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The potential of human embryonic stem (ES) cells as experimental therapies for neuronal replacement has recently received considerable attention. In view of the organization of the mature nervous system into distinct neural circuits, key challenges of such therapies are the directed differentiation of human ES cell-derived neural precursors (NPs) into specific neuronal types and the directional growth of axons along specified trajectories. In the present study, we cultured human NPs derived from the NIH-approved ES line BGO1 on polycaprolactone fiber matrices of different diameter (i.e., nanofibers and microfibers) and orientation (i.e., aligned and random); fibers were coated with poly-L-ornithine/laminin to mimic the extracellular matrix and support the adhesion, viability, and differentiation of NPs. On aligned fibrous meshes, human NPs adopt polarized cell morphology with processes extending along the axis of the fiber, whereas NPs on plain tissue culture surfaces or random fiber substrates form nonpolarized neurite networks. Under differentiation conditions, human NPs cultured on aligned fibrous substrates show a higher rate of neuronal differentiation than other matrices; 62% and 86% of NPs become TUJ1 (+) early neurons on aligned micro- and nanofibers, respectively, whereas only 32% and 27% of NPs acquire the same fate on random micro- and nanofibers. Metabolic cell activity/viability studies reveal that fiber alignment and diameter also have an effect on NP viability, but only in the presence of mitogens. Our findings demonstrate that fibrous substrates serve as an artificial extracellular matrix and provide a microenviroment that influences key aspects of the neuronal differentiation of ES-derived NPs.

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The level of structural detail that can be acquired and incorporated in a finite element (FE) analysis might greatly influence the results of microcomputed tomography (microCT)-based FE simulations, especially when relatively large bones, such as whole vertebrae, are of concern. We evaluated the effect of scanning and reconstruction voxel size on the microCT-based FE analyses of human cancellous tissue samples for fixed- and free-end boundary conditions using different combinations of scan/reconstruction voxel size. We found that the bone volume fraction (BV/TV) did not differ considerably between images scanned at 21 and 50 microm and reconstructed at 21, 50, or 110 microm (-0.5% to 7.8% change from the 21/21 microm case). For the images scanned and reconstructed at 110 microm, however, there was a large increase in BV/TV compared to the 21/21 microm case (58.7%). Fixed-end boundary conditions resulted in 1.8% [coefficient of variation (COV)] to 14.6% (E) difference from the free-end case. Dependence of model output parameters on scanning and reconstruction voxel size was similar between free- and fixed-end simulations. Up to 26%, 30%, 17.8%, and 32.3% difference in modulus (E), and average (VMExp), standard deviation (VMSD) and coefficient of variation (COV) of von Mises stresses, respectively, was observed between the 21/21 microm case and other scan/reconstruction combinations within the same (free or fixed) simulation group. Observed differences were largely attributable to scanning resolution, although reconstruction resolution also contributed significantly at the largest voxel sizes. All 21/21 microm results (taken as the gold standard) could be predicted from the 21/50 (r2adj= 0.91-0.99;p<0.001), 21/110 (r2adj =0.58-0.99;p<0.02) and 50/50 results (r2adj=0.61-0.97;p<0.02). While BV/TV, VMSD, and VMExp/sigma(z) from the 21/21 could be predicted by those from the 50/110 (r2adj =0.63-0.93;p<0.02) and 110/110 (r2adj =0.41-0.77;p<0.05) simulations as well, prediction of E, VMExp, and COV became marginally significant (0.04