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Additive manufacturing guided by computer-aided technologies has enabled new possibilities in tissue engineering. Laser-based cell printing techniques are at the forefront due to the ability to deposit single cells with high accuracy and reproducibility (1 cell, +/−2 μm, 500 cells/sec). Engineered tissue constructs can now be fabricated by layered manufacturing with single cell precision and complete heterogeneous fidelity. As such, researchers are taking aim at fundamental challenges within tissue engineering, including vascularization. Laser direct write techniques are being utilized to fabricate vasculature and investigate vascular interactions with the goal to produce larger tissue substitutes that to this point have been restricted by nutrient flow (<500 μm). This review discusses the recent advancements in laser-based cell printing techniques, their applications, and how they seek to address the vascularization issue.

Abstract

Polymer–ceramic nanocomposites have been thoroughly investigated previously for high energy storage devices. However, the increase in performance of these nanocomposites has proven to be significantly lower than predicted values. Through surface functionalization of high dielectric constant nanoparticles (NP), the flaws that reduce composite performance can be eliminated to form high energy density composite materials. Functionalization methods utilize high throughput printing and curing techniques (i.e., inkjet printing and xenon flash lamp curing) that are crucial for rapid adoption into industrial production. (Ba,Ca) (Zr,Ti)O3NPs (50 nm) are synthesized through the solvothermal method and functionalized with alkene terminated methoxysilanes. A thiol−ene monomer ink system, PTD3 [pentaerythritol tetrakis (3-mercaptopropionate) (PEMP, P), 1,3-Diisopropenylbenzene (DPB, D), 2,4,6-Triallyloxy-1,3,5-triazine (TOTZ, T)], is used as a high breakdown polymer matrix. Neat polymer, alkene terminated NP–polymer composites, and hydrophilic, TBAOH functionalized NP–polymer composites were spin coated onto both copper laminated glass slides and printed onto copper substrates in 1 cm2patterns for testing. Alkene functionalized NPs increased the breakdown strength by ∼38% compared to the nonfunctionalized NPs. Functionalized NPs increased both the breakdown strength and dielectric constant compared to the neat polymer, increasing the energy density nearly 3-fold from 13.3 to 36.1 J·cm–3.

Abstract

Fifteen years ago, the field of cell and organ printing began with a few research groups looking to take newly developed direct-write tools and apply them to living cells. Initial experiments demonstrated cell viability and functionality post-deposition. Recently, research has begun in earnest to create three-dimensional cellular constructs that mimic both the heterogeneous structure and function of natural tissue. Several companies are now marketing cell printers, expanding access to a wider group of scientists and accelerating the pace of development. This article describes the past decade and a half of research by showing examples of some of the most sophisticated work, comparing the approaches and tools used in the field, and predicting the products that will arrive in the not too distant future.

Engineering strategies guided by developmental biology may enhance and accelerate in vitrotissue formation for tissue engineering and regenerative medicine applications. In this study, we looked toward embryonic tendon development as a model system to guide our soft tissue engineering approach. To direct cellular self-assembly, we utilized laser micromachined, differentially adherent growth channels lined with fibronectin. The micromachined growth channels directed human dermal fibroblast cells to form single cellular fibers, without the need for a provisional three-dimensional extracellular matrix or scaffold to establish a fiber structure. Therefore, the resulting tissue structure and mechanical characteristics were determined solely by the cells. Due to the self-assembly nature of this approach, the growing fibers exhibit some key aspects of embryonic tendon development, such as high cellularity, the rapid formation (within 24 h) of a highly organized and aligned cellular structure, and the expression of cadherin-11 (indicating direct cell-to-cell adhesions). To provide a dynamic mechanical environment, we have also developed and characterized a method to apply precise cyclic tensile strain to the cellular fibers as they develop. After an initial period of cellular fiber formation (24 h postseeding), cyclic strain was applied for 48 h, in 8-h intervals, with tensile strain increasing from 0.7% to 1.0%, and at a frequency of 0.5 Hz. Dynamic loading dramatically increased cellular fiber mechanical properties with a nearly twofold increase in both the linear region stiffness and maximum load at failure, thereby demonstrating a mechanism for enhancing cellular fiber formation and mechanical properties. Tissue engineering strategies, designed to capture key aspects of embryonic development, may provide unique insight into accelerated maturation of engineered replacement tissue, and offer significant advances for regenerative medicine applications in tendon, ligament, and other fibrous soft tissues.

AgCl cubes have been fabricated by excimer laser ablation of a silver target in aqueous NaCl solutions with concentration CNaCl≤ 0.01 M. Absorption spectra of the laser ablated solutions indicated that Ag nanoclusters also formed during the ablation. The formation of Ag and AgCl are dependent on CNaCl, laser fluence, and length of ablation time (laser pulses). We consider that the laser ablation of Ag in NaCl solution will produce Ag+ions that combine with Cl−and form insoluble AgCl precipitates. The AgCl could catalyze the reduction of excess Ag+ions resulting in Ag nanoclusters. When CNaCl≥ 0.05 M, soluble complex anions of the type AgClm+1m−will form that reduce the formation of AgCl and Ag. Our research reveals the underlying mechanism of laser ablation of Ag in aqueous NaCl solution and provides a facile route to fabricate cuboidal AgCl particles.

Laser direct-writing provides a method to pattern living cells in vitro, to study various cell–cell interactions, and to build cellular constructs. However, the materials typically used may limit its long-term application. By utilizing gelatin coatings on the print ribbon and growth surface, we developed a new approach for laser cell printing that overcomes the limitations of Matrigel™. Gelatin is free of growth factors and extraneous matrix components that may interfere with cellular processes under investigation. Gelatin-based laser direct-write was able to successfully pattern human dermal fibroblasts with high post-transfer viability (91% ± 3%) and no observed double-strand DNA damage. As seen with atomic force microscopy, gelatin offers a unique benefit in that it is present temporarily to allow cell transfer, but melts and is removed with incubation to reveal the desired application-specific growth surface. This provides unobstructed cellular growth after printing. Monitoring cell location after transfer, we show that melting and removal of gelatin does not affect cellular placement; cells maintained registry within 5.6 ± 2.5 μm to the initial pattern. This study demonstrates the effectiveness of gelatin in laser direct-writing to create spatially precise cell patterns with the potential for applications in tissue engineering, stem cell, and cancer research.

Herein we show how the unique temporal and thermal events occurring during the pulsed excimer laser ablation of an Al target in water result in hollow Al2O3micro/nanoparticles with smooth surfaces and an amorphous structure. We demonstrate that the hollow particles are formed on laser-induced bubbles from laser ionized/evaporated liquid during the ablation. The fabrication of hollow particles can be improved by the addition of ethanol to the water, and the particles contain crystalline Al. Our work and the associated mechanism represent a new paradigm to fabricate hollow particles directly from bulk material; that is, the excimer laser ablation produces nanoclusters from the target and bubbles from the liquid, and the bubble interfaces trap the nanoclusters, resulting in the formation of hollow particles.

We report the fabrication of permalloy nanoparticles by pulsed excimer laser ablation of a permalloy (Fe19Ni81) target in sodium dodecyl sulfate aqueous solution and the subsequent laser-induced assembly, sintering, and fragmentation of the nanoparticles. Specifically, permalloy nanoparticles with diameters of 400−600 nm were observed to assemble into fullerene-like hollow microparticles. Laser irradiation caused sintering of the assembled particles, which showed Wulff construction and ledge growth behavior and finally resulted in smooth shells. Laser−particle interactions also caused fragmentation of the micro/nanoparticles. When the particle concentration is high, laser fragmentation rather than laser ablation dominates due to enhanced light absorption and scattering. The experiments reveal the dynamic process of the nano- and microparticle formation, demonstrating the rich processing environment of laser ablation in liquid.

The survival, proliferation, and differentiation of freshly isolated and cultured cells were studied after absorbing film-assisted laser-induced forward transfer. Rat Schwann and astroglial cells and pig lens epithelial cells were used for transfer and the cells were cultured for 2 weeks after laserpulsed transfer. All three cell types survived, proliferated, and differentiated under cell culture conditions and regained their original phenotype a few days after cell transfer. Time resolution studies have shown that the time required to accelerate the jets and droplets containing the cells was less than 1 µs and that the estimated minimum average acceleration of those ejected cells that reached a constant velocity was approximately 107 × g. This suggests that the majority of studied cells tolerated the extremely high acceleration at the beginning of the ejection and the deceleration during impact on the acceptor plate without significant damage to the original phenotype. These results suggest that the absorbing film-assisted laser-induced forward transfer technique appears to be suitable for several potential applications in tissue engineering and the biomedical tissue repair technologies.

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.

Pulsed laser deposition (PLD) has been used to grow a series of NbNx(0 < x< 1.2) thin-film materials, including a new superconducting phase. The structural and electrical properties of the NbNxfilms were characterized. Structural analysis of the new NbN phase indicates that it is stabilized by heteroepitaxial growth on (100) MgO and is shown to be a primitive cubic distortion from the typical B1, or rock-salt structure. In addition, the NbN phase has a higher superconducting critical temperature and a larger lattice parameter when compared with films of B1-NbN. Growth of this new phase demonstrates that the nonequilibrium synthesis properties of PLD can be used to deposit new, metastable materials.

This paper reports on synthesis of polycrystalline complex perovskite CaCu3Ti4O12(as CCTO) ceramic powders prepared by a sol–gel auto combustion method at different sintering temperatures and sintering times, respectively. The effect of sintering time on the structure, morphology, dielectric and electrical properties of CCTO ceramics is investigated. Tuning the electrical properties via different sintering times is demonstrated for ceramic samples. X-ray diffraction (XRD) studies confirm perovskite-like structure at room temperature. Abnormal grain growth is observed for ceramic samples. Giant dielectric permittivity was realized for CCTO ceramics. High dielectric permittivity was attributed to the internal barrier layer capacitance (IBLC) model associated with the Maxwell–Wagner (MW) polarization mechanism.

Polycrystalline La-doped BaTiO3(Ba(1-x)LaxTiO3)[x=0,0.0005,0.001,0.003] ceramics (denoted as BTO,BLT1,BLT2,BLT3) were synthesized by conventional solid-state reaction method and characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and Raman spectroscopy. XRD and Raman spectra revealed single-phase tetragonal perovskite crystalline structure. Well-saturated polarization–electric field (P–E) hysteresis loops were observed with the measurement frequency of 50 Hz at room temperature and confirmed ferroelectric nature of these ceramics and a high recoverable electrical energy storage density of 0.350 J/cm3with energy efficiency (n)∼9%, which is useful in energy storage capacitor applications. Dielectric studies revealed anomalies around 415–420 K and near the Curie temperature. The latter is attributed to the ferroelectric to paraelectric phase transition. Better dielectric performances were obtained for La-doped samples sintered at 1350°C for 4 h. Grain growth is inhibited with lanthanum (La) incorporation into the BTO lattice. Room temperature semiconducting behavior with positive temperature coefficient of resistivity (PTCR) behavior at TCis attributed to electron compensation mechanism.