Study of Impingement Types and Printing Quality during Laser Printing of Viscoelastic Alginate Solutions.

Laser-induced forward transfer-based laser printing has been being implemented as a promising orifice-free direct-write strategy for different printing applications. The printing quality during laser printing is largely affected by the jet and droplet formation process and subsequential impingement. The objective of this study is to investigate the impingement-based printing type and resulting printing quality during the laser printing of viscoelastic alginate solutions, which are representative inks for soft structure printing such as bioprinting. Three printing types are identified: droplet-impingement printing, jet-impingement printing with multiple breakups, and jet-impingement printing with a single breakup. Printing quality, in terms of printed droplet morphology and size, has been investigated as a function of alginate concentration, laser fluence, and direct-writing height based on a time-resolved imaging approach and microarrays of printed droplets. Of these, the best printing quality is achieved with single-breakup jet-impingement printing, followed by multiple-breakup jet-impingement printing, with droplet-impingement printing producing the lowest quality printing. The printing quality can be improved by using high-concentration alginate solutions. The increase of laser fluence may lead to a well-defined primary droplet for low-concentration alginate solutions; however, this can cause the droplet diameter to increase, which may not be desirable. The direct-writing height (i.e., ribbon coating-receiving substrate distance) also influences the print quality. For example, an increase in direct-writing height can cause the printing type to change from the ideal jet-impingement with a single breakup, to the jet-impingement with multiple breakups, and even the least desired droplet-impingement printing, with only slight variations in droplet diameter.

Time-Resolved Imaging Study of Jetting Dynamics during Laser Printing of Viscoelastic Alginate Solutions.

Matrix-assisted pulsed-laser evaporation direct-write (MAPLE DW) has been successfully implemented as a promising laser printing technology for various fabrication applications, in particular, three-dimensional bioprinting. Since most bioinks used in bioprinting are viscoelastic, it is of importance to understand the jetting dynamics during the laser printing of viscoelastic fluids in order to control and optimize the laser printing performance. In this study, MAPLE DW was implemented to study the jetting dynamics during the laser printing of representative viscoelastic alginate bioinks and evaluate the effects of operating conditions (e.g., laser fluence) and material properties (e.g., alginate concentration) on the jet formation performance. Through a time-resolved imaging approach, it is found that when the laser fluence increases or the alginate concentration decreases, the jetting behavior changes from no material transferring to well-defined jetting to well-defined jetting with an initial bulgy shape to jetting with a bulgy shape to pluming/splashing. For the desirable well-defined jetting regimes, as the laser fluence increases, the jet velocity and breakup length increase while the breakup time and primary droplet size decrease. As the alginate concentration increases, the jet velocity and breakup length decrease while the breakup time and primary droplet size increase. In addition, Ohnesorge, elasto-capillary, and Weber number based phase diagrams are presented to better appreciate the dependence of jetting regimes on the laser fluence and alginate concentration.

Laser printing-enabled direct creation of cellular heterogeneity in lab-on-a-chip devices.

Lab-on-a-chip devices, capable of culturing living cells in continuously perfused, micrometer-sized channels, have been intensively investigated to model physiological microenvironments for cell-related testing and evaluation applications. Various chemical, physical, and/or biological culture cues are usually expected in a designed chip to mimic the in vivo environment with defined spatial heterogeneity of cells and biomaterials. To create such heterogeneity within a given chip, typical methods rely heavily on sophisticated fabrication and cell seeding processes, and chips fabricated with these methods are difficult to readily adapt for other applications. In this study, laser-induced forward transfer (LIFT)-based printing has been implemented to create heterogeneous cellular patterns in a lab-on-a-chip device to achieve the efficiency in creating heterogeneous cellular patterns as well as the flexibility in adapting different evaluation configurations in lab-on-a-chip devices. Two applications, parallel evaluation of cellular behavior and targeted drug delivery to cancer cells, have been implemented as proof-of-concept demonstrations of the proposed fabrication method. For the first application, the morphology of cells in different extracellular matrix (ECM) materials cultured under varying conditions has been investigated. It is found that less stiff ECM and dynamic culturing are preferred for spreading of fibroblasts. For the second application, different drug carriers have been utilized for targeted delivery of anticancer drugs to breast cancer cells. It is found that targeted drug delivery is important to realize effective chemotherapy and drug release rate from drug carriers affects the chemotherapy effect. Consequently, the proposed laser printing-based method enables direct creation of heterogeneous cellular patterns within lab-on-a-chip devices which improves the efficiency and versatility of cell-related sensing and evaluation using lab-on-a-chip devices.

Effects of living cells on the bioink printability during laser printing.

Laser-induced forward transfer has been a promising orifice-free bioprinting technique for the direct writing of three-dimensional cellular constructs from cell-laden bioinks. In order to optimize the printing performance, the effects of living cells on the bioink printability must be carefully investigated in terms of the ability to generate well-defined jets during the jet/droplet formation process as well as well-defined printed droplets on a receiving substrate during the jet/droplet deposition process. In this study, a time-resolved imaging approach has been implemented to study the jet/droplet formation and deposition processes when printing cell-free and cell-laden bioinks under different laser fluences. It is found that the jetting behavior changes from no material transferring to well-defined jetting with or without an initial bulgy shape to jetting with a bulgy shape/pluming/splashing as the laser fluence increases. Under desirable well-defined jetting, two impingement-based deposition and printing types are identified: droplet-impingement printing and jet-impingement printing with multiple breakups. Compared with cell-free bioink printing, the transfer threshold of the cell-laden bioink is higher while the jet velocity, jet breakup length, and printed droplet size are lower, shorter, and smaller, respectively. The addition of living cells transforms the printing type from jet-impingement printing with multiple breakups to droplet-impingement printing. During the printing of cell-laden bioinks, two non-ideal jetting behaviors, a non-straight jet with a non-straight trajectory and a straight jet with a non-straight trajectory, are identified mainly due to the local nonuniformity and nonhomogeneity of cell-laden bioinks.

Printing-induced cell injury evaluation during laser printing of 3T3 mouse fibroblasts.

Three-dimensional bioprinting has emerged as a promising solution for the freeform fabrication of living cellular constructs, which can be used for tissue/organ transplantation and tissue models. During bioprinting, some living cells are unavoidably injured and may become necrotic or apoptotic cells. This study aims to investigate the printing-induced cell injury and evaluates injury types of post-printing cells using the annexin V/7-aminoactinomycin D and FAM-DEVD-FMK/propidium iodide assays during laser printing of NIH 3T3 mouse fibroblasts. As observed, the percentage of post-printing early apoptotic mouse fibroblasts increases with the incubation time, indicating that post-printing apoptotic mouse fibroblasts have different initiation lag times of apoptosis due to different levels of mechanical stress exerted during laser printing. Post-printing necrotic mouse fibroblasts can be detected immediately after printing, while post-printing early apoptotic mouse fibroblasts need time to develop into a late apoptotic stage. The minimum time needed for post-printing early apoptotic mouse fibroblasts to complete their apoptosis pathway and transition into late apoptotic mouse fibroblasts is from 4 h to 5 h post-printing. The resulting knowledge of the evolution of different apoptotic post-printing mouse fibroblasts will help better design future experiments to quantitatively determine, model, and mitigate the post-printing cell injury based on molecular signal pathway modeling.

Study of gelatin as an effective energy absorbing layer for laser bioprinting.

Laser-induced forward transfer printing, also commonly known as laser printing, has been widely implemented for three-dimensional bioprinting due to its unique orifice-free nature during printing. However, the printing quality has the potential to be further improved for various laser bioprinting applications. The objectives of this study are to investigate the feasibility of using gelatin as an energy absorbing layer (EAL) material for laser bioprinting and its effects on the quality of printed constructs, bioink printability, and post-printing cell viability and process-induced DNA damage. The gelatin EAL is applied between the quartz support and the coating of build material, which is to be printed. Printing quality can be improved by EAL-assisted laser printing when using various alginate solutions (1%, 2%, and 4%) and cell-laden bioinks (2% alginate and 5 × 106 cells ml-1 in cell culture medium). The required laser fluence is also reduced due to a higher absorption coefficient of gelatin gel, in particular when to achieve the best printing type/quality. The post-printing cell viability is improved by ∼10% and DNA double-strand breaks are reduced by ∼50%. For all the build materials investigated, the gelatin EAL helps reduce the droplet size and average jet velocity.

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.