Investigation of Polyurea-Crosslinked Silica Aerogels as a Neuronal Scaffold: A Pilot Study

BACKGROUND: Polymer crosslinked aerogels are an attractive class of materials for future implant applications particularly as a biomaterial for the support of nerve growth. The low density and nano-porous structure of this material combined with large surface area, high mechanical strength, and tunable surface properties, make aerogels materials with a high potential in aiding repair of injuries of the peripheral nervous system. however, the interaction of neurons with aerogels remains to be investigated. METHODOLOGY: In this work the attachment and growth of neurons on clear polyurea crosslinked silica aerogels (PCSA) coated with: poly-L-lysine, basement membrane extract (BME), and laminin1 was investigated by means of optical and scanning electron microscopy. After comparing the attachment and growth capability of neurons on these different coatings, laminin1 and BME were chosen for nerve cell attachment and growth on PCSA surfaces. The behavior of neurons on treated petri dish surfaces was used as the control and behavior of neurons on treated PCSA discs was compared against it. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that: 1) untreated PCSA surfaces do not support attachment and growth of nerve cells, 2) a thin application of laminin1 layer onto the PCSA discs adhered well to the PCSA surface while also supporting growth and differentiation of neurons as evidenced by the number of processes extended and b3-tubulin expression, 3) three dimensional porous structure of PCSA remains intact after fixing protocols necessary for preservation of biological samples and 4) laminin1 coating proved to be the most effective method for attaching neurons to the desired regions on PCSA discs. This work provides the basis for potential use of PCSA as a biomaterial scaffold for neural regeneration

SEM images of laminin-aerogel-neuron interface on PCSA surface.

<p>(a) Growth of nerve cells on the laminin-coated section of the PCSA disc compared with growth of neurons directly on the aerogel surface in the absence of laminin (after 7 days). An abundance of cells have been immobilized directly on the aerogel surface but no processes have been extended. The laminin layer is necessary for processes to develop. (b) Higher magnification image of the boundary region highlighting the difference in the behaior of nerve cells on the different surfaces. Desnity of cells in both regions is equal. Dotted line shows boundary between the two regions.</p

Aerogel pores intact after fixing treatment.

<p>(a) SEM image of PCSA after fixation and sputter-coating- off the laminin spot. The image shows the porous structure is still intact after the fixing treatment has been performed. (b) Image of a pore opening post fixing and sputtering stage.</p

SEM image of neurons connecting on laminin-coated PCSA.

<p>(<b>a</b>) A dense array of processes have extended across the area of PCSA covered by laminin. Several layers of nerve cells with crisscrossing neuritis can be observed. The pores of the PCSA can be clearly seen and some processes may have extended into the pores as indicated by the arrows. (b) SEM image of the edge of a PCSA disc+Laminin+neurons showing the grooves created by the saw blade. It is hypothesized that the non homogoenetiy of the surface provides better anchoring and attachment oportunities for the cell body and the proceeses to be extended.</p

Aerogel translucency as a function of material thickness.

<p>Optical images of (a) 2 mm thick, (b) 3 mm thick, (c) 5 mm thick and (d) 10 mm thick PCSA discs with varying diameters, demonstrating the translucency of the PCSA discs as a function of thickness. All have densities close to 0.4 g/cm³.</p

BME restricts nerve cell growth on glass.

<p>Optical microscope images of crystal violet stained with: (a) Poly-L-lysine coating (400× magnification) on glass coverslips not able to support growth and confinement of nerve cells.; (b) A 5 µl drop of BME (40× magnification) restricts nerve cell growth to the area of the drop. Arrows in both images indicating the boundary between the treated and untreated regions. Images were taken 5 days after plating.</p

Effect of laminin on neuron growth.

<p>DRG neurons were plated on aerogel surfaces and grown for 7 days (to confluence) prior to processing for SEM. (a) SEM of nerve growth profile off the laminin dot, directly on the aerogel substrate. Arrows indicating attached nerve cells with no processes extended. (b) SEM of DRGs grown on a laminin drop on PCSA disc post-fixation and sputtering. DRGs cell bodies (*) are extending processes ( ) that interact with those of other nerves. Sample processing (freezing and sputter-coating) has lead to the fracturing of the lamimin layer (#). Images taken post-fixation and sputtering stage.</p

Neuron contact pattern.

<p>SEM images of neighboring neurons extending processes on: (a) surface of laminin-coated PCSA. Image was taken from the edge of PCSA disc capturing the interaction of several neurons. (b) Wide view of two neurons extending processes on a petri dish surface. Fewer processes have been extended in the latter case.</p

Mapping coastal Great Lakes wetlands and adjacent land use through hybrid optical-infrared and radar image classification techniques

A mapping effort is underway through GLRI funding to produce an international contemporary baseline map of wetland type, extent and adjacent land use in the Great Lakes Basin. This includes all area within 10 kilometers of the U.S. and Canadian coasts. Improved mapping and monitoring of the Great Lakes coastal wetlands will be achieved through a fusion of Synthetic Aperture Radar (SAR) and optical/infrared satellite imagery. The combination of sensor frequencies complement one another in the identification of wetlands and adjacent land use. MTRI developed techniques to merge optical/IR and SAR for the mapping of Great Lakes coastal wetlands and adjacent land use in demonstration areas in 2004 for the Great Lakes Coastal Wetlands Consortium (GLCWC). That pilot study demonstrated the improved capabilities of merging multi-sensor SAR and Landsat data for better wetland condition monitoring. A significant database of Japanese ALOS-PALSAR imagery and field validation/training sites has already been constructed for use in monitoring Phragmites australis on the U.S. side of the Great Lakes coastal zone. These data will be combined with Landsat 5 data and new field collections to achieve the final mapped product. Hybrid algorithms are currently being refined through analysis of a series of pilot study areas