Changxue Xu

Scaffold-free inkjet printing of three-dimensional zigzag cellular tubes†

The capability to print three‐dimensional (3D) cellular tubes is not only a logical first step towards successful organ printing but also a critical indicator of the feasibility of the envisioned organ printing technology. A platform‐assisted 3D inkjet bioprinting system has been proposed to fabricate 3D complex constructs such as zigzag tubes. Fibroblast (3T3 cell)‐based tubes with an overhang structure have been successfully fabricated using the proposed bioprinting system. The post‐printing 3T3 cell viability of printed cellular tubes has been found above 82% (or 93% with the control effect considered) even after a 72‐h incubation period using the identified printing conditions for good droplet formation, indicating the promising application of the proposed bioprinting system. Particularly, it is proved that the tubular overhang structure can be scaffold‐free fabricated using inkjetting, and the maximum achievable height depends on the inclination angle of the overhang structure. As a proof‐of‐concept study, the resulting fabrication knowledge helps print tissue‐engineered blood vessels with complex geometry. Biotechnol. Bioeng. 2012; 109: 3152–3160.

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.

Study of droplet formation process during drop-on-demand inkjetting of living cell-laden bioink.

Biofabrication offers a great potential for the fabrication of three-dimensional living tissues and organs by precisely layer-by-layer placing various tissue spheroids as anatomically designed. Inkjet printing of living cell-laden bioink is one of the most promising technologies enabling biofabrication, and the bioink printability must be carefully examined for it to be a viable biofabrication technology. In this study, the cell-laden bioink droplet formation process has been studied in terms of the breakup time, droplet size and velocity, and satellite formation using a time-resolved imaging approach. The bioink has been prepared using fibroblasts and sodium alginate with four different cell concentrations: without cells, 1 × 10(6), 5 × 10(6), and 1 × 10(7) cells/mL to appreciate the effect of cell concentration on the droplet formation process. Furthermore, the bioink droplet formation process is compared with that during the inkjetting of a comparable polystyrene microbead-laden suspension under the identical operating conditions to understand the effect of particle physical properties on the droplet formation process. It is found that (1) as the cell concentration of bioink increases, the droplet size and velocity decrease, the formation of satellite droplets is suppressed, and the breakup time increases, and (2) compared to the hard bead-laden suspension, the bioink tends to have a less ejected fluid volume, lower droplet velocity, and longer breakup time.

Effects of fluid properties and laser fluence on jet formation during laser direct writing of glycerol solution

Laser-induced forward transfer (LIFT) has been widely studied to print various structures. It is important to investigate the jet and droplet formation process under different LIFT operating conditions. The resulting knowledge will help to better control the resulting printing quality and feature resolution. This study aims to better understand the effects of fluid properties and laser fluence on the jet formation process using time resolved imaging analysis during LIFT of glycerol solutions. It is found that if the laser fluence is too low and/or the glycerol concentration is too high, it is less likely for a bubble to fully form and/or grow before it diminishes. If the laser fluence is too high and/or the glycerol concentration is too low, it is also difficult to form a well-developed jet since dramatic bubble expansion may lead to a bulgy shape and even splashing. Only under certain combinations of glycerol concentration and laser fluence, can a well-defined jet form. When a jetting fluid is given, its...

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]

Electric field-assisted droplet formation using piezoactuation-based drop-on-demand inkjet printing

Droplets with diameters from a few to hundreds of micrometers have found increasing applications in various fields. For inkjet printing, there is always a great need to control and reduce the droplet size for a given nozzle diameter and print viscous fluids by avoiding clogging. This study investigates the electric field-assisted droplet formation process under piezoactuation-based drop-on-demand (DOD) inkjet printing. For better control of droplet monodispersity, the Taylor cone is intentionally suppressed to avoid undesirable satellite droplets. The droplet formation process of deionized water is investigated, and some main conclusions are drawn as follows: (1) with an increase of applied voltage, the droplet velocity increases and the droplet size decreases, (2) the pinch-off location may be different depending on the applied voltage; and (3) the combination effect of the electric field and meniscus oscillation can be utilized to significantly reduce the droplet diameter to less than one-fifth of the orifice diameter. The electric field also demonstrates its capability in facilitating the DOD inkjet printing of high-concentration cell-alginate suspensions.

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.

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.

Predictive Modeling of Droplet Formation Processes in Inkjet-Based Bioprinting

Additive manufacturing is driving major innovations in many areas such as biomedical engineering. Recent advances have enabled three-dimensional (3D) printing of biocompatible materials and cells into complex 3D functional living tissues and organs using bio-printable materials (i.e., bioink). Inkjet-based bioprinting fabricates the tissue and organ constructs by ejecting droplets onto a substrate. Compared with microextrusionbased and laser-assisted bioprinting, it is very difficult to predict and control the droplet formation process (e.g., droplet velocity and volume). To address this issue, this paper presents a new data-driven approach to predicting droplet velocity and volume in the inkjet-based bioprinting process. An imaging system was used to monitor the droplet formation process. To investigate the effects of polymer concentration, excitation voltage, dwell time, and rise time on droplet velocity and volume, a full factorial design of experiments (DOE) was conducted. Two predictive models were developed to predict droplet velocity and volume using ensemble learning. The accuracy of the two predictive models was measured using the root-mean-square error (RMSE), relative error (RE), and coefficient of determination (R). Experimental results have shown that the predictive models are capable of predicting droplet velocity and volume with sufficient accuracy. [DOI: 10.1115/1.4040619]