Gamma-protocadherin localization at the synapse is associated with parameters of synaptic maturation.

Clustered protocadherins (Pcdhs) are a family of ~60 cadherin-like proteins (divided into subclasses α, ß, and γ) that regulate dendrite morphology and neural connectivity. Their expression is controlled through epigenetic regulation at a gene cluster encoding the molecules. During neural development, Pcdhs mediate dendrite self-avoidance in some neuronal types through an uncharacterized anti-adhesive mechanism. Pcdhs are also important for dendritic complexity in cortical neurons likely through a pro-adhesive mechanism. Pcdhs have also been postulated to participate in synaptogenesis and connectivity. Some synaptic defects were noted in knockout animals, including synaptic number and physiology, but the role of these molecules in synaptic development is not understood. The effect of Pcdh knockout on dendritic patterning may present a confound to studying synaptogenesis. We showed previously that Pcdh-γs are highly enriched in intracellular compartments in dendrites and spines with localization at only a few synaptic clefts. To gain insight into how Pcdh-γs might affect synapses, we compared synapses that harbored Pcdh-γs versus those that did not for parameters of synaptic maturation including pre- and postsynaptic size, postsynaptic perforations, and spine morphology by light microscopy in cultured hippocampal neurons and by serial section immuno-electron microscopy in hippocampal CA1. In mature neurons, synapses immunopositive for Pcdh-γs were larger in diameter with more frequent perforations. Analysis of spines in cultured neurons revealed that mushroom spines were more frequently immunopositive for Pcdh-γs at their tips than thin spines. These results suggest that Pcdh-γ function at the synapse may be related to promotion of synaptic maturation and stabilization.

Rediscovering Chemical Gardens: Self-Assembling Cytocompatible Protein-Intercalated Silicate-Phosphate Sponge-Mimetic Tubules.

The classic chemical garden experiment is reconstructed to produce protein-intercalated silicate-phosphate tubules that resemble tubular sponges. The constructs were synthesized by seeding calcium chloride into a solution of sodium silicate-potassium phosphate and gelatin. Sponge-mimetic tubules were fabricated with varying percentages of gelatin (0-15% w/v), in diameters ranging from 200 µm to 2 mm, characterized morphologically and compositionally, functionalized with biomolecules for cell adhesion, and evaluated for cytocompatibility. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy analysis (EDS) experiments showed that the external surface of the tubules was relatively more amorphous in texture and carbon/protein-rich in comparison to the interior surface. Transmission electron microscopy (TEM) images indicate a network composed of gelatin incorporated into the inorganic scaffold. The presence of gelatin in the constructs was confirmed by infrared spectroscopy. Powder X-ray diffraction (XRD) was used to identify inorganic crystalline phases in the scaffolds that are mainly composed of Ca(OH)2, NaCl, and Ca2SiO4 along with a band corresponding to amorphous gelatin. Bioconjugation and coating protocols were developed to program the scaffolds with cues for cell adhesion, and the resulting constructs were employed for 3D cell culture of marine (Pyrocystis lunula) and mammalian (HeLa and H9C2) cell lines. The cytocompatibility of the constructs was demonstrated by live cell assays. We have successfully shown that these biomimetic materials can indeed support life; they serve as scaffolds that facilitate the attachment and assembly of individual cells to form multicellular entities, thereby revisiting the 350-year-old effort to link chemical gardens with the origins of life. Hybrid chemical garden biomaterials are programmable, readily fabricated and could be employed in tissue engineering, biomolecular materials development, 3D mammalian cell culture and by researchers investigating the origins of multicellular life.

High throughput, high resolution enzymatic lithography process: effect of crystallite size, moisture, and enzyme concentration.

By bringing enzymes into contact with predefined regions of a surface, a polymer film can be selectively degraded to form desired patterns that find a variety of applications in biotechnology and electronics. This so-called "enzymatic lithography" is an environmentally friendly process as it does not require actinic radiation or synthetic chemicals to develop the patterns. A significant challenge to using enzymatic lithography has been the need to restrict the mobility of the enzyme in order to maintain control of feature sizes. Previous approaches have resulted in low throughput and were limited to polymer films only a few nanometers thick. In this paper, we demonstrate an enzymatic lithography system based on Candida antartica lipase B (CALB) and poly(ε-caprolactone) (PCL) that can resolve fine-scale features, (<1 µm across) in thick (0.1-2.0 µm) polymer films. A Polymer Pen Lithography (PPL) tool was developed to deposit an aqueous solution of CALB onto a spin-cast PCL film. Immobilization of the enzyme on the polymer surface was monitored using fluorescence microscopy by labeling CALB with FITC. The crystallite size in the PCL films was systematically varied; small crystallites resulted in significantly faster etch rates (20 nm/min) and the ability to resolve smaller features (as fine as 1 µm). The effect of printing conditions and relative humidity during incubation is also presented. Patterns formed in the PCL film were transferred to an underlying copper foil demonstrating a "Green" approach to the fabrication of printed circuit boards.

Fine-tuning the degree of stem cell polarization and alignment on ordered arrays of high-aspect-ratio nanopillars.

Nanobiomaterials are introducing new capabilities to coordinate cell selection, growth, morphology, and differentiation. Herein, we report that tuning the geometry of ordered arrays of nanopillars (NP) elicits specialized morphologies in adherent cells. Systematic analysis of the effects of the NP radius, height, and spacing reveals that stem cells assume either flattened, polarized, or stellate morphologies in direct response to interpillar spacing. Notably, on NPs of pitch near a critical spacing (d(crit) ≈ 2 µm for C3H10T1/2 cells), cells exhibit rounding of the cell body, pronounced polarization, and extension of narrow axon-like cell projections aligned with the square lattice of the NP array and extending hundreds of micrometers. Furthermore, increasing the NPs' aspect ratio from 12:1 to 50:1 to produce NPs with a corresponding reduction in the NP bending stiffness of 2 orders of magnitude amplified the cellular response and resulted in a previously unseen degree of cell polarization and alignment. The rapid morphological transformation is reproducible on surfaces that maintain key parameters of the NP geometry and spacing, is influenced by the cell seeding density, and persists for different stem cell lines and primary mesenchymal stem cells. The demonstrated ability to support various morphogenetic trends in stem cells by simply tuning the geometry of the NP substrates provides a stepping-stone for the future design of scaffolds where cellular morphology and alignment are crucial.

Numerical modeling of oxygen distributions in cortical and cancellous bone: oxygen availability governs osteonal and trabecular dimensions.

Whereas recent work has demonstrated the role of oxygen tension in the regulation of skeletal cell function and viability, the microenvironmental oxemic status of bone cells remains unknown. In this study, we have employed the Krogh cylinder model of oxygen diffusion to predict the oxygen distribution profiles in cortical and cancellous bone. Under the assumption of saturation-type Michaelis-Menten kinetics, our numerical modeling has indicated that, under steady-state conditions, there would be oxygen gradients across mature osteons and trabeculae. In Haversian bone, the calculated oxygen tension decrement ranges from 15 to 60%. For trabecular bone, a much shallower gradient is predicted. We note that, in Haversian bone, the gradient is largely dependent on osteocyte oxygen utilization and tissue oxygen diffusivity; in trabecular bone, the gradient is dependent on oxygen utilization by cells lining the bone surface. The Krogh model also predicts dramatic differences in oxygen availability during bone development. Thus, during osteon formation, the modeling equations predict a steep oxygen gradient at the initial stage of development, with the gradient becoming lesser as osteonal layers are added. In contrast, during trabeculum formation, the oxygen gradient is steepest when the diameter of the trabeculum is maximal. Based on these results, it is concluded that significant oxygen gradients exist within cortical and cancellous bone and that the oxygen tension may regulate the physical dimensions of both osteons and bone trabeculae.

Tunable liquid optics: electrowetting-controlled liquid mirrors based on self-assembled Janus tiles.

In this paper, we describe a tunable, high-reflectivity optofluidic device based on self-assembly of anisotropically functionalized hexagonal micromirrors (Janus tiles) on the surface of an oil droplet to create a concave liquid mirror. The liquid mirror is deposited on a patterned transparent electrode that allows the focal length and axial position to be electrically controlled. The mirror is mechanically robust and retains its integrity even at high levels of vibrational excitation of the interface. The use of reflection instead of refraction overcomes the limited available refractive-index contrast between pairs of density-matched liquids, allowing stronger focusing than is possible for a liquid lens of the same geometry. This approach is compatible with optical instruments that could provide novel functionality-for example, a dynamic 3D projector, i.e., a light source which can scan an image onto a moving, nonplanar focal surface. Janus tiles with complex optical properties can be manufactured using our approach, thus potentially enabling a wide range of novel optical elements.

Oxygen tension regulates preosteocyte maturation and mineralization.

Oxygen availability is a critical signal for proper development of many tissues, however there is limited knowledge of its role in the maturation of bone cells. To test the hypothesis that low pO2 regulates bone cell mineralization, MLO-A5 and MLO-Y4 cells were cultured in monolayer and three-dimensional alginate scaffolds in hypoxia (2% O2) or normoxia (20% O2). Hypoxia reduced mineralization and decreased alkaline phosphatase activity of preosteocyte-like MLO-A5 cells in both monolayer and alginate cultures. Similar changes in osteogenic activity were seen when the were subjected to chemical hypoxia. Likewise, Osteocyte-like MLO-Y4 cells also exhibited reduced osteogenic activity in hypoxia relative to normoxic controls. Based on these observations, it is concluded that a low pO2 decreased the mineralization potential of bone cells at both early and late stages of maturation. Since the oxemic state is transduced by the transcription factor, HIF-1alpha, experiments were performed to determine if this protein was responsible for the observed changes in mineral formation. It was noted that when HIF-1alpha was silenced, mineralization activities were not restored. Indeed, in hypoxia, in relationship to wild type controls, the mineralization potential of the knockdown cells was further reduced. Based on these findings, it is concluded that the osteogenic activity of preosteocyte-like cells is dependent on both the O2 tension and the expression of HIF-1alpha.