The long-term goal of our research is to engineer an acellular nerve graft for clinical nerve repair and for use as a model system with which to study nerve-extracellular matrix interactions during nerve regeneration. To develop this model acellular nerve graft we (1) examined the effects of detergents on peripheral nerve tissue, and (2) used that knowledge to create a nerve graft devoid of cells with a well-preserved extracellular matrix. Using histochemistry and Western analysis, the impact of each detergent on cellular and extracellular tissue components was determined. An optimized protocol was created with the detergents Triton X-200, sulfobetaine-16, and sulfobetaine-10. This study represents the most comprehensive examination to date of the effects of detergents on peripheral nerve tissue morphology and protein composition. Also presented is an improved chemical decellularization protocol that preserves the internal structure of native nerve more than the predominant current protocol.

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A living cell microarray consists of an orderly arrangement of cells attached to a solid support such as a glass microscope slide. The chief difficulty of obtaining such arrays is the fabrication of substrates patterned with micro-wells, adhesive spots, or other features to guide orderly cell attachment. Here we report a novel method using woven Nylon mesh to micropattern three-dimensional alginate hydrogel grids on glass slides. The Nylon mesh is both inexpensive and off-the-shelf. By using Nylon mesh we have eliminated any need for lithography, clean room equipment, and microarray printers to generate microarray patterns; thus, this technique can be easily adopted by biological research labs that may lack microfabrication expertise and facilities. We have demonstrated that glass slides micropatterned with hydrogel grids guide the orderly attachment of single fibroblast cells and Schwann cell clusters in microarrays. The fibroblast arrays consisted of 70 microm square compartments at a density of 21,000 compartments per cm(2). The Schwann cell arrays consisted of 100 microm square compartments at a density of 6000 per cm(2). This patterning technique addresses the need for a simple, inexpensive, benchtop method for micro-patterning glass slides and obtaining living cell microarrays.

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