Douglas B. Chrisey

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.

Laser-based direct-write techniques for cell printing

Fabrication of cellular constructs with spatial control of cell location (+/-5 microm) is essential to the advancement of a wide range of applications including tissue engineering, stem cell and cancer research. Precise cell placement, especially of multiple cell types in co- or multi-cultures and in three dimensions, can enable research possibilities otherwise impossible, such as the cell-by-cell assembly of complex cellular constructs. Laser-based direct writing, a printing technique first utilized in electronics applications, has been adapted to transfer living cells and other biological materials (e.g., enzymes, proteins and bioceramics). Many different cell types have been printed using laser-based direct writing, and this technique offers significant improvements when compared to conventional cell patterning techniques. The predominance of work to date has not been in application of the technique, but rather focused on demonstrating the ability of direct writing to pattern living cells, in a spatially precise manner, while maintaining cellular viability. This paper reviews laser-based additive direct-write techniques for cell printing, and the various cell types successfully laser direct-written that have applications in tissue engineering, stem cell and cancer research are highlighted. A particular focus is paid to process dynamics modeling and process-induced cell injury during laser-based cell direct writing.

Barium zirconate-titanate/barium calcium-titanate ceramics via sol–gel process: novel high-energy-density capacitors

Lead-free barium zirconate-titanate/barium calcium-titanate, [(BaZr0.2Ti0.80)O3]1?x?[(Ba0.70Ca0.30)TiO3]x (x = 0.10, 0.15, 0.20) (BZT?BCT) ceramics with high dielectric constant, low dielectric loss and moderate electric breakdown field were prepared by the sol?gel synthesis technique. X-ray diffraction patterns revealed tetragonal crystal structure and this was further confirmed by Raman spectra. Well-behaved ferroelectric hysteresis loops and moderate polarizations (spontaneous polarization, Ps ~ 3?6??C?cm?2) were obtained in these BZT?BCT ceramics. Frequency-dependent dielectric spectra confirmed that ferroelectric diffuse phase transition (DPT) exists near room temperature. Scanning electron microscope images revealed monolithic grain growth in samples sintered at 1280??C. 1000/? versus (T) plots revealed ferroelectric DPT behaviour with estimated ? values of ~1.52, 1.51 and 1.88, respectively, for the studied BZT?BCT compositions. All three compositions showed packing-limited breakdown fields of ~47?73?kV?cm?1 with an energy density of 0.05?0.6?J?cm?3 for thick ceramics (>1?mm). Therefore these compositions might be useful in Y5V-type capacitor applications.

Study of Impact-Induced Mechanical Effects in Cell Direct Writing Using Smooth Particle Hydrodynamic Method

Biomaterial direct-write technologies have been receiving more and more attention as rapid prototyping innovations in the area of tissue engineering, regenerative medicine, and biosensor/actuator fabrication based on computer-aided designs. However, cell damage due to the mechanical impact during cell direct writing has been observed and is a possible hurdle for broad applications of fragile cell direct writing. The objective of this study is to investigate the impact-induced cell mechanical loading profile in cell landing in terms of stress, acceleration, and maximum shear strain component during cell direct writing using a mesh-free smooth particle hydrodynamic method. Such cell mechanical loading profile information can be used to understand and predict possible impact-induced cell damage. It is found that the cell membrane usually undergoes a relatively severe deformation and the cell mechanical loading profile is dependent on the cell droplet initial velocity and the substrate coating thickness. Two important impact processes may occur during cell direct writing: the first impact between the cell droplet and the substrate coating and the second impact between the cell and the substrate. It is concluded that the impact-induced cell damage depends not only on the magnitudes of stress, acceleration, and/or shear strain but also the loading history that a cell experiences.

The Maintenance of Pluripotency Following Laser Direct-Write of Mouse Embryonic Stem Cells

The ability to precisely pattern embryonic stem (ES) cells in vitro into predefined arrays/geometries may allow for the recreation of a stem cell niche for better understanding of how cellular microenvironmental factors govern stem cell maintenance and differentiation. In this study, a new gelatin-based laser direct-write (LDW) technique was utilized to deposit mouse ES cells into defined arrays of spots, while maintaining stem cell pluripotency. Results obtained from these studies showed that ES cells were successfully printed into specific patterns and remained viable. Furthermore, ES cells retained the expression of Oct4 in nuclei after LDW, indicating that the laser energy did not affect their maintenance of an undifferentiated state. The differentiation potential of mouse ES cells after LDW was confirmed by their ability to form embryoid bodies (EBs) and to spontaneously become cell lineages representing all three germ layers, revealed by the expression of marker proteins of nestin (ectoderm), Myf-5 (mesoderm) and PDX-1 (endoderm), after 7 days of cultivation. Gelatin-based LDW provides a new avenue for stem cell patterning, with precision and control of the cellular microenvironment.

Droplet formation in matrix-assisted pulsed-laser evaporation direct writing of glycerol-water solution

Matrix-assisted pulsed-laser evaporation direct-write (MAPLE DW) is emerging as a promising technique for printing microelectronics as well as fabricating biological constructs. For disparate MAPLE DW-based microfabrication applications, the droplet formation during MAPLE DW should be first carefully understood. Toward this goal, this study aims to study the effects of laser fluence and material properties of material to be transferred on the formed droplet in direct writing glycerol-water droplets using MAPLE DW. It was found that (1) at a given glycerol concentration ratio, the droplet diameter was linearly dependent on the laser fluence, and the slope of this relationship was dependent on the glycerol concentration, and (2) the droplet diameter had no systematic relationship with the glycerol concentration ratio. This study reveals important phenomena for droplet formation in MAPLE DW; further theoretical modeling is expected to further explain these observations.Matrix-assisted pulsed-laser evaporation direct-write (MAPLE DW) is emerging as a promising technique for printing microelectronics as well as fabricating biological constructs. For disparate MAPLE DW-based microfabrication applications, the droplet formation during MAPLE DW should be first carefully understood. Toward this goal, this study aims to study the effects of laser fluence and material properties of material to be transferred on the formed droplet in direct writing glycerol-water droplets using MAPLE DW. It was found that (1) at a given glycerol concentration ratio, the droplet diameter was linearly dependent on the laser fluence, and the slope of this relationship was dependent on the glycerol concentration, and (2) the droplet diameter had no systematic relationship with the glycerol concentration ratio. This study reveals important phenomena for droplet formation in MAPLE DW; further theoretical modeling is expected to further explain these observations.

Medical prototyping using two photon polymerization

Two photon polymerization involves nearly simultaneous absorption of ultrashort laser pulses for selective curing of photosensitive material. This process has recently been used to create small-scale medical devices out of several classes of photosensitive materials, such as acrylate-based polymers, organically-modified ceramic materials, zirconium sol-gels, and titanium-containing hybrid materials. In this review, the use of two photon polymerization for fabrication of several types of small-scale medical devices, including microneedles, artificial tissues, microfluidic devices, pumps, sensors, and valves, from computer models is described. Necessary steps in the development of two photon polymerization as a commercially viable medical device manufacturing method are also considered.

Freeform drop-on-demand laser printing of 3D alginate and cellular constructs.

Laser printing is an orifice-free printing approach and has been investigated for the printing of two-dimensional patterns and simple three-dimensional (3D) constructs. To demonstrate the potential of laser printing as an effective bioprinting technique, both straight and Y-shaped tubes have been freeform printed using two different bioinks: 8% alginate solution and 2% alginate-based mouse fibroblast suspension. It has been demonstrated that 3D cellular tubes, including constructs with bifurcated overhang structures, can be adequately fabricated under optimal printing conditions. The post-printing cell viabilities immediately after printing as well as after 24 h incubation are above 60% for printed straight and Y-shaped fibroblast tubes. During fabrication, overhang and spanning structures can be printed using a dual-purpose crosslinking solution, which also functions as a support material. The advancement distance of gelation reaction front after a cycle time of the receiving platform downward motion should be estimated for experimental planning. The optimal downward movement step size of receiving platform should be chosen to be equal to the height of ungelled portion of a previously printed layer.

In Vitro/In Vivo Toxicity Evaluation and Quantification of Iron Oxide Nanoparticles.

Increasing biomedical applications of iron oxide nanoparticles (IONPs) in academic and commercial settings have alarmed the scientific community about the safety and assessment of toxicity profiles of IONPs. The great amount of diversity found in the cytotoxic measurements of IONPs points toward the necessity of careful characterization and quantification of IONPs. The present document discusses the major developments related to in vitro and in vivo toxicity assessment of IONPs and its relationship with the physicochemical parameters of IONPs. Major discussion is included on the current spectrophotometric and imaging based techniques used for quantifying, and studying the clearance and biodistribution of IONPs. Several invasive and non-invasive quantification techniques along with the pitfalls are discussed in detail. Finally, critical guidelines are provided to optimize the design of IONPs to minimize the toxicity.

Cobalt ferrite nanoparticles: Achieving the superparamagnetic limit by chemical reduction

An unanticipated superparamagnetic response has been observed in cobalt ferrite materials after thermal treatment under inert atmosphere. Cobalt ferrite particles were prepared via normal micelle precipitation that typically yields CoxFe3−xO4 nanoparticles (x=0.6−1.0). While samples thermally treated under oxygen show majority spinel phase formation, annealing in nitrogen gas yields materials consisting of Co-Fe alloy, FeS, and CoFe2O4 spinel. After thermal treatment, thermomagnetic studies reveal composition-insensitive, but highly treatment-sensitive, saturation magnetization, coercivity, blocking temperature, and Verwey transition temperature dependence. Extremely high saturation magnetization (159 emu/g) with low coercivity (31 Oe) was observed for one of the treated compositions, which drastically deviates from prototypical cobalt ferrite with large magnetocrystalline anisotropy. We attribute such unique magnetic response to Co-Fe alloy coexisting with FeS and CoFe2O4 spinel where the diameter of the...