Gelatin Bioink Research Articles

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells.

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 µm in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves' direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

Granular gel support-enabled extrusion of three-dimensional alginate and cellular structures

Freeform fabrication of soft structures has been of great interest in recent years. In particular, it is viewed as a critical step toward the grand vision of organ printing—the on-demand design and fabrication of three-dimensional (3D) human organ constructs for implantation and regenerative medicine. The objective of this study is to develop a novel granular gel support material-enabled, two-step gelation-based ‘printing-then-gelation’ approach to fabricate 3D alginate structures using filament extrusion. Specifically, a granular Carbopol microgel bath holds the ungelled alginate structure being extruded, avoiding the instantaneous gelation of each printed layer as well as resultant surface tension-induced nozzle clogging. Since Carbopol microgels react with multivalent cations, which are needed for alginate crosslinking, gelatin is introduced as a sacrificial material to make an alginate and gelatin bioink for extrusion, which gels thermally (step-one gelation) to initially stabilize the printed structure for removal from Carbopol. Then gelatin is melted and diffused away while alginate is ionically crosslinked in a 37 °C calcium chloride bath (step-two gelation), resulting in an alginate structure. The proposed ‘printing-then-gelation’ approach works for alginate structure fabrication, and it is also applicable for the printing of cellular constructs and other similar homogeneous soft structures using a two-step or even multi-step approach. The main conclusions are: (1) 0.8% (w/v) Carbopol bath with a neutral pH value may be most suitable for soft structure printing; (2) it is most effective to use a 0.9% (w/v) NaCl solution to facilitate the removal of residual Carbopol; and (3) alginate structures fabricated using the proposed approach demonstrate better mechanical properties than those fabricated using the conventional ‘gelation-while-printing’ approach.

Bioprinting technique incorporating topographical guidance for stem cell alignment, precision deposition and promoting cardiomyocyte differentiation

Event Abstract Back to Event Bioprinting technique incorporating topographical guidance for stem cell alignment, precision deposition and promoting cardiomyocyte differentiation Ramya Bhuthalingam1, Pei Qi Lim1, Scott A. Irvine1, Animesh Agrawal1, Priyadarshini S. Mhaisalkar1, An Jia2, Chee Kai Chua2 and Subbu S. Venkatraman1 1 Nanyang Technological University, School of Material Science & Engineering, Singapore 2 Nanyang Technological University, School of Mechanical & Aerospace Engineering, Singapore Introduction: Bioprinting is greatly aided by the use of automated robotic dispensing systems for the delivery of biomaterials and cells. The robotic dispensing systems allow precision control for placement and patterning down to 10s of µm. This precision can be exploited not only for dispensing but also for surface patterning. Surface patterning aids the orientation and differentiation of stem cells seeded onto the biomaterial[2]. In particular, the alignment of Mesenchymal stem cells (MSCs) within channels or grooves on the surface promotes differentiation towards a cardiomyocyte phenotype[1],[3]. There are many complex methods for the introduction of grooves into a polymer surface, which can be time consuming and lack the flexibility to create more complex patterns. We tested the robotic dispensing systems ability to create cell aligning surface channels following the multiple patterns programmed into the controlling CAD software. Furthermore, stem cells aligned to these grooves were examined for lineage commitment towards cardiomyocytes. Finally the dispensing system was assessed for the potential for delivering stem cells within a gelatin bioink directly to the surface grooves[4]. The precision delivery and capability of the cells to sense the grooves was observed. Method: Polystyrene films were patterned with grooves, etched using pre-programmed designs by a Janome 2300N pressure controlled robotic dispensing system. A sharpened stylus was fitted to the dispensing head, thus allowing surface etching. MSCs were seeded onto the surface and their ability to align along the grooves was judged. The lineage commitment of the stem cells was also assessed by FACS for cardiomyocyte markers. Subsequently, a 2% gelatin/DMEM bioink containing MSCs was loaded into the printing head and the bioink was delivered to the pre-etched grooves following the identical pattern used for etching. Results and Discussion: The sharpened stylus allowed etching of the polystyrene surface following preprogrammed coordinates. The MSCs seeded onto the surface orientated along the grooves with an elongated morphology. FACS demonstrated an increase in the cardiomyocyte gene marker GATA4 and a decrease in the stem cell marker CD29[5]. The robot dispenser system could deliver cells in based gelatin bioink directly to the channels by following identical programming coordinates used for etching. The bioink delivered cells could sense the grooves and become aligned.

3D printing structures with a cell bearing gelatin bioink and transglutaminase crosslinking.

3D printing structures with a cell bearing gelatin bioink and transglutaminase crosslinking.

A novel 3D printing method for cell alignment and differentiation

The application of bioprinting allows precision deposition of biological materials for bioengineering applications. Here we propose a 2 stage methodology for bioprinting using a back pressure-driven, automated robotic dispensing system. This apparatus can prepare topographic guidance features for cell orientation and then bioprint cells directly onto them. Topographic guidance features generate cues that influence adhered cell morphology and phenotype. The robotic dispensing system was modified to include a sharpened stylus that etched on a polystyrene surface. The same computer-aided design (CAD) software was used for both precision control of etching and bioink deposition. Various etched groove patterns such as linear, concentric circles, and sinusoidal wave patterns were possible. Fibroblasts and mesenchymal stem cells (MSC) were able to sense the grooves, as shown by their elongation and orientation in the direction of the features. The orientated MSCs displayed indications of lineage commitment as detected by fluorescence-activated cell sorting (FACS) analysis. A 2% gelatin bioink was then used to dispense cells onto the etched features using identical, programmed co-ordinates. The bioink allows the cells to contact sense the pattern while containing their deposition within the printed pattern.

Bioprintable, cell-laden silk fibroin–gelatin hydrogel supporting multilineage differentiation of stem cells for fabrication of three-dimensional tissue constructs

Bioprinting has exciting prospects for printing three-dimensional (3-D) tissue constructs by delivering living cells with appropriate matrix materials. However, progress in this field is currently extremely slow due to limited choices of bioink for cell encapsulation and cytocompatible gelation mechanisms. Here we report the development of clinically relevant sized tissue analogs by 3-D bioprinting, delivering human nasal inferior turbinate tissue-derived mesenchymal progenitor cells encapsulated in silk fibroin–gelatin (SF–G) bioink. Gelation in this bioink was induced via in situ cytocompatible gelation mechanisms, namely enzymatic crosslinking by mushroom tyrosinase and physical crosslinking via sonication. Mechanistically, tyrosinases oxidize the accessible tyrosine residues of silk and/or gelatin into reactive o-quinone moieties that can either condense with each other or undergo nonenzymatic reactions with available amines of both silk and gelatin. Sonication alters the hydrophobic interaction and accelerates self-assembly of silk fibroin macromolecules to form β-sheet crystals, which physically crosslink the hydrogel. However, sonication has no effect on the conformation of gelatin. The effect of optimized rheology, secondary conformations of silk–gelatin bioink, temporally controllable gelation strategies and printing parameters were assessed to achieve maximum cell viability and multilineage differentiation of the encapsulated human nasal inferior turbinate tissue-derived mesenchymal progenitor cells. This strategy offers a unique path forward in the direction of direct printing of spatially customized anatomical architecture in a patient-specific manner.