CONCEPTUAL DESIGN AND EVALUATION OF A NOVEL MULTI-MATERIAL BIOPRINTING SYSTEM

Millions of people suffer from damaged tissues or organs. The gold standard for treatment is tissue/organ transplantation; however, the demand for donated tissues and organs eclipses the number of donors. Scaffold-based tissue engineering aims to produce tissue/organ substitutes or scaffolds for transplants or implantation for promoting tissue regeneration. Extrusion-based bioprinting has recently emerged to create scaffolds by printing biomaterials with living cells in a layer-by-layer pattern. A significant limitation of existing extrusion-based techniques is their material distribution, e.g., printing scaffolds from only one material. Multi-material bioprinting is essential to mimic the complex anisotropic and heterogeneous features of native tissues. Researchers have taken steps towards making multi-material scaffolds; however, current methods are limited in terms of the material distribution and longitudinal/circumferential organization in the printed filaments. This M.Sc. work aims to study the design of a multi-material bioprinting system with spatial control of material in longitudinal and circumferential directions. The design was conceived through a methodical approach, from technical specifications to conceptual-, embodiment-, and detail-design stages. The system will employ a combination of a desktop 3D printer for x-y-z control, a multi-channel pressure controller for on-the-fly adjustments, a custom printhead for organizing multiple inlets to a single outlet, and a carriage to affix the printhead assembly to the x-y-z- controller. For the proposed system, spatial control of material comes from several configurations of the custom modular printhead and the flow controller. Axiomatic Design principles are then used to compare and evaluate the proposed systems against existing systems in terms of material control and ease of configurability. Specified functional requirements and design ranges quantify longitudinal and circumferential material control and ease of configurability. Then, the system ranges for the functional requirements were built using the reported data of the existing systems. Axiom 1 shows that side-by-side, core-and-shell, and advanced techniques lack functional independence. Then, Axiom 2 shows that the proposed technique has the probability of completing the specified functions, granting it as the single-best design. The resulting design, justified by the evaluation and comparison, shows that this work is promising in helping researchers realize intricate scaffold designs with specific material control

Development of 3D bioprinting techniques based on supportive media

The production of anatomically complex tissues and organs with high biological function requires bioinks to have contradictory material properties. Properties that enable bioinks to be mechanically self-sufficient and accurate in terms of geometric fidelity may not be inherently compatible for cell viability and vice versa. Such is the practical dilemma of bioprinting, leading to the development of bioinks with balanced mechanical and biological properties that do not excel in either respect. In this thesis, the development of a customised, modular, extrusion-based 3D bioprinter and two novel supportive bath strategies is described. This custom bioprinter is able to extrude low-concentration, low-viscosity bioinks deep into the developed support baths and suspend the extruded bioink in 3D space. Printing structures in this manner reduces the demand for mechanically strong bioinks during the fabrication process as the structure’s weight is supported by the bath in all dimensions. These supportive strategies enable the production of larger and geometrically more complex anatomical structures whilst using a low-concentration, low-viscosity alginate hydrogel bioink. Therefore the material’s mechanical needs for bioprinting are addressed in such a way that encourages the use of bioinks with qualities that can be biologically more favourable. The support baths detailed in this thesis includes a quiescently gelled gelatine-based approach and a fluidised-agar fluid gel approach. The gelatine baths are prepared in a very simple, reliable, and repeatable two-step manner, and printed structures embedded within the gel are removed gently and easily by utilising gelatine’s physiologically relevant melting temperature to liquefy the support. Blood vessel-like structures and noses were fabricated in this manner. Agar fluid gel support baths are also simple to produce and only require a gelled puck of agar be blended prior to its application as a supportive material. Agar fluid gel baths have been used successfully to support the fabrication of geometrically challenging structures such as bucky balls and Eiffel towers as well as replicate anatomical models such as ears, noses, brains, and hearts, which are easily separated from their supports by washing away the residual fluid gel.Engineering and Physical Sciences Research Council (EPSRC

Development of a fluidic mixing nozzle for 3D bioprinting

3D bioprinting is a relatively new and very promising field that uses conventional 3D printing techniques and adapts them to print biological materials that are suited for use with cells. These bioprinters can be used to print cells encapsulated within biological ink (bio-ink) to create and customize complex three-dimensional tissues and organs. Our work has focused on developing a new bioprinter nozzle that addresses critical gaps with present-day bioprinters, namely, the lack of standardized, physiologically-relevant biomaterials, and their one nozzle per composition printing capacity. These shortcomings preclude printing a range of cellular and biomaterial compositions (including gradients of cells and matrix components) within a single tissue construct. Type I collagen oligomers, a new soluble collagen subdomain that falls between molecular and fibrillar size scales, are ideally suited for tissue fabrication. This collagen formulation, which is produced according to an ASTM voluntary consensus standard, i) exhibits rapid suprafibrillar self-assembly yielding highly interconnected collagen-fibril matrices resembling those found in the body\u27s tissues, ii) supports cell encapsulation, and iii) allows customized, multi-scale design across the broadest range of tissue architectures and physical properties. These properties, along with its superior physiologic relevance, support the use of this biomaterial in the development of a bioprinting nozzle that is able to address the key gaps in the field of 3D bioprinting. After researching microfluidic mixing devices and current bioprinters, early iterations of a 3D bioprinting nozzle were designed and machined to mix three fundamental reagents required to form a broad array of collagen-fibril matrix compositions, namely oligomeric type I collagen (oligomer), oligomer diluent (diluent), and self-assembly reagent (S.A.R). The nozzle was designed to mix specified proportions of these solutions using a combination of hydrodynamic focusing and twisted channel mixing mechanisms before depositing the selfassembling collagen. Three syringe pumps were used to continuously drive varying flow rates of the three reagents to the nozzle, which allowed for the creation of a broad array of cell and matrix compositions, including fibril-density gradients. To validate nozzle performance, three experiments were conducted to define dispensing volume accuracy and precision, mixing quality, and functional performance of dispensed materials, including cells and matrix. In summary, the integration of standardized self-assembling collagens with this innovative fluidic mixer effectively minimizes the number of printing reservoirs, employs a single dispensing nozzle, and most importantly supports on demand fabrication of various tissue compositions. This advanced 3D bioprinting technology, together with our mechanistic-based tissue engineering design principles, is expected to support customized design and fabrication of complex and scalable tissues for both research and medical applications

Investigation of Cell Behavior in 3D Printed Lumen Structures for Capillary Regeneration

This thesis work successfully generated a coaxial printing setup and process to generate 3D printed tubules. A dual syringe pump mechanism was developed to print tubular structures to investigate cell behavior with a goal to generate future capillary beds. The mechanism involves the use of collagen–alginate tubules and the use of EDTA to increase the porosity of the tubule structures to study their interaction with media and incubation techniques as well as behavior and morphology. Tubules of 1.7% sodium alginate and 0.4% collagen I reacting with 3.2% CaCl2 solution proved to be more stable in both the printing process and the incubation process than 0.25% bioink mixes. Migration conforming to the edges of tubule walls. and cell morphology was observed in and outside the tubules showing cells EDTA and collagen were not statistically significant in determining cell viability. Cell behavior within and outside the tubule structures varied and maintained the possibility of utilizing coaxially printed tubules within the designed device to further maintain the tubule and advance the complexity of the cell populations that interact with it

3D Bioprinting in Microgravity: Opportunities, Challenges, and Possible Applications in Space

: 3D bioprinting has developed tremendously in the last couple of years and enables the fabrication of simple, as well as complex, tissue models. The international space agencies have recognized the unique opportunities of these technologies for manufacturing cell and tissue models for basic research in space, in particular for investigating the effects of microgravity and cosmic radiation on different types of human tissues. In addition, bioprinting is capable of producing clinically applicable tissue grafts, and its implementation in space therefore can support the autonomous medical treatment options for astronauts in future long term and far-distant space missions. The article discusses opportunities but also challenges of operating different types of bioprinters under space conditions, mainly in microgravity. While some process steps, most of which involving the handling of liquids, are challenging under microgravity, this environment can help overcome problems such as cell sedimentation in low viscous bioinks. Hopefully, this publication will motivate more researchers to engage in the topic, with publicly available bioprinting opportunities becoming available at the International Space Station (ISS) in the imminent future

Recent progress in extrusion 3D bioprinting of hydrogel biomaterials for tissue regeneration: a comprehensive review with a focus on advanced fabrication techniques

Over the last decade, 3D bioprinting has received immense attention from research communities for developing functional tissues. Thanks to the complexity of tissues, various bioprinting methods are exploited to figure out the challenges of tissue fabrication, in which hydrogels are widely adopted as a bioink in cell printing technologies based on the extrusion principle. Thus far, there is a wealth of the literature proposing the crucial parameters of extrusion-based bioprinting of hydrogel biomaterials (e.g., hydrogel properties, printing conditions, and tissue scaffold design) toward enhancing performance. Despite the growing research in this field, numerous challenges that hinder advanced applications still exist. Herein, the most recently reported hydrogel-based bioprinted scaffolds, i.e., skin, bone, cartilage, vascular, neural, and muscular (including skeletal, cardiac, and smooth), are systematically discussed with an emphasis on the advanced fabrication techniques from tissue engineering perspective. Methods covered include the multiple-dispenser, coaxial, and hybrid 3D bioprinting. The present work is a unique study to figure out the opportunities of the novel techniques to fabricate complicated constructs with structural and functional heterogeneity. Finally, the principal challenges of current studies and a vision of future research are presented

3D Printing for Tissue Regeneration

Tissue engineering is an interdisciplinary field and 3D bioprinting has emerged to be the holy grail to fabricate artificial organs. This chapter gives an overview of the latest advances in 3D bioprinting technology in the commercial space and academic research sector. It explores the commercially available 3D bioprinters and commercially printed products that are currently available in the market. It provides a brief introduction to bioinks and the latest developments in 3D bioprinting various organs. The chapter also discusses the advancements in tissue regeneration from 3D printing to 4D printing

Development of a cell encapsulation technology for the production of functional, micro-encapsulated pancreatic islets for transplantation

Previously held under moratorium from 8 August 2019 until 9 December 2021Diabetes type 1 is an autoimmune disease in which the patient’s own immune system destroys the insulin producing β-cells, located in the pancreatic islets. Without enough insulin production, the blood glucose levels of the patient rise, which can lead to damages of blood vessels and nerves, blindness or even seizures and comas. For some patients that have trouble maintaining normoglycaemia allogeneic islet transplantation has become an alternative treatment option. Patients with these transplanted islets are no longer prone to hypoglycaemic episodes and can sometimes become completely insulin independent. However, this success is not long-lived. The life span of the transplanted islets is limited due to the host’s immune responses and the toxicity of modern immunosuppressive agents. In this thesis, islet encapsulation for clinical transplantation is investigated and further developed. Islet encapsulation can protect the islets from the immune system, without the aid of the immunosuppressants. The construction and optimization of a micro-encapsulator that can be used to create encapsulations is described, as well as the multiple parameters to create small, uniform encapsulations. To further enhance the biocompatibility and immunoprotective properties of alginate hydrogel, alginate was purified to eliminate most of the impurities and tested for its permeability. Encapsulating pancreatic islets in this purified alginate showed encouraging results, with the islets remaining viable and functional longer than their control counterparts. Larger islets can develop necrotic cores within encapsulations, due to the lack of vascularization. To create smaller islets out of dissociated larger islets, a single-step encapsulation and aggregation method was developed, that unfortunately was not suitable for islet cells, but was capable of developing functional hepatic organoids out of HepaRG cells, that could be used for drug testing. Finally, a proof of principle was given for the creation of pancreatic islet patches using 3D bioprinting methods.Diabetes type 1 is an autoimmune disease in which the patient’s own immune system destroys the insulin producing β-cells, located in the pancreatic islets. Without enough insulin production, the blood glucose levels of the patient rise, which can lead to damages of blood vessels and nerves, blindness or even seizures and comas. For some patients that have trouble maintaining normoglycaemia allogeneic islet transplantation has become an alternative treatment option. Patients with these transplanted islets are no longer prone to hypoglycaemic episodes and can sometimes become completely insulin independent. However, this success is not long-lived. The life span of the transplanted islets is limited due to the host’s immune responses and the toxicity of modern immunosuppressive agents. In this thesis, islet encapsulation for clinical transplantation is investigated and further developed. Islet encapsulation can protect the islets from the immune system, without the aid of the immunosuppressants. The construction and optimization of a micro-encapsulator that can be used to create encapsulations is described, as well as the multiple parameters to create small, uniform encapsulations. To further enhance the biocompatibility and immunoprotective properties of alginate hydrogel, alginate was purified to eliminate most of the impurities and tested for its permeability. Encapsulating pancreatic islets in this purified alginate showed encouraging results, with the islets remaining viable and functional longer than their control counterparts. Larger islets can develop necrotic cores within encapsulations, due to the lack of vascularization. To create smaller islets out of dissociated larger islets, a single-step encapsulation and aggregation method was developed, that unfortunately was not suitable for islet cells, but was capable of developing functional hepatic organoids out of HepaRG cells, that could be used for drug testing. Finally, a proof of principle was given for the creation of pancreatic islet patches using 3D bioprinting methods

3D Bioprinting Tissue Scaffolds with Living Cells for Tissue Engineering Applications

In tissue engineering, tissue scaffolds are used as temporary supports to promote regeneration of dysfunctional tissues. Of the available strategies, scaffolds produced from hydrogels and living cells show the great potential for their enhanced biological properties. To produce such scaffolds, three-dimensional (3D) bioprinting has evolved and is showing promise as a fabrication technique. However, its applications for fabricating customized hydrogel scaffolds containing living cells is still in its infancy. The major challenge with this approach is to print scaffolds while preserving cell viability and functionality as well as ensuring the structural integrity of the scaffold. To overcome this challenge, the present thesis aims to investigate the influences of hydrogel properties and the bioprinting process on cell viability and functionality, while also ensuing structural integrity, and on this basis, to develop bioprinting processes to produce tissue scaffolds with living cells for potential tissue engineering applications. This thesis first examined the influence of the mechanical properties of hydrogel on cell viability and functionality, utilizing alginate hydrogels and Schwann cells (the major glial cells of peripheral nervous system). Due to its poor cell adhesion, the alginate hydrogel was modified in this study with cell-adhesion supplements, including fibronectin, poly-l-lysine (PLL), and RGD (Arg-Gly-Asp) peptides. The RGD-modified alginate substrates were prepared with varying alginate concentrations in order to alter the mechanical properties of hydrogels, which were then seeded and encapsulated with Schwann cells. Cell viability and functionality, including proliferation, morphology, and expression of the extracellular matrix protein, were examined and correlated to the hydrogel mechanical properties. The results demonstrate that the viability and functionality of Schwann cells within alginate-based hydrogel vary with hydrogel mechanical properties, thus highlighting the importance of regulating the mechanical properties of hydrogel for improved cell viability and functionality in scaffold bioprinting. During the bioprinting process, cells are subject to process-induced forces, such as shear and extensional stresses, which can result in cell damage and therefore loss of cell function and even cell death. A method was developed to study the cell damage introduced by the shear and extensional stresses in the bioprinting process. A plate-and-cone rheometer was adopted to examine the effect of shear stress on cell damage. In these experiments, the relationship of cell damage to the shear stress was examined and quantified, which was then applied to identify the cell damage attributed to shear stress in bioprinting. On this basis, the damage to cells caused by extensional stress was inferred from the difference between the total cell damage occurring during the bioprinting process and the cell damage attributed to shear stress. This developed method allowed a relationship to be established between cell damage and both shear and extensional stresses during bioprinting. The experiments on this method provide insight into both the cell damage that occurs during bioprinting and the effect on cell viability and proliferative ability thereafter, which can be used to optimize the bioprinting process so as to preserve cell functionality. Based on the previous investigations, bioprinting processes were developed to fabricate tissue scaffolds containing Schwann cells for potential applications in nerve tissue engineering. Composite hydrogels consisting of alginate, fibrin, hyaluronic acid, and RGD peptide were prepared, and their hydrogel microstructures, mechanical stiffness after gelation, and capability to support the Schwann cell spreading were examined for identifying appropriate composite hydrogel for bioprinting processes. The flow behavior of composite hydrogel solutions and bioprinting process parameters (e.g., dispensing pressure, dispensing head speed, crosslinking process) were then examined with regard to their influence on the structure of the printed scaffolds and on this basis, bioprinting process were developed to fabricate scaffolds with Schwann cells. The functionality of Schwann cells within the printed scaffolds were assessed in terms of cell viability, proliferation, morphology, orientation, and protein expression, demonstrating that the printed scaffolds have potential for nerve tissue engineering applications. This thesis presents a comprehensive study on the bioprinting of scaffolds with living cells. The method developed and the study results will pave the way to fabricate scaffolds with living cells for more tissue engineering applications

Three-dimensional bioprinting of tissue constructs with live cells

Tissue engineering is an emerging multidisciplinary field to regenerate damaged or In this research work, novel bioprinting methodologies are developed to fabricate 3D artificial biological structures directly from computer models using live multicellular aggregates. Multicellular aggregates made out of at least two cell types from fibroblast, endothelial and smooth muscle cells are prepared and optimized. A semi-continuous bioprinting approach is proposed in order to extrude cylindrical multicellular aggregates through the bioprinter’s gladiseased tissues and organs. Traditional tissue engineering strategies involve seeding cells into porous scaffolds to regenerate tissue or organs. Bioprinting is a relatively new technology where living cells with or without biomaterials are printed layer-by-layer in order to create 3D living structures. ss micro-capillaries. The multicellular pellets are first aspirated into a capillary and then compressed to form a continuous cylindrical multicellular bioink. To overcome surface tension-driven droplet formation, the required compression ratio is calculated based on viscosity of cell suspensions. Using the developed biomodeling and path-planning methods, example vascular structures are bioprinted biomimetically from medical images. Based on the developed bioprinting strategies, multicellular aggregates and their support structures are bioprinted to form 3D tissue constructs with predefined shapes. The results show that the bioprinted 3D constructs fuse rapidly and have high cell viability after printing