Isolated node engineering of neuronal systems using laser direct write.

Current limitations to the engineering of ex vivo and in vitro neural environments are hampering the ability to understand underlying neurophysiology. High levels of spatial specificity, reproducibility and viability have been previously reported using laser direct write (LDW) to print cells. However, despite the significant need no one has yet reported laser assisted printing of primary mammalian neuronal cells, an inherently sensitive but critically important population. Herein, we describe the use of LDW to reproducibly and accurately pattern viable dorsal root ganglion (DRG) neurons and supportive cells capable of neural outgrowth and network formation. Our demonstrated ability to engineer and control distinct micro-environmental components unlocks the potential for high throughput experiments to both understand underlying physiology and investigate therapeutic interventions.

Fabrication of magnetite-based core-shell coated nanoparticles with antibacterial properties.

We report the fabrication of biofunctionalized magnetite core/sodium lauryl sulfate shell/antibiotic adsorption-shell nanoparticles assembled thin coatings by matrix assisted pulsed laser evaporation for antibacterial drug-targeted delivery. Magnetite nanoparticles have been synthesized and subsequently characterized by transmission electron microscopy and x-ray diffraction. The obtained thin coatings have been investigated by FTIR and scanning electron microscope, and tested by in vitro biological assays, for their influence on in vitro bacterial biofilm development and cytotoxicity on human epidermoid carcinoma (HEp2) cells.

Generating size-controlled embryoid bodies using laser direct-write.

Embryonic stem cells (ESCs) have the potential to self-renew and differentiate into any specialized cell type. One common method to differentiate ESCs in vitro is through embryoid bodies (EBs), three-dimensional cellular aggregates that spontaneously self-assemble and generally express markers for the three germ layers, endoderm, ectoderm, and mesoderm. It has been previously shown that both EB size and 2D colony size each influence differentiation. We hypothesized that we could control the size of the EB formed by mouse ESCs (mESCs) by using a cell printing method, laser direct-write (LDW), to control both the size of the initial printed colony and the local cell density in printed colonies. After printing mESCs at various printed colony sizes and printing densities, two-way ANOVAs indicated that the EB diameter was influenced by printing density after three days (p = 0.0002), while there was no effect of the printed colony diameter on the EB diameter at the same timepoint (p = 0.74). There was no significant interaction between these two factors. Tukey's honestly significant difference test showed that high-density colonies formed significantly larger EBs, suggesting that printed mESCs quickly aggregate with nearby cells. Thus, EBs can be engineered to a desired size by controlling printing density, which will influence the design of future differentiation studies. Herein, we highlight the capacity of LDW to control the local cell density and colony size independently, at prescribed spatial locations, potentially leading to better stem cell maintenance and directed differentiation.

Single-step laser-based fabrication and patterning of cell-encapsulated alginate microbeads.

Alginate can be used to encapsulate mammalian cells and for the slow release of small molecules. Packaging alginate as microbead structures allows customizable delivery for tissue engineering, drug release, or contrast agents for imaging. However, state-of-the-art microbead fabrication has a limited range in achievable bead sizes, and poor control over bead placement, which may be desired to localize cellular signaling or delivery. Herein, we present a novel, laser-based method for single-step fabrication and precise planar placement of alginate microbeads. Our results show that bead size is controllable within 8%, and fabricated microbeads can remain immobilized within 2% of their target placement. Demonstration of this technique using human breast cancer cells shows that cells encapsulated within these microbeads survive at a rate of 89.6%, decreasing to 84.3% after five days in culture. Infusing rhodamine dye into microbeads prior to fluorescent microscopy shows their 3D spheroidal geometry and the ability to sequester small molecules. Microbead fabrication and patterning is compatible with conventional cellular transfer and patterning by laser direct-write, allowing location-based cellular studies. While this method can also be used to fabricate microbeads en masse for collection, the greatest value to tissue engineering and drug delivery studies and applications lies in the pattern registry of printed microbeads.

Functionalized magnetite silica thin films fabricated by MAPLE with antibiofilm properties.

We report on the fabrication of magnetite/salicylic acid/silica shell/antibiotics (Fe(3)O(4)/SA/SiO(2)/ATB) thin films by matrix-assisted pulsed laser evaporation (MAPLE) to inert substrates. Fe(3)O(4)-based powder have been synthesized and investigated by XRD and TEM. All thin films were studied by FTIR, SEM and in vitro biological assays using Staphylococcus aureus and Pseudomonas aeruginosa reference strains, as well as eukaryotic HEp-2 cells. The influence of the obtained nanosystems on the microbial biofilm development as well as their biocompatibility has been assessed. For optimum deposition conditions, we obtained uniform adherent films with the composition identical with the raw materials. Fe(3)O(4)/SA/SiO(2)/ATB thin films had an inhibitory activity on the ability of microbial strains to initiate and develop mature biofilms, in a strain- and antibiotic-dependent manner. These magnetite silica thin films are promising candidates for the development of novel materials designed for the inhibition of medical biofilms formed by different pathogenic agents on common substrates, frequently implicated in the etiology of chronic and hard to treat infections.

Cellular motors for molecular manufacturing.

Cells are composed of macromolecular structures of various sizes that act individually or collectively to maintain their viability and perform their function within the organism. This review focuses on one structure, the microtubule, and one of the motor proteins that move along it, conventional kinesin (kinesin 1). Recent work on the cellular functions of kinesins, such as the organization of microtubules during cellular division and the movement of the organelles and vesicles, offers insights into how biological motors might prove useful for organizing structures in engineered environments.

Laser microfabrication of hydroxyapatite-osteoblast-like cell composites.

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.

Two photon induced polymerization of organic-inorganic hybrid biomaterials for microstructured medical devices.

Three-dimensional microstructured medical devices, including microneedles and tissue engineering scaffolds, were fabricated by two photon induced polymerization of Ormocer organic-inorganic hybrid materials. Femtosecond laser pulses from a titanium:sapphire laser were used to break chemical bonds on Irgacure 369 photoinitiator within a small focal volume. The radicalized starter molecules reacted with Ormocer US-S4 monomers to create radicalized polymolecules. The desired structures are fabricated by moving the laser focus in three dimensions using a galvano-scanner and a micropositioning system. Ormocer surfaces fabricated using two photon induced polymerization demonstrated acceptable cell viability and cell growth profiles against B35 neuroblast-like cells and HT1080 epithelial-like cells. Lego-like interlocking tissue engineering scaffolds and microneedle arrays with unique geometries were created using two photon induced polymerization. These results suggest that two photon induced polymerization is able to create medical microdevices with a larger range of sizes, shapes, and materials than chemical isotropic etching, injection molding, reactive ion etching, surface micromachining, bulk micromachining, polysilicon micromolding, lithography-electroforming-replication, or other conventional microfabrication techniques.

Three-dimensional direct writing of B35 neuronal cells.

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.

Picoliter-scale protein microarrays by laser direct write.

We demonstrate the accurate picoliter-scale dispensing of active proteins using a novel laser transfer technique. Droplets of protein solution are dispensed onto functionalized glass slides and into plastic microwells, activating as small as 50-microm diameter areas on these surfaces. Protein microarrays fabricated by laser transfer were assayed using standard fluorescent labeling techniques to demonstrate successful protein and antigen binding. These results indicate that laser transfer does not damage the active site of the dispensed protein and that this technique can be used to successfully fabricate a functioning protein microarray. Also, as a result of the efficient nature of the process, material usage is reduced by two to four orders of magnitude compared to conventional pin dispensing methods for protein spotting.

Generation of mesoscopic patterns of viable Escherichia coli by ambient laser transfer.

We have generated mesoscopic patterns of viable Escherichia coli on Si(1 1 1), glass, and nutrient agar plates by using a novel laser-based transfer process termed matrix assisted pulsed laser evaporation direct write (MAPLE DW). We observe no alterations to the E. coli induced by the laser-material interaction or the shear forces during the transfer. Transferred E. coli patterns were observed by optical and electron microscopes, and cell viability was shown through green fluorescent protein (GFP) expression and cell culturing experiments. The transfer mechanism for our approach appears remarkably gentle and suggests that active biomaterials such as proteins, DNA and antibodies could be serially deposited adjacent to viable cells. Furthermore, this technique is a direct write technology and therefore does not involve the use of masks, etching, or other lithographic tools.

MATERIALS PROCESSING: The Power of Direct Writing.

In a direct-write approach, micrometer-scale structures are built directly without the use of masks, allowing rapid prototyping. Direct-write approaches are enabling faster, cheaper manufacture of electronic components and are also used for tissue engineering and array-based biosensors. In his Perspective, Chrisey provides a short overview of current research in the area of direct-write technologies, focusing on the materials science aspects.

Pulsed laser deposition of thin film hydroxyapatite. Applications for flexible catheters.

Skin exit site infections are a major source of morbidity in patients with indwelling percutaneous catheters. Ceramic materials, such as hydroxyapatite (HA) and alumina, have demonstrated excellent biocompatibility and low rates of infection in soft tissues. Previous attempts to design ceramic materials for use as percutaneous connectors have resulted in rigid discs or solid cylindrical tubes. In order to take advantage of the inherent properties of HA without reducing patient comfort or mobility, the feasibility of applying a thin film of HA directly onto a flexible polymeric catheter was studied. The coating was applied by pulsed laser deposition (PLD). The beam from a KrF excimer laser impinged upon a target of pressed and sintered HA, producing a plume of ablated material that was deposited onto the catheter tubing. By rotating the tubing, an even coating of HA was applied to the catheter at a thickness of approximately 0.50 microm. The coating did not compromise the flexibility of the catheter tubing. Hence, PLD of a thin film of HA at the exit site of percutaneous catheters may be a means of incorporating the bioactive and biocompatible properties of HA with the mobility and patient comfort that characterize polymeric catheters.