Soluble factors from neocortical astrocytes enhance neuronal differentiation of neural progenitor cells from adult rat hippocampus on micropatterned polymer substrates

Rat adult hippocampal progenitor cells (AHPCs) are self-renewing, multipotent neural progenitors that have the ability to differentiate into neurons and glia. Previously, we demonstrated that coculture of AHPCs with postnatal day two, type 1 cortical astrocytes on laminin-coated micropatterned polymer substrates facilitates selective neuronal differentiation of the AHPCs 1. Under this condition, multi-dimensional cell-cell and/or cell-extracellular matrix interactions, as well as possible soluble factors released from astrocytes provided spatial and temporal control selectively enhancing neuronal differentiation and neurite alignment on topographically different regions of the same substrate. To investigate the potential role of astrocyte-derived soluble factors as cues involved in neuronal differentiation, a non-contact co-culture system was used. Under control conditions, approximately 14% of the AHPCs were immunoreactive (IR) for the neuronal marker, class III β-tubulin (TUJ1-IR). When co-cultured in physical contact with astrocytes, neuronal differentiation increased significantly to about 25%, consistent with our previous results. Moreover, under non-contact co-culture conditions using Transwell insert cultures, neuronal differentiation was dramatically increased to approximately 64%. Furthermore, neurite outgrowth from neuronal cell bodies was considerably greater on the patterned substrate, compared to the non-patterned planar substrate under non-contact co-culture conditions. Taken together, our results demonstrate that astrocyte-derived soluble factors provide cues for specific neuronal differentiation of AHPCs cultured on micropatterned substrates. In addition, a suppressive influence on neuronal differentiation appears to be mediated by contact with co-cultured astrocytes. These results provide important insights into mechanisms for controlling neural progenitor/stem cell differentiation and facilitate development of strategies for CNS repair

Gelatin-based 3D conduits for transdifferentiation of mesenchymal stem cells into Schwann cell-like phenotypes

In this study, gelatin-based 3D conduits with three different microstructures (nanofibrous, macroporous and ladder-like) were fabricated for the first time via combined molding and thermally induced phase separation (TIPS) technique for peripheral nerve regeneration. The effects of conduit microstructure and mechanical properties on the transdifferentiation of bone marrow-derived mesenchymal stem cells (MSCs) into Schwann cell (SC) like phenotypes were examined to help facilitate neuroregeneration and understand material-cell interfaces. Results indicated that 3D macroporous and ladder-like structures enhanced MSC attachment, proliferation and spreading, creating interconnected cellular networks with large numbers of viable cells compared to nanofibrous and 2D-tissue culture plate counterparts. 3D-ladder-like conduit structure with complex modulus of ∼0.4 × 106 Pa and pore size of ∼150 μm provided the most favorable microenvironment for MSC transdifferentiation leading to ∼85% immunolabeling of all SC markers. On the other hand, the macroporous conduits with complex modulus of ∼4 × 106 Pa and pore size of ∼100 μm showed slightly lower (∼65% for p75, ∼75% for S100 and ∼85% for S100β markers) immunolabeling. Transdifferentiated MSCs within 3D-ladder-like conduits secreted significant amounts (∼2.5 pg/mL NGF and ∼0.7 pg/mL GDNF per cell) of neurotrophic factors, while MSCs in macroporous conduits released slightly lower (∼1.5 pg/mL NGF and 0.7 pg/mL GDNF per cell) levels. PC12 cells displayed enhanced neurite outgrowth in media conditioned by conduits with transdifferentiated MSCs. Overall, conduits with macroporous and ladder-like 3D structures are promising platforms in transdifferentiation of MSCs for neuroregeneration and should be further tested in vivo. Statement of Significance This manuscript focuses on the effect of microstructure and mechanical properties of gelatin-based 3D conduits on the transdifferentiation of mesenchymal stem cells to Schwann cell-like phenotypes. This work builds on our recently accepted manuscript in Acta Biomaterialia focused on multifunctional 2D films, and focuses on 3D microstructured conduits designed to overcome limitations of current strategies to facilitate peripheral nerve regeneration. The comparison between conduits fabricated with nanofibrous, macroporous and ladder-like microstructures showed that the ladder-like conduits showed the most favorable environment for MSC transdifferentiation to Schwann-cell like phenotypes, as seen by both immunolabeling as well as secretion of neurotrophic factors. This work demonstrates the importance of controlling the 3D microstructure to facilitate tissue engineering strategies involving stem cells that can serve as promising approaches for peripheral nerve regeneration.US Army Medical Research and Materiel Command (W81XWH-11-1-0700); Stem Cell Biology Fund; Stanley Endowed Chai

Enabling nanomaterial, nanofabrication and cellular technologies for nanoneuromedicines

Nanoparticulate delivery systems represent an area of particular promise for nanoneuromedicines. They possess significant potential for desperately needed therapies designed to combat a range of disorders associated with aging. As such, the field was selected as the focus for the 2014 meeting of the American Society for Nanomedicine. Regenerative, protective, immune modulatory, anti-microbial and anti-inflammatory products, or imaging agents are readily encapsulated in or conjugated to nanoparticles and as such facilitate the delivery of drug payloads to specific action sites across the blood-brain barrier. Diagnostic imaging serves to precisely monitor disease onset and progression while neural stem cell replacement can regenerate damaged tissue through control of stem cell fates. These, taken together, can improve disease burden and limit systemic toxicities. Such enabling technologies serve to protect the nervous system against a broad range of degenerative, traumatic, metabolic, infectious and immune disorders

Atomistic Studies of Defect Nucleation during Nanoindentation of Au (001)

Atomistic studies are carried out to investigate the formation and evolution of defects during nanoindentation of a gold crystal. The results in this theoretical study complement the experimental investigations [J. D. Kiely and J. E. Houston, Phys. Rev. B, v57, 12588 (1998)] extremely well. The defects are produced by a three step mechanism involving nucleation, glide and reaction of Shockley partials on the {111} slip planes noncoplanar with the indented surface. We have observed that slip is in the directions along which the resolved shear stress has reached the critical value of approximately 2 GPa. The first yield occurs when the shear stresses reach this critical value on all the {111} planes involved in the formation of the defect. The phenomenon of strain hardening is observed due to the sessile stair-rods produced by the zipping of the partials. The dislocation locks produced during the second yield give rise to permanent deformation after retraction.Comment: 11 pages, 13 figures, submitted to Physical Review

Biomimetic self-assembling copolymer-hydroxyapatite nanocomposites with the nanocrystal size controlled by citrate

Citrate binds strongly to the surface of calcium phosphate (apatite) nanocrystals in bone and is thought to prevent crystal thickening. In this work, citrate added as a regulatory element enabled molecular control of the size and stability of hydroxyapatite (HAp) nanocrystals in synthetic nanocomposites, fabricated with self-assembling block copolymer templates. The decrease of the HAp crystal size within the polymer matrix with increasing citrate concentration was documented by solid-state nuclear magnetic resonance (NMR) techniques and wide-angle X-ray diffraction (XRD), while the shapes of HAp nanocrystals were determined by transmission electron microscopy (TEM). Advanced NMR techniques were used to characterize the interfacial species and reveal enhanced interactions between mineral and organic matrix, concomitant with the size effects. The surface-to-volume ratios determined by NMR spectroscopy and long-range 31P{1H} dipolar dephasing show that 2, 10, and 40 mM citrate changes the thicknesses of the HAp crystals from 4 nm without citrate to 2.9, 2.8, and 2.3 nm, respectively. With citrate concentrations comparable to those in body fluids, HAp nanocrystals of sizes and morphologies similar to those in avian and bovine bones have been produced

In vitro phosphorylation as tool for modification of silk and keratin fibrous materials

An overview is given of the recent work on in vitro enzymatic phosphorylation of silk fibroin and human hair keratin. Opposing to many chemical "conventional" approaches, enzymatic phosphorylation is in fact a mild reaction and the treatment falls within "green chemistry" approach. Silk and keratin are not phosphorylated in vivo, but in vitro. This enzyme-driven modification is a major technological breakthrough. Harsh chemical chemicals are avoided, and mild conditions make enzymatic phosphorylation a real "green chemistry" approach. The current communication presents a novel approach stating that enzyme phosphorylation may be used as a tool to modify the surface charge of biocompatible materials such as keratin and silk