Investigation of Cell Aggregation on the Printing Performance in Inkjet-Based Bioprinting of Cell-Laden Bioink.

During 3D bioprinting, when the gravitational force exceeds the buoyant force, cell sedimentation will be induced, resulting in local cell concentration change and cell aggregation which affect the printing performance. This paper aims at studying and quantifying cell aggregation and its effects on the droplet formation process during inkjet-based bioprinting and cell distribution after inkjet-based bioprinting. The major conclusions of this study are as follows: (1) Cell aggregation is a significant challenge during inkjet-based bioprinting by observing the percentage of individual cells after different printing times. In addition, as polymer concentration increases, the cell aggregation is suppressed. (2) As printing time and cell aggregation increase, the ligament length and droplet velocity generally decrease first and then increase due to the initial increase and subsequent decrease of the viscous effect. (3) As the printing time increases, both the maximum number of cells within one microsphere and the mean cell number have a significant increase, especially for low polymer concentrations such as 0.5% (w/v). In addition, the increased rate is the highest using the lowest polymer concentration of 0.5% (w/v) because of its highest cell sedimentation velocity.

A review on cell damage, viability, and functionality during 3D bioprinting.

Three-dimensional (3D) bioprinting fabricates 3D functional tissues/organs by accurately depositing the bioink composed of the biological materials and living cells. Even though 3D bioprinting techniques have experienced significant advancement over the past decades, it remains challenging for 3D bioprinting to artificially fabricate functional tissues/organs with high post-printing cell viability and functionality since cells endure various types of stress during the bioprinting process. Generally, cell viability which is affected by several factors including the stress and the environmental factors, such as pH and temperature, is mainly determined by the magnitude and duration of the stress imposed on the cells with poorer cell viability under a higher stress and a longer duration condition. The maintenance of high cell viability especially for those vulnerable cells, such as stem cells which are more sensitive to multiple stresses, is a key initial step to ensure the functionality of the artificial tissues/organs. In addition, maintaining the pluripotency of the cells such as proliferation and differentiation abilities is also essential for the 3D-bioprinted tissues/organs to be similar to native tissues/organs. This review discusses various pathways triggering cell damage and the major factors affecting cell viability during different bioprinting processes, summarizes the studies on cell viabilities and functionalities in different bioprinting processes, and presents several potential approaches to protect cells from injuries to ensure high cell viability and functionality.

Cell-laden bioink circulation-assisted inkjet-based bioprinting to mitigate cell sedimentation and aggregation.

Three-dimensional (3D) bioprinting precisely deposits picolitre bioink to fabricate functional tissues and organs in a layer-by-layer manner. The bioink used for 3D bioprinting incorporates living cells. During printing, cells suspended in the bioink sediment to form cell aggregates through cell-cell interaction. The formation of cell aggregates due to cell sedimentation have been widely recognized as a significant challenge to affect the printing reliability and quality. This study has incorporated the active circulation into the bioink reservoir to mitigate cell sedimentation and aggregation. Force and velocity analysis were performed, and a circulation model has been proposed based on iteration algorithm with the time step for each divided region. It has been found that (a) the comparison of the cell sedimentation and aggregation with and without the active bioink circulation has demonstrated high effectiveness of active circulation to mitigate cell sedimentation and aggregation for the bioink with both a low cell concentration of 1 × 106cells ml-1and a high cell concentration of 5 × 106cells ml-1; and (b) the effect of circulation flow rate on cell sedimentation and aggregation has been investigated, showing that large flow rate results in slow increments in effectiveness. Besides, the predicted mitigation effectiveness percentages on cell sedimentation by the circulation model generally agrees well with the experimental results. In addition, the cell viability assessment at the recommended maximum flow rate of 0.5 ml min-1has demonstrated negligible cell damage due to the circulation. The proposed active circulation approach is an effective and efficient approach with superior performance in mitigating cell sedimentation and aggregation, and the resulting knowledge is easily applicable to other 3D bioprinting techniques significantly improving printing reliability and quality in 3D bioprinting.

Effect of topography parameters on cellular morphology during guided cell migration on a graded micropillar surface.

PURPOSE: Guided cell migration refers to the engineering of local cell environment to specifically direct cell migration and has important applications such as utilization in cell sorting and wound healing assays. Graded micropillar surfaces have been utilized for achieving guided cell migration. Topographic parameters such as micropillar diameter and spacing gradient may have effects on the morphology of attached cells. It is critical to understand this interaction between the cells and the underlying microscale structures. METHODS: In this study, a graded micropillar substrate has been fabricated to investigate the effects of the microtopography on the cell morphology in terms of the cell aspect ratio and cell circularity. RESULTS: It is found that 1) with the increase of the micropillar diameter, the cell aspect ratio has no significance change. At the small spacing gradients, the aspect ratio is smaller than that at the large spacing gradients; 2) statistical analysis shows both the micropillar diameter and spacing gradient have no significant effect on the cell aspect ratio compared to the flat surface; 3) the cell circularity at the small micropillar diameters is higher than that at the large micropillar diameters. The cell circularity at the micropillar gradient of 0.1 µm is higher than those at the other micropillar gradients; 4) three microtopographic conditions are considered to have statistically significant effect on the cell circularity compared to the flat surface, including the micropillar diameters of 5 µm and 10 µm and the spacing gradient of 0.1 µm.

Effects of Irgacure 2959 and lithium phenyl-2,4,6-trimethylbenzoylphosphinate on cell viability, physical properties, and microstructure in 3D bioprinting of vascular-like constructs.

Photocrosslinkable polymers such as gelatin methacrylate (GelMA) have various 3D bioprinting applications. These polymers crosslink upon exposure to UV irradiation with the existence of an appropriate photoinitiator. Two photoinitiators, Irgacure 2959 and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) are commonly used. This study systematically investigates the effects of photoinitiator types on the cell viability, physical properties, and microstructure in 3D bioprinting of GelMA-based cellular constructs. The main conclusions are: (1) during the 3D bioprinting, the cell viability generally decreases as the photoinitiator concentration and printing time increase using both Irgacure 2959 and LAP. At the low photoinitiator concentrations (such as 0.3% and 0.5% (w/v)), the overall cell viability is good within the printing time of 60 min using both Irgacure 2959 and LAP. However, at the high photoinitiator concentrations (such as 0.7% and 0.9% (w/v)), the overall cell viability using LAP is much higher than that using Irgacure 2959 within the printing time of 60 min; (2) after the 3D bioprinting, the photoinitiator types, either Irgacure 2959 or LAP, have negligible effects on the post-printing cell viability after crosslinking; (3) after the 3D bioprinting, GelMA samples cured with Irgacure 2959 have slightly larger pore size, faster degradation rate, and greater swelling ratio compared to those cured with LAP; (4) 3D GelMA-based vascular-like constructs have been fabricated using dynamic optical projection stereolithography, and the measured dimensions have been compared with the designed dimensions showing good shape fidelity.

Effect of hyperbranched poly(trimellitic glyceride) paired with different metal ions on the physicochemical properties of starch.

The novel hyperbranched poly(trimellitic glyceride) (PTG) starch plasticizer synthesized in our previous study was neutralized with different alkaline metal hydroxides. Mixed with starch, the effects of different alkaline metal cations, M+, on gelatinization of starch suspensions and thermal behaviors of the films were analyzed using RVA and DMA, respectively. The structures of the starch suspensions, films and freeze-dried samples (S/PTG-M) were investigated using DSC, XRD and FTIR spectroscopy, respectively. M+ increased the gelatinization temperature of starch suspensions in the order of S/PTG-Li > S/PTG-Na > S/PTG-K. The formation of a complex between M+ and starch in the films observed using FTIR spectroscopy improved the stability of the starch paste and gel, and increased the gel temperature of starch dispersions. The corresponding starch gel was relatively thermostable, but not shear-resistant. PTG decreased the Tg of starch films with different paired M+. PTG-Li and PTG-K, but not PTG-Na, strengthened the mechanical properties of starch films.

Effects of Encapsulated Cells on the Physical-Mechanical Properties and Microstructure of Gelatin Methacrylate Hydrogels.

Gelatin methacrylate (GelMA) has been gaining popularity in recent years as a photo-crosslinkable biomaterial widely used in a variety of bioprinting and tissue engineering applications. Several studies have established the effects of process-based and material-based parameters on the physical-mechanical properties and microstructure of GelMA hydrogels. However, the effect of encapsulated cells on the physical-mechanical properties and microstructure of GelMA hydrogels has not been fully understood. In this study, 3T3 fibroblasts were encapsulated at different cell densities within the GelMA hydrogels and incubated over 96 h. The effects of encapsulated cells were investigated in terms of mechanical properties (tensile modulus and strength), physical properties (swelling and degradation), and microstructure (pore size). Cell viability was also evaluated to confirm that most cells were alive during the incubation. It was found that with an increase in cell density, the mechanical properties decreased, while the degradation and the pore size increased.

Digital light processing (DLP) 3D-printing technology and photoreactive polymers in fabrication of modified-release tablets.

In this study, we assessed the feasibility of using digital light processing (DLP) 3D printers (3DP) in fabrication of solid oral dosage forms. The DLP technology uses a digital micromirror device (DMD) that reflects and focuses ultraviolet (UV) light on the surfaces of photoreactive materials that polymerize in a layer-by-layer fashion. Using poly(ethylene glycol) diacrylate (PEGDA) and poly(ethylene glycol) dimethacrylate (PEGDMA) as photoreactive polymers and theophylline as a model drug, we deployed a DLP printer to fabricate tablets. After optimizing various printing parameters including UV intensity and exposure time, layer thickness, and polymer concentration, we printed various types of tablets with and without perforation. We then assessed the tablets for drug content, mechanical strengths, swellability, weight variation, microscopic features, drug-polymer interactions and drug release profiles. The loading of theophylline was 1%, which was independent of tablet weights. The drug content and weight variation were within the acceptable range, as recommended by the United States Pharmacopeia (USP). Scanning electronic microscopic (SEM) pictures showed tablets with distinct layers and smooth outer surfaces. The spectral scans, obtained using Fourier Transform Infrared Spectroscopy (FTIR), showed no chemical interactions between the drug and polymers. Similarly, drug content determined using a UV spectrophotometer was the same as that determined by a high performance liquid chromatography (UPLC). The extent of drug release increased with the increase in the number of perforations in the tablets. Overall, this study demonstrates that DLP 3DP can be used as a platform for fabricating oral tablets with well-defined shapes and different release profiles.

Biofabrication of 3D cell-encapsulated tubular constructs using dynamic optical projection stereolithography.

It has been widely recognized that one of the critical limitations in biofabrication of functional tissues/organs is lack of vascular networks which provide tissues and organs with oxygen and nutrients. Biofabrication of 3D vascular-like constructs is a reasonable first step towards successful printing of functional tissues and organs. In this paper, a dynamic optical projection stereolithography system has been implemented to successfully fabricate 3D Y-shaped tubular constructs with living cells encapsulated. The effects of operating conditions on the cure depth of a single layer have been investigated, such as UV intensity, exposure time, and cell density. A phase diagram has been constructed to identify optimal operating conditions. Cell viability immediately after printing has been measured to be around 75%. Post-printing mechanical properties, swelling properties, and microstructures of the gelatin methacrylate hydrogels have been characterized. The resulting fabrication knowledge helps to effectively and efficiently print tissue-engineered vascular networks with complex geometries.

Biofabrication of three-dimensional cellular structures based on gelatin methacrylate-alginate interpenetrating network hydrogel.

Hydrogels have been widely used as extracellular matrix materials in various three-dimensional bioprinting applications. However, they possess limitations such as insufficient mechanical integrity and strength, especially in the vascular applications requiring suture retention and tolerance of systemic intraluminal pressure. Interpenetrating network hydrogels are unique mixtures of two separate hydrogels with enhanced properties. This paper has demonstrated the fabrication of three-dimensional cellular constructs based on gelatin methacrylate-alginate interpenetrating network hydrogels using a microgel-assisted bioprinting method. Filament formation was investigated in terms of the filament diameter under different nozzle speed and dispensing pressure, and a phase diagram to identify the optimal conditions for continuous and uniform filaments was prepared. Three-dimensional hollow cellular constructs were fabricated and the cell viability was 75% after 24-hour incubation. The post-printing properties were characterized including mechanical properties, degradation and swelling properties, and pore size. The interpenetrating network hydrogels with different concentrations were compared with their individual components. It is found that the interpenetrating network hydrogels exhibit stronger mechanical properties, faster degradation and larger pore sizes than their individual components.

Multi-purposable filaments of HPMC for 3D printing of medications with tailored drug release and timed-absorption.

Three-dimensional printing (3DP), though developed for nonmedical applications and once regarded as futuristic only, has recently been deployed for the fabrication of pharmaceutical products. However, the existing feeding materials (inks and filaments) that are used for printing drug products have various shortcomings, including the lack of biocompatibility, inadequate extrudability and printability, poor drug loading, and instability. Here, we have sought to develop a filament using a single pharmaceutical polymer, with no additives, which can be multi-purposed and manipulated by computational design for the preparation of tablets with desired release and absorption patterns. As such, we have used hydroxypropyl-methylcellulose (HPMC) and diltiazem, a model drug, to prepare both drug-free and drug-impregnated filaments, and investigated their thermal and crystalline properties, studied the cytotoxicity of the filaments, designed and printed tablets with various infill densities and patterns. By alternating the drug-free and drug-impregnated filaments, we fabricated various types of tablets, studied the drug release profiles, and assessed oral absorption in rats. Both diltiazem and HPMC were stable at extrusion and printing temperatures, and the drug loading was 10% (w/w). The infill density, as well as infill patterns, influenced the drug release profile, and thus, when the infill density was increased to 100%, the percentage of drug released dramatically declined. Tablets with alternating drug-free and drug-loaded layers showed delayed and intermittent drug release, depending on when the drug-loaded layers encountered the dissolution media. Importantly, the oral absorption patterns accurately reproduced the drug release profiles and showed immediate, extended, delayed and episodic absorption of the drug from the rat gastrointestinal tract (GIT). Overall, we have demonstrated here that filaments for 3D printers can be prepared from a pharmaceutical polymer with no additives, and the novel computational design allows for fabricating tablets with the capability of producing distinct absorption patterns after oral administration.

3D Printing Hierarchical Silver Nanowire Aerogel with Highly Compressive Resilience and Tensile Elongation through Tunable Poisson's Ratio.

Metallic aerogels have attracted intense attention due to their superior properties, such as high electrical conductivity, ultralow densities, and large specific surface area. The preparation of metal aerogels with high efficiency and controllability remains challenge. A 3D freeze assembling printing technique integrated with drop-on-demand inkjet printing and freeze casting are proposed for metallic aerogels preparation. This technique enables tailoring both the macrostructure and microstructure of silver nanowire aerogels (SNWAs) by integrating programmable 3D printing and freeze casting, respectively. The density of the printed SNWAs is controllable, which can be down to 1.3 mg cm-3 . The ultralight SNWAs reach high electrical conductivity of 1.3 S cm-1 and exhibit excellent compressive resilience under 50% compressive strain. Remarkably, the printing methodology also enables tuning aerogel architectures with designed Poisson's ratio (from negative to positive). Moreover, these aerogel architechtures with tunable Poisson's ratio present highly electromechanical stability under high compressive and tensile strain (both strain up to 20% with fully recovery).

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.

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.

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.

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.