3D Carbon Scaffolds for Neural Stem Cell Culture and Magnetic Resonance ImagingErwin Fuhrer, Anne Bäcker, Stephanie Kraft, Friederike J. Gruhl, Matthias Kirsch, Neil MacKinnon, Jan G. Korvink, and Swati Sharma* DOI: 10.1002/adhm.201700915 the dimensionality of the culture system. Many in vitro assays are 2D substrate systems, which raises the question of the relevance to tissues that thrive in a 3D environment. Since it has been recognized that the scaffold topology imparts important cues for cell development and function, there has been a concerted effort to develop 3D scaffold systems that better reproduce the in vivo environment.Common materials used for 3D scaffold fabrication for various cell differentiation, angiogenesis, and tumor growth studies include commercially available Matrigel, hydrogels, and cryogels. [2] Matrigel is a gelatinous protein mixture that contains components of the basement membrane, such as laminin, collagen type IV, and entactin. [3] Hydrogels (water-containing polymer networks) are composed of either natural polymers such as collagen, hyaluronates, fibrin, and chitosan or synthetic polymers such as polyvinyl caprolactam and polyethylene glycol. [4] These materials are generally advantageous since a 3D hydrated network can be produced together with the desired chemical cues required for cell development (e.g., growth factors and cell adhesion promoters). In addition, the porous structure (size and shape) of the polymer network can be tuned by varying the freezing conditions and concentration of the cross-linker in the case of cryogels. [5,6] However, several disadvantages can be identified. Matrigel is often poorly chemically defined and thus culture-to-culture variation has been observed. [7] Hydrogels may require further chemical processing to introduce the important cellular chemical cues. [8] This introduces the potential for residual chemicals to be entrapped within the gel, influencing the reproducibility of cell development. These materials additionally have the potential to degrade after prolonged exposure to culture media. While not always an issue, in cases of extended culture times, cell cultures require a scaffold that will maintain its mechanical integrity. In this work, we propose glassy carbon as a material capable of overcoming these challenges.Conversion of polymer structures into glassy carbon using pyrolysis is a widespread process that is extensively used for the fabrication of carbon MEMS and NEMS, [9,10] battery and supercapacitor anodes, [11] and carbon nanofibers. [12,13] One very attractive yet rather unexplored feature of glassy carbon is its excellent cytocompatibility, [14][15][16][17][18] which, in combination with its mechanical strength, [9] inertness, and patternability, [10] makes it a very suitable material for the fabrication of 3D cell culture platforms.

The natural neurosteroid allopregnanolone exerts beneficial effects in animal models of neurodegenerative diseases, nervous system injury and peripheral neuropathies. It not only has anti-apoptotic activity, but also promotes proliferation of progenitor cells. With respect to using it as a therapeutic tool, such pleiotropic actions might create unwanted side effects. Therefore, we have synthesized allopregnanolone analogs and analyzed their neuroprotective and proliferative effects to identify compounds with higher efficiency and less ambiguous biological actions. Proliferation-promoting effects of 3α and 3β isomers of 3-O-allyl-allopregnanolone and 12 oxo-allopregnanolone were studied in adult subventricular zone stem cell cultures and in primary hippocampal cultures by measuring 5-ethynyl-2'-deoxyuridine incorporation. Neuroprotective activity against amyloid beta 42-induced cell death was determined by quantifying caspase 3/7 activity. The 3α isomers significantly stimulated proliferation in all culture systems, whereas the 3β isomers were ineffective. The stimulatory effect of 3α-O-allyl-allopregnanolone was significantly higher than that of allopregnanolone. In neural stem cell cultures, 3α-O-allyl-allopregnanolone specifically enhanced proliferation of Nestin-positive progenitors. In addition, it promoted the differentiation of doublecortin-positive neurons. In neural stem cell cultures treated with amyloid beta 42, both the α and β isomers of O-allyl- allopregnanolone showed increased neuroprotective activity as compared to allopregnanolone, completely preventing amyloid-induced caspase 3/7 activation. The 12 oxo-allopregnanolone analogs were ineffective. These results identify structural allopregnanolone analogs with higher anti-apoptotic and proliferation-promoting activity than the natural neurosteroid. Interestingly, stereoisomers of the analogs were found to have distinct profiles of activity raising the possibility of exploiting the neuroprotective properties of neurosteroids with or without simultaneously stimulating neurogenesis. Cover Image for this issue: doi: 10.1111/jnc.13344.

The versatile structure of smectites can exhibit large variations in chemical compositions and cationic substitutions in different crystallographic sites, resulting in various locations of layer charge. Natural smectites can contain various amounts of structural iron, the chemical form of which can influence the reactivity of these minerals. The variety of Fe crystal chemistry in smectite was explored for eight natural smectites of distinct chemical compositions and charge locations, together with two synthetic ferric saponites used as reference compounds for tetrahedral Fe(III). All samples were identified as dioctahedral or trioctahedral smectite by X-ray diffraction and Fourier-transform infrared spectroscopy. The extent of [4] Al for [4] Si substitution was determined by 27 Al and 29 Si magic angle spinning nuclear magnetic resonance spectroscopy. The Fe local chemical environment was probed by polarized X-ray absorption spectroscopy. Only Fe(III) could be detected in all samples, with no evidence of cluster formation. The O shell at 1.86 Å in synthetic saponites suggests Fe insertion in tetrahedral sites, and the absence of detected octahedral Fe implies quantitative substitution of [4] Fe 3+ for [4] Si 4+ . In natural smectites, Fe(III) is bound to six O atoms at ~2.00 Å, suggesting insertion in octahedral sites. This inference is also supported by the detection of in-plane Mg/Al/Fe atoms at ~3.05 Å and out-of-plane Si/Al atoms at ~3.25 Å. In one Fe-rich nontronite, the detection of an O subshell at ~1.88 Å suggests a concomitant insertion of Fe(III) in tetrahedral sites. Low numbers of octahedral neighbors were detected in natural saponite and hectorite, presumably because of the presence of vacancies and/or Li(I) in adjacent octahedral sites balancing the local charge excess originating from the substitution of Fe(III) for Mg(II). The substitution of [4] Fe 3+ for [4] Si 4+ can be readily obtained under defined conditions in the laboratory, but seems more rare in natural samples, or present in amounts below the detection limit of spectroscopic methods used in this study.

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