Zhengyi Zhang

Freeform inkjet printing of cellular structures with bifurcations

Organ printing offers a great potential for the freeform layer‐by‐layer fabrication of three‐dimensional (3D) living organs using cellular spheroids or bioinks as building blocks. Vascularization is often identified as a main technological barrier for building 3D organs. As such, the fabrication of 3D biological vascular trees is of great importance for the overall feasibility of the envisioned organ printing approach. In this study, vascular‐like cellular structures are fabricated using a liquid support‐based inkjet printing approach, which utilizes a calcium chloride solution as both a cross‐linking agent and support material. This solution enables the freeform printing of spanning and overhang features by providing a buoyant force. A heuristic approach is implemented to compensate for the axially‐varying deformation of horizontal tubular structures to achieve a uniform diameter along their axial directions. Vascular‐like structures with both horizontal and vertical bifurcations have been successfully printed from sodium alginate only as well as mouse fibroblast‐based alginate bioinks. The post‐printing fibroblast cell viability of printed cellular tubes was found to be above 90% even after a 24 h incubation, considering the control effect. Biotechnol. Bioeng. 2015;112: 1047–1055.

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.

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.

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.

Freeform Vertical and Horizontal Fabrication of Alginate-Based Vascular-Like Tubular Constructs Using Inkjetting

Organ printing, among different tissue engineering innovations, is a freeform fabrication approach for making three-dimensional (3D) tissue and organ constructs using cellular spheroids or bioinks as building blocks. The capability to fabricate vascular-like tubular constructs is an important indicator of the overall feasibility of envisioned organ printing technology. In this study, vascular-like alginate tubes, which mimic typical vascular constructs, are fabricated both vertically and horizontally using drop-on-demand (DOD) inkjetting. Manufacturing-related challenges are different for the vertical and horizontal printing configurations. In general, the vertical printing configuration has instability or collapse/buckling problems and may experience some difficulty in fabricating complex constructs such as Yor K-shaped constructs if there is no supporting material. The horizontal printing configuration may easily result in a deformed hollow cross section and may require extra effort to mitigate the undesired deformation. It is envisioned that the combination of vertical and horizontal printing provides an efficient and effective way to fabricate complex tubular constructs with both vertical and horizontal branching features. [DOI: 10.1115/1.4028578]

Bubble Formation Modeling During Laser Direct Writing of Glycerol Solutions

Laser direct writing, a noncontact modified laser-induced forward transfer (LIFT) technique, has emerged as a promising technology for various applications from microelectronics printing to biofabrication. For it to be a viable technology, the bubble formation process during laser direct writing should be carefully examined. In this study, the bubble formation process during the laser direct writing of glycerol–water solutions has been studied using a nucleation-based phase explosion modeling approach. The effects of laser fluence and material properties of glycerol solution on the resulting bubble geometry have been examined both analytically and experimentally. Overall, a satisfactory modeling accuracy has been achieved, while the proposed modeling approach slightly underestimates the bubble diameter. Both the measured and predicted bubble diameters increase when the laser fluence increases. Interestingly, the measured and predicted diameters first decrease, then increase, and decrease again with the increase of glycerol concentration. Furthermore, it is noted that the bubble diameter is more sensitive to the laser fluence than the glycerol concentration.

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 Pinch-Off Locations during Drop-on-Demand Inkjet Printing of Viscoelastic Alginate Solutions

The ligament pinch-off process of viscoelastic fluids during jetting is a key step in various biotechnology and dropwise three-dimensional printing applications. Various pinch-off locations have been investigated as a function of material properties and operating conditions during the drop-on-demand (DOD) inkjet printing of viscoelastic alginate solutions. Four breakup types are identified on the basis of the location of the first pinch-off position: front pinching is mainly governed by a balance of inertial and capillary effects, exit pinching is affected by the external actuation-induced hydrodynamic instability and mainly governed by a balance of elastic and capillary effects, middle pinching usually occurs any place along a uniform thin ligament under dominant viscous and elastic effects, and hybrid pinching happens when front pinching and exit pinching occur simultaneously as a special case.