem cells (hBM-MSCs). The produced scaffolds are subjected to mechanical stretching to recapitulate the strains occurring in native tendon tissue. Stem cell differentiation is promoted by addition of bone morphogenetic protein 12 (BMP-12) in the culture medium. The aligned orientation of the fibers combined with mechanical stimulation results in highly preferential longitudinal cell orientation and demonstrates enhanced collagen type I and III expression. Additionally, the combination of biochemical and mechanical stimulations promotes the expression of specific tenogenic markers, signatures of efficient cell differentiation towards tendon. The obtained results suggest that the proposed 3D cell-laden aligned system can be used for engineering of scaffolds for tendon regeneration.

A search for the in trans role of GraL, an Escherichia coli small RNA.

Small RNA are very important post-transcriptional regulators in both, bacteria and eukaryotes. One of such sRNA is GraL, encoded in the greA leader region and conserved among enteric bacteria. Here, we conducted a bioinformatics search for GraL's targets in trans and validated our findings in vivo by constructing fusions of probable targets with lacZ and measuring their activity when GraL was overexpressed. Only one target's activity (nudE) decreased under those conditions and was thus selected for further analysis. In the absence of GraL and greA, the nudE::lacZ fusion's ß-galactosidase activity was increased. However, a similar effect was also visible in the strain deleted only for greA. Furthermore, overproduction of GreA alone increased the nudE::lacZ fusion's activity as well. This suggests existence of complex regulatory loop-like interactions between GreA, GraL and nudE mRNA. To further dissect this relationship, we performed in vitro EMSA experiments employing GraL and nudE mRNA. However, stable GraL-nudE complexes were not detected, even though the detectable amount of unbound GraL decreased as increasing amounts of nudE mRNA were added. Interestingly, GraL is being bound by Hfq, but nudE easily displaces it. We also conducted a search for genes that are synthetic lethal when deleted along with GraL. This revealed 40 genes that are rendered essential by GraL deletion, however, they are involved in many different cellular processes and no clear correlation was found. The obtained data suggest that GraL's mechanism of action is non-canonical, unique and requires further research.