Objectives: Three-dimensional (3D) bioprinting is a manufacturing process that incorporates viable cells into a 3D matrix by adding layer upon layer of material. The objectives of this study are to characterize a novel matrix of collagen and hydroxyapatite and to assess the effects of the 3D bioprinting process on cytotoxicity, proliferation rate, and cytokine expression of Homo sapiens palatal mesenchyme (HEPM) cells. Methods: For this, we prepared a 3D matrix of collagen and hydroxyapatite without and with cells. We used light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) to characterize the structure and arrangement of the collagen fibers. We then incubated the matrix with known standards of cytokines to measure adsorption. We assessed the proliferation and viability of HEPM cells in the presence of the 3D construct and after 3D bioprinting. Finally, we assessed the cytotoxicity of this matrix for HEPM cells and assessed its effect on the production of chemokines and cytokines. A one-way fixed effect ANOVA was fit to concentrations of cytokines and pairwise group comparisons were conducted using Tukey’s Honest Significant Differences test (p< 0.05). Results: The matrix was found to contain interwoven strands of collagen and some hydroxyapatite crystals that did not absorb cytokines except for MIP-1a (p< 0.05). The matrix was found to be non-cytotoxic using an AlamarBlue® assay. We found that the cell proliferation rate was lower when seeded on the 3D construct than in 2D culture. We also found that the proliferation rate was very low for the HEPM cells in the 3D bioprinted constructs. In the presence of the 3D construct, the HEPM cells had similar expression profiles of the cytokines measured (P > 0.05 for GM-CSF, IL-6, IL-8, and RANTES). Conclusion: 3D-bioprinting has the potential to be used in dentistry as a novel osteogenic bone grafting material. Here we show that a novel matrix of collagen and hydroxyapatite is non-cytotoxic to HEPM cells and does not induce a proinflammatory response.

The use of 3D printing in biological applications is a new field of study given that 3D printing technology has become more available and user friendly. Possible uses include using existing 3D printing polymers to use in extracorporeal or in vitro devices, like Lab-on-a-Chip, and the development of new biologically derived materials to print cell-containing constructs. The latter concept is what is more commonly known as bioprinting. Our research had the goal of developing a bioprinting system including the printer, a bioink, and a feedback system for printing parameter optimization which could be done cheaply and within the reach of nearly any research lab. To make the bioprinter, we were able to take a popular plastic 3D printer and convert it to a bioprinter with 3D printed parts and the addition of a new motherboard. This came with great contribution from Carnegie Melon University. We were also able to improve upon the original design and, along with the new bioprinting capabilities, maintain the original capabilities of the plastic 3D printer. A new bioink was developed to work in coordination with this bioprinting system. Our lab has the luxury of having access to decellularized tissue, which provided a unique material to create a bioink which is derived from the extra-cellular matrix of porcine hearts. The final bioink protocol allows the users to make their own bioink, from easily obtainable tissue and determine their own concentration of the extra-cellular matrix/collagen within a range. Lastly, a feedback system was developed using a Raspberry Pi and camera module to provide real-time visual feedback of the bioprinting process which is otherwise very difficult to see and optimize parameters from. A protocol was developed to sequentially optimize the parameters for an open-source slicing software which governs the resolution of the bioprinter itself. In related research, the cytotoxicity and cell adherence properties of a printing resin for a microfluidic 3D printer were evaluated for use in Lab-on-a-Chip applications. The existing resin was tested and determined to be cytotoxic to cells and therefore not suitable for biological applications. We showed that a simple ethanol washing step and plasma treatment pulled the cytotoxic elements out of the polymer and modified the surface such that cells could attach and proliferate on the printed resin. Another printed resin was also tested which was determined to have no natural cytotoxicity, but the same plasma treatment was needed to allow for cell adherence.

Organ transplantation has made great progress since the first successful kidney transplant in 1953 and now more than one million tissue transplants are performed in the United States every year (www.organdonor.gov/statistics-stories, 2015). However, the hope and success of organ transplants are often overshadowed by their reputation as being notoriously difficult to procure because of donor-recipient matching and availability. In addition, those that are fortunate enough to receive a transplant are burdened with a lifetime of immunosuppressants. The field of regenerative medicine is currently making exceptional progress toward making it possible for a patient to be their own donor. Cells from a patient can be collected, reprogrammed into stem cells, and then differentiated into specific cell types. This technology combined with recent advances in 3D printing provides a unique opportunity. Cells can now be accurately deposited with computerized precision allowing tissue engineering from the inside out (Gill, 2016). However, more work needs to be done as these techniques have yet to be perfected. Bioprinters can cost hundreds of thousands of dollars, and the bioink they consume costs thousands per liter. The resulting cost in development of protocols required for effective tissue printing can thus be cost-prohibitive, limiting the research to labs which can afford this exorbitant cost and in turn slowing the progress made in the eventual creation of patient derived stem cell engineered organs. The objective of my research is to develop a simple and low-cost introductory system for biological additive manufacturing (Otherwise known as 3D bioprinting). To create an easily accessible and cost-effective system several design constraints were implemented. First, the system had to use mechanical components that could be purchased “off-the-shelf” from commonly available retailers. Second, any mechanical components involved had to be easily sterilizable, modifiable, and compatible with open-source software. Third, any customized components had to be fabricated using only 3D printing and basic tools (i.e. saw, screwdriver, and wrench). Fourth, the system and any expendable materials should be financially available to underfunded school labs, in addition to being sterilizable, biocompatible, customizable, and biodegradable. Finally, all hardware and expendables had to be simple enough as to be operated by high school science students.

Three-dimensional microextrusion bioprinting has attracted great interest for fabrication of hierarchically structured, functional tissue substitutes with spatially defined cell distribution. Despite considerable progress, several significant limitations remain such as a lack of suitable bioinks which combine favorable cell response with high shape fidelity. Therefore, in this work a novel bioink of alginate-methylcellulose (AlgMC) blend functionalized with egg white (EW) was developed with the aim of solving this limitation. In this regard, a stepwise strategy was proposed to improve and examine the cell response in low-viscosity alginate inks (3%, w/v) with different EW concentrations, and in high-viscosity inks after gradual methylcellulose addition for enhancing printability. The rheological properties and printability of these cell-responsive bioinks were characterized to obtain an optimized formulation eliciting balanced physicochemical and biological properties for fabrication of volumetric scaffolds. The bioprinted AlgMC + EW constructs exhibited excellent shape fidelity while encapsulated human mesenchymal stem cells showed high post-printing viability as well as adhesion and spreading within the matrix. In a proof-of-concept experiment, the impact of these EW-mediated effects on osteogenesis of bioprinted primary human pre-osteoblasts (hOB) was evaluated. Results confirmed a high viability of hOB (93.7 ± 0.15%) post-fabrication in an EW-supported AlgMC bioink allowing cell adhesion, proliferation and migration. EW even promoted the expression of osteogenic genes, coding for bone sialoprotein (integrin binding sialoprotein/bone sialoprotein precursor (IBSP)) and osteocalcin (BGLAP) on mRNA level. To demonstrate the suitability of the novel ink for future fabrication of multi-zonal bone substitutes, AlgMC + EW was successfully co-printed together with a pasty calcium phosphate bone cement biomaterial ink to achieve a partly mineralized 3D volumetric environment with good cell viability and spreading. Along with the EW-mediated positive effects within bioprinted AlgMC-based scaffolds, this highlighted the promising potential of this novel ink for biofabrication of bone tissue substitutes in clinically relevant dimensions.

Bioprinting is one of the most promising technologies for future healthcare as it may benefit the repairing of wounds and injuries, disease modeling and development, transplantation of organs and reduce animal testing. This thesis aim to investigate this industry further, as there is no excessive literature on how to handle the innovation in regards to entrepreneurial and biotechnological knowledge. Hence, a research gap can be spotted and the purpose of the conducted research questions should contribute to this gap. In order to fully understand the bioprinting industry, an outline of the technology is made as part of the research. In addition to this, secondary data for patents, market valuation and annual growth rates are collected to support arguments from previous literature. Also, interviews are conducted to gather specific knowledge. As a result, bioprinting may be presented as a disruptive innovation in an uncertain market, which places certain demands on companies to act more in line with the complexity of the technology. Such companies must think more strategically and design more complex and long-term strategies. The patent data shows that there has been a decline in the technological development as patent applications have decreased significantly. Even though the technology (regarding the patents) has started to slowly decline, there is still hope for some technological improvements to come. It can be concluded that developments in bioink, scaffolds, expansion of cells and diffusion is expected, and that the use of bioprinting is increasing and will most likely continue to do so.

The present thesis aimed to fabricate a liver sinusoid model by combining different components and techniques to closely mimic the physiological microenvironment of the hepatic cells. Since liver is a complex multicellular organ, 3D extrusion bioprinting was employed as well as core-shell 3D bioprinting for creating more complex constructs with relevant physiological microarchitecture to the in vivo liver sinusoid. Figure 1 illustrates the concept of the aimed model fabrication and the combination of components required to achieve this model. To create an initial model, HepG2, a human carcinoma-derived liver cell line was used as a model cell line for hepatocytes. Biocompatible inks based on a printable alginate-methylcellulose (algMC) blend were aimed to be developed for the encapsulation of hepatocytes by functionalization with bioactive molecules to better recapitulate the hepatocytes biochemical microenvironment, supporting cellular functions. Towards tissue complexity, a further aim was to employ core-shell bioprinting to establish a coculture model of hepatocytes and fibroblasts, which acted as supportive cells; by coaxially printing HepG2 encapsulated in the shell with fibroblasts in the core of a single core-shell strand. Different bioinks were investigated as core for the fibroblasts encapsulation, whereby plasma and fibrin were utilized to functionalize the algMC blends with the aim of enhancing the fibroblasts attachment, proliferation and spreading. Moreover, the influence of functionalized core bioinks on the hepatocytes performance and function would be demonstrated, as well as the influence of the coculture with the fibroblasts. As a final step towards integrating vascularized structures in the liver sinusoid model, endothelial cells (EC) were to be cocultured with the supporting fibroblasts in the core of the core-shell constructs to create a triple-culture model with the hepatocytes in the shell. Based on collagen-fibrin matrices, suitable to support angiogenesis, a natural extracellular matrix-like Introduction 5 core bioink, which is independently printable and allows for the printing of stable core-shell vascularized constructs is aimed to be developed. The culture and coculture parameters of the ECs will be optimized and evaluated. Optimization of the shell bioink encapsulating the hepatocytes is aimed to be investigated with the goal of enhancing the HepG2 microenvironment. Printing parameters and crosslinking procedures as well as culture conditions for all the cells were to be optimized for the model. The final triple-culture core-shell printed in vitro model is aimed to characterize the ability of this triple-culture construct to support vascularization by the HUVECs printed in the core, the physiological functions of the hepatocytes printed in the shell bioink, as well as to evaluate the cellular interactions between core and shell compartments. Through engineering and modifying the bioinks which represent the extra-cellular matrix and adjusting the culture conditions for the cells, cell-cell and cell-matrix interactions can be studied in such coculture models, providing new insights towards clinical and therapeutic biomedical applications.:Abbreviations 1 1 Motivation 3 2 Introduction and state of the art 6 2.1 Liver and its microarchitecture 6 2.2 Tissue Engineering 9 2.2.1 Liver Tissue Engineering 9 2.3 From two- to three-dimensional cell cultures 10 2.3.1 2D and 3D hepatocytes coculture models 12 2.4 3D Bioprinting 14 2.4.1 Biomaterial inks and bioinks for 3D bioprinting 15 2.4.2 Core-shell 3D bioprinting 17 2.4.3 3D bioprinting of in vitro liver models 18 3 Materials and methods 20 3.1 Biomaterials for ink preparation 20 3.2 Cell lines used for bioink encapsulation 20 3.3 Cell culture media 21 3.4 3D bioprinting 22 3.5 Characterization assays 23 3.5.1 Rheological and mechanical characterization of the inks and printed scaffolds 23 3.5.2 Characterization of cell viability 23 3.5.3 Characterization of cell metabolic activity 24 3.5.4 Determination of cell number and proliferation 24 3.5.5 Quantitative analysis of hepatocytes functionality 25 3.5.6 Immunostaining 26 3.5.7 Imaging 27 3.5.8 Statistics 28 3.6 Specific experimental procedures and characterizations 28 3.6.1 Bioink development for bioprinting of hepatocytes 28 3.6.1.1 Bioink preparation 3.6.1.2 Bioprinting and crosslinking 3.6.1.3 Rheological and mechanical characterization 3.6.1.4 Biological characterization 3.6.2 Coculture of hepatocytes (HepG2) and fibroblasts (NIH 3T3) in core-shell bioprinted scaffolds 3.6.2.1 Bioink preparation 3.6.2.2 Core-shell bioprinting and crosslinking 3.6.2.3 Rheological and mechanical characterization 3.6.2.4 Biological characterization 3.6.3 Vascularization of bioprinted HepG2-laden constructs 3.6.3.2 Collagen : Fibrin (CF)-based core bioinks preparation and characterization 32 3.6.3.3 Optimization of shell bioink for HepG2 encapsulation 34 3.6.3.4 Influence of shell bioink on endothelial tube formation in the core 35 3.6.3.5 Transwell experiment for analysis of triple-cultures 35 3.6.3.6 Core-shell bioprinting of triple-culture in vitro liver sinusoid model 37 4 Results and Discussion 38 4.1 Bioink development for encapsulation of hepatocytes by 3D bioprinting 38 4.2 Spatially defined pattern of hepatocyte-fibroblast co-culture in a core-shell bioprinted system 46 4.2.1 Establishment of core-shell bioprinting 47 4.2.2 Simultaneous embedding of HepG2 and NIH 3T3 cells in core-shell strand scaffolds 49 4.2.3 Functionalization of the core bioink – enhancing fibroblast network formation 51 4.2.4 Influence of the microenvironment on expression of hepatic marker proteins in the core-shell bioprinted co-culture system 56 4.3 Vascularization strategies of an in vitro bioprinted liver sinusoid model 61 4.3.1 Preliminary investigation of prevascular-tube formation in fibrin gels 63 4.3.2 Optimization of culture conditions for HUVECs pre-vascular network formation 65 4.3.2.1 Collagen : Fibrin composite gels 65 4.3.2.2 Optimization of CF network density and influence of supportive cells on HUVECs pre-vascular network formation 70 4.3.3 Core-shell bioprinting: development of a core bioink to support formation of pre-vascular structures 75 4.3.3.1 Collagen : fibrin-based ink development for printability 75 4.3.3.2 Formation of pre-vascular structures in 3D bioprinted CFG core 83 4.3.4 Optimization of the shell bioink: HepG2 encapsulation in Plasma vs. Matrigel functionalized algMC 85 4.3.5 Vascularization in different shell biomaterial inks 94 4.3.6 Triple-culture of HepG2, HUVECs and NHDFs: analysis of shell and core bioinks in transwell-coculture 100 4.3.7 Core-shell bioprinting of a triple-culture 3D in vitro liver sinusoid model 111 5 Conclusions and Outlook 120 5.1 Bioink development for hepatocytes encapsulation 120 5.2 Establishment of core-shell bioprinting to fabricate a liver sinusoid model 120 5.3 Coculture of hepatocytes with supportive fibroblasts 121 5.4 Establishment of a vascularized liver sinusoid model 121 5.4.1 Optimization of culture conditions for endothelial cells 121 5.4.2 Development of CF-based printable bioink for endothelial cells encapsulation in the core 122 5.4.3 Optimizing hepatocytes encapsulation bioink 124 5.4.4 Establishment of the complex triple-culture liver sinusoid model 124 5.5 Outlook and future prospects 125 Summary 129 Zusammenfassung 132 References 135 List of Figures 157 List of Tables 159 List of publications

In the past decade, there has been significant progress in 3D printing research for tissue engineering (TE) using biomaterial inks made from natural and synthetic compounds. These constructs can aid in the regeneration process after tissue loss or injury, but achieving high shape fidelity is a challenge as it affects the construct's physical and biological performance with cells. In parallel with the growth of 3D bioprinting approaches, some marine-origin polymers have been studied due to their biocompatibility, biodegradability, low immunogenicity, and similarities to human extracellular matrix components, making them an excellent alternative to land mammal-origin polymers with reduced disease transmission risk and ethical concerns. In this research, collagen from shark skin, chitosan from squid pens, and fucoidan from brown algae were effectively blended for the manufacturing of an adequate biomaterial ink to achieve a printable, reproducible material with a high shape fidelity and reticulated using four different approaches (phosphate-buffered saline, cell culture medium, 6% CaCl2, and 5 mM Genipin). Materials characterization was composed by filament collapse, fusion behavior, swelling behavior, and rheological and compressive tests, which demonstrated favorable shape fidelity resulting in a stable structure without deformations, and interesting shear recovery properties around the 80% mark. Additionally, live/dead assays were conducted in order to assess the cell viability of an immortalized human mesenchymal stem cell line, seeded directly on the 3D printed constructs, which showed over 90% viable cells. Overall, the Roswell Park Memorial Institute cell culture medium promoted the adequate crosslinking of this biopolymer blend to serve the TE approach, taking advantage of its capacity to hamper pH decrease coming from the acidic biomaterial ink. While the crosslinking occurs, the pH can be easily monitored by the presence of the indicator phenol red in the cell culture medium, which reduces costs and time.

Tissue engineering (TE) uses a combination of biomaterial scaffolds, cells, and drug delivery systems (DDS) to create tissues that resemble the human physiology. Such engineered tissues could be used to treat, repair, replace, and augment damaged tissues or organs, for disease modeling, and drug screening purposes. This work describes the development and use of novel strategies for engineering neural tissue using a combination of drug delivery systems (DDS), human induced pluripotent stem cells (hiPSCs), and bioprinting technologies for the generation of a drug screening tool to be used in the process of drug discovery and development. The DDS consisted of purmorphamine (puro) loaded microspheres that were fabricated using an oil-in-water single emulsion with 84% encapsulation efficiency and showed the slow release of puro for up to 46 days in vitro. Puro and retinoic acid (RA)-loaded microspheres were combined with hiPSCs-derived neural aggregates (NAs) that differentiated into neural tissues expressing βT-III and showed increased neural extension. hiPCS-derived neural progenitor cells (NPCs) were bioprinted on a layer-by-layer using a fibrin based-bioink and extrusion based- bioprinting. The bioprinted structures showed >81% cellular viability after 7 days of culture in vitro and the expression of the mature motor neuron (MN) markers HB9 and CHAT. Lastly, hiPCS-derived NPCs were bioprinted in combination with puro and RA-loaded microspheres and cultured for 45 days in vitro. The microspheres slowly released the drug and after 30 and 45 days the tissues contained mature neurons, astrocytes and oligodendrocytes expressing CHAT, GFAP, and O4, respectively. Changes in membrane potential indicated tissue responsiveness to different types of treatments such as acetylcholine and gamma-aminobutyric acid (GABA). In the future the bioprinted tissues could contain localized regions of varied drug releasing microspheres using a concentration gradient to promote differentiation into specific cell types in order to create more complex tissues. Moreover, these tissues will benefit from the presence of a neurovascular unit (NVU). Upon validation, the engineered tissues could be used as preclinical tools to test potential drugs and be used for personalized medicine by using patient specific hiPSCs. / Graduate / 2020-11-19

Bioprinting is considered a key technology for future space missions and is currently being established on the International Space Station (ISS). With the aim to perform bioink production as a critical and resource-consuming preparatory step already on Earth and transport a bioink cartridge “ready to use” to the ISS, the storability of bioinks is investigated. Hydrogel blends based on alginate and methylcellulose are laden with either green microalgae of the species Chlorella vulgaris or with different human cell lines including immortilized human mesenchymal stem cells, SaOS-2 and HepG2, as well as with primary human dental pulp stem cells. The bioinks are filled into printing cartridges and stored at 4°C for up to four weeks. Printability of the bioinks is maintained after storage. Viability and function of the cells embedded in constructs bioprinted from the stored bioinks are investigated during subsequent cultivation: The microalgae survive the storage period very well and show no loss of growth and functionality, however a significant decrease is visible for human cells, varying between the different cell types. The study demonstrates that storage of bioinks is in principle possible and is a promising starting point for future research, making complex printing processes more effective and reproducible.

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