Microextrusion-based Bioprinting Research Articles

Rheology, simulation and data analysis toward bioprinting cell viability awareness

Microextrusion-based bioprinting consists of extruding a cellularized hydrogel through a nozzle of known geometry. As a consequence of this travelling through the nozzle, shear forces will inevitably be generated upon cells during the extrusion process and have a negative effect on cell viability, proliferation and biological fate of the tissue. In the present study and for the first time in the bioprinting field, flow simulation and cells fate (lysis, necrosis, apoptosis and viable) were consolidated to establish mathematical relationships. We demonstrated that for constant residence time at wall, the higher the wall shear stress values, the lower the viable populations. Furthermore, at constant maximum wall shear stress, the longer the residence time at wall, the lower the viable populations. Regarding the quantification of the damaged fibroblasts (lysis, necrosis and apoptosis) during the microextrusion, lysis was shown to be the prevailing cell death pathway compared to necrosis and apoptosis. Finally, partial least square (PLS) modelling was able to correctly predicted viable cells’ population with a high correlation (R2 of 0.859; error of 9.2%) between predicted and measured viable cells percentage. This model was shown to be predictive over a large range of cells viability (38.5–94%) with a prediction error of only 9.2%.

3D bioprinting of bicellular liver lobule-mimetic structures via microextrusion of cellulose nanocrystal-incorporated shear-thinning bioink

3D bioprinting of living cellular constructs with heterogeneity in cell types and extra cellular matrices (ECMs) matching those of biological tissues remains challenging. Here, we demonstrate that, through bioink material design, microextrusion-based (ME) bioprinting techniques have the potential to address this challenge. A new bioink employing alginate (1%), cellulose nanocrystal (CNC) (3%), and gelatin methacryloyl (GelMA) (5%) (namely 135ACG hybrid ink) was formulated for the direct printing of cell-laden and acellular architectures. The 135ACG ink displayed excellent shear-thinning behavior and solid-like properties, leading to high printability without cell damage. After crosslinking, the ACG gel can also provide a stiff ECM ideal for stromal cell growth. By controlling the degree of substitution and polymer concentration, a GelMA (4%) bioink was designed to encapsulate hepatoma cells (hepG2), as GelMA gel possesses the desired low mechanical stiffness matching that of human liver tissue. Four different versions of to-scale liver lobule-mimetic constructs were fabricated via ME bioprinting, with precise positioning of two different cell types (NIH/3T3 and hepG2) embedded in matching ECMs (135ACG and GelMA, respectively). The four versions allowed us to exam effects of mechanical cues and intercellular interactions on cell behaviors. Fibroblasts thrived in stiff 135ACG matrix and aligned at the 135ACG/GelMA boundary due to durotaxis, while hepG2 formed spheroids exclusively in the soft GelMA matrix. Elevated albumin production was observed in the bicellular 3D co-culture of hepG2 and NIH/3T3, both with and without direct intercellular contact, indicating that improved hepatic cell function can be attributed to soluble chemical factors. Overall, our results showed that complex constructs with multiple cell types and varying ECMs can be bioprinted and potentially useful for both fundamental biomedical research and translational tissue engineering.

A Versatile Open-Source Printhead for Low-Cost 3D Microextrusion-Based Bioprinting.

Three-dimensional (3D) bioprinting promises to be essential in tissue engineering for solving the rising demand for organs and tissues. Some bioprinters are commercially available, but their impact on the field of Tissue engineering (TE) is still limited due to their cost or difficulty to tune. Herein, we present a low-cost easy-to-build printhead for microextrusion-based bioprinting (MEBB) that can be installed in many desktop 3D printers to transform them into 3D bioprinters. We can extrude bioinks with precise control of print temperature between 2–60 °C. We validated the versatility of the printhead, by assembling it in three low-cost open-source desktop 3D printers. Multiple units of the printhead can also be easily put together in a single printer carriage for building a multi-material 3D bioprinter. Print resolution was evaluated by creating representative calibration models at different temperatures using natural hydrogels such as gelatin and alginate, and synthetic ones like poloxamer. Using one of the three modified low-cost 3D printers, we successfully printed cell-laden lattice constructs with cell viabilities higher than 90% after 24-h post printing. Controlling temperature and pressure according to the rheological properties of the bioinks was essential in achieving optimal printability and great cell viability. The cost per unit of our device, which can be used with syringes of different volume, is less expensive than any other commercially available product. These data demonstrate an affordable open-source printhead with the potential to become a reliable alternative to commercial bioprinters for any laboratory.

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs.

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Accurate Calibration in Multi-Material 3D Bioprinting for Tissue Engineering.

Most of the studies in three-dimensional (3D) bioprinting have been traditionally based on printing a single bioink. Addressing the complexity of organ and tissue engineering, however, will require combining multiple building and sacrificial biomaterials and several cells types in a single biofabrication session. This is a significant challenge, and, to tackle that, we must focus on the complex relationships between the printing parameters and the print resolution. In this paper, we study the influence of the main parameters driven multi-material 3D bioprinting and we present a method to calibrate these systems and control the print resolution accurately. Firstly, poloxamer hydrogels were extruded using a desktop 3D printer modified to incorporate four microextrusion-based bioprinting (MEBB) printheads. The printed hydrogels provided us the particular range of printing parameters (mainly printing pressure, deposition speed, and nozzle z-offset) to assure the correct calibration of the multi-material 3D bioprinter. Using the printheads, we demonstrated the excellent performance of the calibrated system extruding different fluorescent bioinks. Representative multi-material structures were printed in both poloxamer and cell-laden gelatin-alginate bioinks in a single session corroborating the capabilities of our system and the calibration method. Cell viability was not significantly affected by any of the changes proposed. We conclude that our proposal has enormous potential to help with advancing in the creation of complex 3D constructs and vascular networks for tissue engineering.

Simulating image-guided in situ bioprinting of a skin graft onto a phantom burn wound bed

Deep skin thermal wounds require skin excision and engraftment. Clinical treatment for deep skin wounds is the use of autologous split-thickness skin grafts. However, timely coverage of large-area burn wounds remains a significant challenge. Engineered skin tissue constructs aim to overcome the limitations of traditional clinical intervention. In this study, an in situ bioprinting-based methodological workflow is advanced to directly fabricate a custom engineered skin graft onto a skin burn phantom. To illustrate this modular approach, a burn phantom is first created by mold casting gelatin-alginate hydrogel material to simulate a burn wound bed with arbitrary 2D shape and uniform depth. The cast hydrogel phantom is then placed on the printer platform to host the to-be-printed skin graft. Next, a color image-based module is proposed to extract the contour of the burn wound. This is followed by implementing a contour calibration process based on fiducial markers to yield the real dimension and pose of the burn phantom. A new directed toolpath generation algorithm is detailed to generate a burn-specific toolpath for the microextrusion-based bioprinting process. Based on this method, the bioprinted cell-laden gelatin-alginate hydrogel filaments are precisely arranged in a meshed pattern that is bound by the burn phantom contour. Internal geometries defined by the filament and pore dimensional characteristics of the printed construct design can be controlled to promote cell viability, proliferation, and nutrient delivery. Printed cell-laden multi-layered constructs are evaluated for single filament and pore dimensional precision, alignment of filaments between layers, and positional accuracy of the filaments within the extracted contour. Finally, a 24-hour time course incubation study reveals that the printed constructs preserve their structural properties while cells proliferate and maintain their spatial positioning.

Printability Study of Bioprinted Tubular Structures Using Liquid Hydrogel Precursors in a Support Bath

Microextrusion-based bioprinting within a support bath material is an emerging additive manufacturing paradigm for complex three-dimensional (3D) tissue construct fabrication. Although a support bath medium enables arbitrary in-process geometries to be printed, a significant challenge lies in preserving the shape fidelity upon the extraction of the support bath material. Based on the bioprinting in a support bath paradigm, this paper advances quantitative analyses to systematically determine the printability of cell-laden liquid hydrogel precursors towards filament-based tissue constructs. First, a yield stress nanoclay material is judiciously selected as the support bath medium owing to its insensitivity to temperature and ionic variations that are considered in the context of the current gelatin-alginate bio-ink material formulation. Furthermore, phenomenological observations for the rheology-mediated print outcomes enable the compositions for the bio-ink material (10% gelatin, 3% alginate), in tandem with the support bath medium (4% nanoclay, 0.5% CaCl2), to be identified. To systematically evaluate the performance outcomes for bioprinting within a support bath, this paper advances an experimental parametric study to optimize the 3D structural shape fidelity by varying parameters such as the layer height, extrusion flowrate, printing temperature, and printhead speed, towards fabricating complex 3D structures with the stabilization of the desired shape outcome. Specifically, it is found that the layer height and printhead speed are determinant parameters for the extent of successive layer fusion. Moreover, maintenance of an optimal bath temperature is identified as a key parameter for establishing the printability for the hydrogel bio-ink. Studying this effect is enabled by the custom design of a PID temperature control system with integration with the bioprinter for real-time precision control of the support bath temperature. In order to qualify the printed construct, a surface irregularity metric, defined as the average height difference between consecutive local maximum and minimum points of the binary image contour for the printed structure, is advanced to evaluate the quality of the printed constructs. Complex one-to-four bifurcating tubular structure prints demonstrate the applicability of the optimized bioprinting parameter space to create exemplar 3D human vessel-like structures. Finally, a cell viability assay and perfusion test for a printed cell-laden tubular element demonstrates high cell survival rates and leakage-free flow, respectively.