Barry J. Spargo

Laser Printing of Pluripotent Embryonal Carcinoma Cells

A technique by which to print patterns and multilayers of scaffolding and living cells could be used in tissue engineering to fabricate tissue constructs with cells, materials, and chemical diversity at the micron scale. We describe here studies using a laser forward transfer technology to print single-layer patterns of pluripotent murine embryonal carcinoma cells. This report focuses on verifying cell viability and functionality as well as the ability to differentiate cells after laser transfer. We find that when cells are printed onto model tissue scaffolding such as a layer of hydrogel, greater than 95% of the cells survive the transfer process and remain viable. In addition, alkaline comet assays were performed on transferred cells, showing minimal single-strand DNA damage from potential ultraviolet-cell interaction. We also find that laser-transferred cells express microtubular associated protein 2 after retinoic acid stimulus and myosin heavy chain protein after dimethyl sulfoxide stimulus, indicating successful neural and muscular pathway differentiation. These studies provide a foundation so that laser printing may next be used to build heterogeneous multilayer cellular structures, enabling cell growth and differentiation in heterogeneous three-dimensional environments to be uniquely studied.

Growth of organic thin films by the matrix assisted pulsed laser evaporation (MAPLE) technique

Abstract A novel variation of conventional pulsed laser evaporation, known as matrix assisted pulsed laser evaporation, or MAPLE, has been successfully used to deposit highly uniform thin films of a variety of organic materials including a number of polymers. The MAPLE technique is carried out in a vacuum chamber and involves directing a pulsed laser beam (λ=193 or 248 nm; fluence=0.01 to 0.5 J/cm2) onto a frozen target (100–200 K) consisting of a solute polymeric or organic compound dissolved in a solvent matrix. The laser beam evaporates the surface layers of the target, with both solvent and solute molecules being released into the chamber. The volatile solvent is pumped away, whereas the polymer/organic molecules coat the substrate. Thin uniform films ( nm) of various materials, such as functionalized polysiloxanes and carbohydrates, have been deposited on Si(111) and NaCl substrates. The films prepared using this method have been examined by optical microscopy, scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy and electrospray mass spectrometry. Careful control of the processing conditions allowed the complex polymer/organic molecules to be transferred to the substrate as uniform films without any significant chemical modification. Using MAPLE, large or small regions within a substrate can be discretely coated with submonolayer thickness control. The use of MAPLE films for chemical sensor applications has been investigated and the potential of this technique for producing high quality thin films of other organic compounds will be discussed.

The deposition, structure, pattern deposition, and activity of biomaterial thin-films by matrix-assisted pulsed-laser evaporation (MAPLE) and MAPLE direct write

Two techniques, Matrix-Assisted Pulsed-Laser Evaporation (MAPLE) and MAPLE Direct Write (MDW) were developed to deposit biomaterial thin-films. MAPLE involves dissolving or suspending the biomaterial in a volatile solvent, freezing the mixture to create a solid target, and using a low fluence pulsed laser to evaporate the target for deposition of the solute inside a vacuum system. Using simple shadow masks, i.e. lines, dots and arrays, pattern features with length scales as small as 20 μm can be deposited using multiple materials on different types of substrates. MDW uses pulsed laser to directly transfer material from a ribbon to a substrate. Patterns with a spatial resolution of ∼10 μm can be written directly. Biomaterials ranging from polyethylene glycol to eukaryotic cells, i.e. Chinese hamster ovaries, were deposited with no measurable damage to their structures or genotype. Deposits of immobilized horseradish peroxidase, an enzyme, in the form of a polymer composite with a protective coating, i.e. polyurethane, retained their enzymatic functions. A dopamine electrochemical sensor was fabricated by MDW using a natural tissues/graphite composite. These examples and the unique features of MAPLE and MDW for biosensor fabrication have been discussed.

Laser transfer of biomaterials: Matrix-assisted pulsed laser evaporation (MAPLE) and MAPLE Direct Write

Two techniques for transferring biomaterial using a pulsed laser beam were developed: matrix-assisted pulsed laser evaporation (MAPLE) and MAPLE direct write (MDW). MAPLE is a large-area vacuum based technique suitable for coatings, i.e., antibiofouling, and MDW is a localized deposition technique capable of fast prototyping of devices, i.e., protein or tissue arrays. Both techniques have demonstrated the capability of transferring large (mol wt>100 kDa) molecules in different forms, e.g., liquid and gel, and preserving their functions. They can deposit patterned films with spatial accuracy and resolution of tens of μm and layering on a variety of substrate materials and geometries. MDW can dispense volumes less than 100 pl, transfer solid tissues, fabricate a complete device, and is computed aided design/computer aided manufacturing compatible. They are noncontact techniques and can be integrated with other sterile processes. These attributes are substantiated by films and arrays of biomaterials, e.g., polymers, enzymes, proteins, eucaryotic cells, and tissue, and a dopamine sensor. These examples, the instrumentation, basic mechanisms, a comparison with other techniques, and future developments are discussed.

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.

Fabrication and selective surface modification of 3‐dimensionally textured biomedical polymers from etched silicon substrates

A new method is described for producing biomedically relevant polymers with precisely defined micron scale surface texture in the x, y, and z planes. Patterned Si templates were fabricated using photolithography to create a relief pattern in photoresist with lateral dimensions as small as 1 micron. Electroless Ni was selectively deposited in the trenches of the patterned substrate. The Ni served as a resilient mask for transferring the patterns onto the Si substrate to depths of up to 8.5 microns by anisotropic reactive ion etching with a fluorine-based plasma. The 3-dimensional (3-D) textured silicon substrates were used as robust, reusable molds for pattern transfer onto poly (dimethyl siloxane), low density poly (ethylene), poly (L-lactide), and poly (glycolide) by either casting or injection molding. The fidelity of the pattern transfer from the silicon substrates to the polymers was 90 to 95% in all three planes for all polymers for more than 60 transfers from a single wafer, as determined by scanning electron microscopy and atomic force microscopy. Further, the 3-D textured polymers were selectively modified to coat proteins either in the trenches or on the mesas by capillary modification or selective coating techniques. These selectively patterned 3-D polymer substrates may be useful for a variety of biomaterial applications.

Controlled release of transforming growth factor-β from lipid-based microcylinders

The release of transforming growth factor-beta (TGF-beta) from a lipid microstructure has been demonstrated. Lipid microcylinders, with dimensions of 100 x 0.5 microns and composed of a diacetylenic lipid, have been loaded with 25 ng TGF-beta/mg lipid. Physical and bioactive release characteristics of TGF-beta from these microcylinders and from microcylinders embedded in an agarose hydrogel are reported. Release of TGF-beta from lipid microcylinders follows typical diffusion-limited characteristics, where 10-12% of the TGF is released in the first 10 h at 37 degrees C. The release rate is shown to be temperature controlled and dependent on the integrity of the lipid microcylinder. Immobilization of the lipid microcylinder in a hydrogel matrix composed of agarose and gelatin does not impair the diffusion of TGF-beta from the lipid microcylinders. The utilization of microcylinders as release vehicles in wound repair is discussed.

Dissolved oxygen saturation controls PAH biodegradation in freshwater estuary sediments.

Polycyclic aromatic hydrocarbons (PAHs) are common contaminants in terrestrial and aquatic environments and can represent a significant constituent of the carbon pool in coastal sediments. We report here the results of an 18-month seasonal study of PAH biodegradation and heterotrophic bacterial production and their controlling biogeochemical factors from 186 sediment samples taken in a tidally influenced freshwater estuary. For each sampling event, measurements were averaged from 25–45 stations covering ∼250 km2. There was a clear relationship between bacterial production and ambient temperature, but none between production and bottom water dissolved oxygen (DO) % saturation or PAH concentrations. In contrast with other studies, we found no effect of temperature on the biodegradation of naphthalene, phenanthrene, or fluoranthene. PAH mineralization correlated with bottom water DO saturation above 70% (r2 > 0.99). These results suggest that the proportional utilization of PAH carbon to natural organic carbon is as much as three orders of magnitude higher during cooler months, when water temperatures are lower and DO % saturation is higher. Infusion of cooler, well-oxygenated water to the water column overlying contaminated sediments during the summer months may stimulate PAH metabolism preferentially over non-PAH organic matter.

Transport, Deposition and Biodegradation of Particle Bound Polycyclic Aromatic Hydrocarbons in a Tidal Basin of an Industrial Watershed

Polycylic aromatic hydrocarbons (PAHs) are commoncontaminants in industrial watersheds. Their origin,transport and fate are important to scientists,environmental managers and citizens. The Philadelphia NavalReserve Basin (RB) is a small semi-enclosed embayment nearthe confluence of the Schuylkill and Delaware Rivers inPennsylvania (USA). We conducted a study at this site todetermine the tidal flux of particles and particle-boundcontaminants associated with the RB. Particle traps wereplaced at the mouth and inside the RB and in the Schuylkilland Delaware Rivers. There was net particle deposition intothe RB, which was determined for three seasons. Spring andfall depositions were highest (1740 and 1230 kg ofparticles, respectively) while winter deposition wasinsignificant. PAH concentrations on settling particlesindicated a net deposition of 12.7 g PAH in fall and 2.1 gPAH in spring over one tidal cycle. There was nosignificant PAH deposition in the winter. Biodegradationrates, calculated from 14C-labeled PAH substratemineralization, could attenuate only about 0.25% of the PAHdeposited during a tidal cycle in fall. However, in thespring, biodegradation could be responsible for degrading50% of the settling PAHs. The RB appears to be a sink forPAHs in this watershed.

Recruitment of tissue resident cells to hydrogel composites: in vivo response to implant materials.

A model of local cellular recruitment was established using hydrogel matrices composed of alginate implanted subcutaneously into mice. Cells which trafficked to the matrix blocks were recovered and characterized for surface phenotype using fluorescently labelled antibodies and flow cytometry (fluorescence activated cell sorting). Temporal information of the differential recruitment of cells was determined. The basic pattern of recruitment in response to the hydrogels was established and mimicked that seen in a local inflammatory response. Neutrophils (PMN) were rapidly recruited (1 d) followed by macrophages and lymphocytes (1-3 d). Cell surface phenotype studies included the determination of CD3+, CD4+ and CD8+ cells, Mac-1+ cells, and immunoglobulin bearing cells. Microscopic analysis revealed numerous activated PMNs and monocyte derived foamy macrophages. Fluorescence immunocytochemistry of frozen sections of the block revealed that macrophages, CD3+ and natural killer cells were all recruited to the interior of the block. Ultrastructural analysis (transmission electron microscopy) showed highly activated macrophages, with abundant rough endoplasmic reticulum and secretory vesicles. Cells which remained on the surface of the matrix block were CD44 positive migratory cells. Electron microscopic evidence showed foamy macrophages with a varying degree of involvement with the hydrogel material. Surface scanning electron microscopy revealed numerous fibroblast-like cells coating the surface of the block. We suggest that these methods may be used to address the inflammatory response elicited with a a variety of implanted materials such as hydrogels, silicones, ceramics and metals. Furthermore, this model has been useful in determining cellular responses to cytokines and growth factors under similar conditions.