We have developed a novel approach for layer-by-layer growth of tissue-engineered materials using a direct writing process known as matrix assisted pulsed laser evaporation direct write (MAPLE DW). Unlike conventional cell-seeding methods, this technique provides the possibility for cell-material integration prior to artificial tissue fabrication. This process also provides greater flexibility in selection and processing of scaffold materials. In addition, MAPLE DW offers rapid computer-controlled deposition of mesoscopic voxels at high spatial resolutions. We have examined MAPLE DW processing of zirconia and hydroxyapatite scaffold materials that can provide a medical device with nearly inert and bioactive implant-tissue interfaces, respectively. We have also demonstrated codeposition of hydroxyapatite, MG 63 osteoblast-like cells, and extracellular matrix using MAPLE DW. We have shown that osteoblast-like cells remain viable and retain the capacity for proliferation when codeposited with bioceramic scaffold materials. Our results on MG 63-hydroxyapatite composites can be extended to develop other integrated cell-scaffold structures for medical and dental applications.
We have demonstrated two-dimensional and three-dimensional transfer of B35 neuronal cells onto and within polymerized Matrigel substrates, using matrix-assisted pulsed laser evaporation-direct write (MDW). The B35 cells were transferred from a quartz ribbon to depths of up to 75 microm by systematically varying the fluence emitted from the ArF (lambda = 193 nm) laser source. MDW-transferred cells were examined using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), 4',6-diamidino-2-phenylindole (DAPI), and alpha-tubulin staining. Confocal microscopy has shown that the transferred B35 cells extended their axons outward in three dimensions within the polymerized Matrigel substrate. The B35 cells made axonal connections and formed a three-dimensional neural network within 72 h after MDW transfer. In addition, TUNEL staining demonstrated that only 3% of the B35 cells underwent apoptosis after being transferred using the MDW process. MDW and other emergent direct write processes may provide unique approaches for creating layered, heterogeneous, three-dimensional cell-seeded scaffolds for use in peripheral nerve repair.
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