No abstract

been used as 3D tissue structures in newly discovered drugs and personalized medicines and are more relevant to in vivo microenvironmental conditions than 2D substrates. [2] Recently, a variety of micro/nanoscale biofabrication methods have been used to build complex 3D biomedical structures. Unlike conventional processes, which can incur unpredictable properties, such as non-homogeneous physicochemical and mechanical properties, within the structure, 3D bioprinting provides precise control of various biophysical and biochemical properties. It can also be used to print different cell types that are appropriate to the region of tissue restoration.However, despite advances in 3D bioprinting of scaffolds for various tissue engineering applications, developing functional bioprinting of cell-laden structures remains challenging due to several limitations. A common shortcoming of bioprinted structures is the low degree of cell-cell interaction in matrix hydrogels, which are critical for tissue restoration. Additionally, relatively high cell densities in bioink can cause significant cell damage during the printing process. Even fabricated structures with high cell densities cannot sustain printed 3D structures due to their relatively low physical strength.However, although the appropriate cell density for a given application is highly dependent on the cell type and hydrogel physicochemical properties, high cell density induces efficient cell-to-cell signaling and regulates stem cell differentiation. [3][4][5] According to Maia et al., high densities of human mesenchymal stem cells cultured in Arg-Gly-Asp (RGD)-alginate 3D matrixes allow the development of multicellular structures and effectively stimulates osteogenic differentiation of the implanted cells. [6] Thus, cell spheroids, high density 3D cell aggregations, can be considered microtissues that mimic natural microenvironmental conditions by providing sufficient cell-to-cell or cell-to-ECM interactions to increase various cellular activities, including cell differentiation. [7] In general, spheroids are used to treat or regenerate damaged tissues through an injection process. However, to obtain the intricately designed configurations necessary for some applications, spheroids must be mixed with hydrogels and spheroid-laden bioink printed into 3D mesh structures.Cell-laden structures are widely applied for a variety of tissue engineering applications, including tissue restoration. Cell-to-cell interactions in bioprinted structures are important for successful tissue restoration, because cell-cell signaling pathways can regulate tissue development and stem cell fate. However, the low degree of cell-cell interaction in conventional cellladen bioprinted structures is challenging for the therapeutic application of this modality. Herein, a microfluidic device with cell-laden methacrylated gelatin (GelMa) bioink and alginate as a matrix hydrogel is used to fabricate a functional hybrid structure laden with cell-aggregated microbeads. This approach effectively increases t...

Mechanistic studies of axon growth during development are beneficial to the search for neuron-intrinsic regulators of axon regeneration. Here, we discovered that, in the developing neuron from rat, Akt signaling regulates axon growth and growth cone formation through phosphorylation of serine 14 (S14) on Inhibitor of DNA binding 2 (Id2). This enhances Id2 protein stability by means of escape from proteasomal degradation, and steers its localization to the growth cone, where Id2 interacts with radixin that is critical for growth cone formation. Knockdown of Id2, or abrogation of Id2 phosphorylation at S14, greatly impairs axon growth and the architecture of growth cone. Intriguingly, reinstatement of Akt/Id2 signaling after injury in mouse hippocampal slices redeemed growth promoting ability, leading to obvious axon regeneration. Our results suggest that Akt/Id2 signaling is a key module for growth cone formation and axon growth, and its augmentation plays a potential role in CNS axonal regeneration.DOI: http://dx.doi.org/10.7554/eLife.20799.001

ErbB3-binding protein 1 (EBP1) is implicated in diverse cellular functions, including apoptosis, cell proliferation, and differentiation. Here, by generating genetic inactivation of Ebp1 mice, we identified the physiological roles of EBP1 in vivo. Loss of Ebp1 in mice caused aberrant organogenesis, including brain malformation, and death between E13.5 and 15.5 owing to severe hemorrhages, with massive apoptosis and cessation of cell proliferation. Specific ablation of Ebp1 in neurons caused structural abnormalities of brain with neuron loss in [Nestin-Cre; Ebp1flox/flox] mice. Notably, global methylation increased with high levels of the gene-silencing unit Suv39H1/DNMT1 in Ebp1-deficient mice. EBP1 repressed the transcription of Dnmt1 by binding to its promoter region and interrupted DNMT1-mediated methylation at its target gene, Survivin promoter region. Reinstatement of EBP1 into embryo brain relived gene repression and rescued neuron death. Our findings uncover an essential role for EBP1 in embryonic development and implicate its function in transcriptional regulation.

Neurite growth is controlled by a complex molecular signaling network that regulates filamentous actin (F-actin) dynamics at the growth cone. The evolutionarily conserved ezrin, radixin, and moesin family of proteins tether F-actin to the cell membrane when phosphorylated at a conserved threonine residue and modulate neurite outgrowth. Here we show that Akt binds to and phosphorylates a threonine 573 residue on radixin. Akt-mediated phosphorylation protects radixin from ubiquitin-dependent proteasomal degradation, thereby enhancing radixin protein stability, which permits proper neurite outgrowth and growth cone formation. Conversely, the inhibition of Akt kinase or disruption of Akt-dependent phosphorylation reduces the binding affinity of radixin to F-actin as well as lowers radixin protein levels, resulting in decreased neurite outgrowth and growth cone formation. Our findings suggest that Akt signaling regulates neurite outgrowth by stabilizing radixin interactions with F-actin, thus facilitating local F-actin dynamics.