The goal of nerve tissue engineering is to promote and guide axon growth across a site of nerve injury without misdirection. Bioengineered tissue scaffolds have been shown to be promising for the regeneration of damaged peripheral nerves. Schwann cells play a pivotal role following nerve injury by forming aligned “bands of Büngner” that promote and guide axon regeneration into the distal nerve segment. The incorporation of living Schwann cells into various hydrogels has therefore been urged during the fabrication of tissue engineered nerve scaffolds. The aim of this research is to characterize biomaterials suitable for 3D bioplotting of nerve repair scaffolds. Here a novel technique of scaffold fabrication has been optimized to print alginate-based three-dimensional tissue scaffolds containing hyaluronic acid and living Schwann cells. Alginate/hyaluronic acid scaffolds were successfully fabricated with good printability and cell viability. Addition of the polycation polyethyleneimine (PEI) during the fabrication process stabilized the structure of alginate through the formation of a polyelectrolyte complex and had a significant influence on the degree of swelling, degradation rate, mechanical property, and release kinetics of incorporated protein within the scaffolds. A preliminary in vivo study showed the feasibility of implanting 3D printed alginate/hyaluronic acid scaffolds as nerve conduits in Sprague-Dawley (SD) rats with resected sciatic nerves. However alginate/hyaluronic acid scaffolds were found to be unsuitable for axonal regeneration. Further in vitro culture of Schwann cells was performed in collagen type-I, fibrin, fibrin/hyaluronic acid, and their combination with alginate. It was found that Schwann cells had more favorable cell morphology in fibrin/hyaluronic acid or collagen without alginate. Schwann cell proliferation and alignment were better in fibrin/hyaluronic acid. Therefore fibrin/hyaluronic acid is more ideal than most other hydrogel formulations for use in the bioprinting of nerve repair tissue engineering scaffolds, which incorporate cellular elements. As Schwann cells also align along the long axis of the printed fibrin/hyaluronic acid strands, 3D bioprinting of multiple layers of crosslinked fibrin strands can be used to fabricate a nerve conduit mimicking the bands of Büngner.

In most cases new technology emergence does not guarantee overnight success nor is it developed overnight, rather it is a result of industry expertise spanning over several fields of research. 3D bioprinting is an industry that promises revolutionary implications for healthcare and scholars are discussing application areas such as tissue for oncology research, tissue for patient specific drug development and lastly complex organs for implantation. The promises and strides of the industry pose new prospects in healthcare, for instance printing new skin for burn victims & drug development all while being patient specific. The promises of bioprinting are practices that will lead to improved quality of life whether it be in the form of medicine or a new organ. The industry is characterized by fast pace and rapid technological advancements, research has explored these technological advancements and explained them in detail, but information on the market remains scarce. To contribute to filling this gap the thesis explores what the state of the market currently is and what the stage of market development is.  In order to explore how the market has developed and the current state of it a longitudinal case study, in Sweden which is one of the most innovative countries in the world was conducted. Newspaper articles from three news outlets were collected, two of which are Swedens most reputable publishers regarding new technology and business and economic development, the third is an industry specific outlet to biotechnology advancement. This resulted in finding that while the dialog of bioprinting is promising, the degree of readiness remains the same, furthermore it shows that the industry is heavily dependent on external actors to further develop the market and technology. It was also found that there is no shortage in actors spanning across various industries that collaborate with bioprinting firms since not all industries need the full capacity of the technology that is necessary to print complex organ structures. It can be stated that while the bioprinting market is evolving the development of it is in early stages and is far from being established.

The interactions between cells and their microenvironment influence their morphological features and regulate important cellular processes. To understand deleterious neurological disorders such as Parkinson’s disease, there is an immense need to develop efficient in vitro 3D models that can recapitulate complex organs such as the brain. Ultrashort self- assembling peptides offer a revolutionary tool for generating tunable and well-defined 3D in vitro neural tissues capable of recreating complex cellular characteristics, and tissue-level responses. Herein, we describe the use of ultrashort self-assembling peptide-based scaffolds for the development of functional 3D neuronal models including an in vitro model for Parkinson’s disease. Both primary mouse embryonic dopaminergic neurons and human dopaminergic neurons derived from human embryonic stem cells were found biocompatible in our peptide-based models. Using microelectrode arrays, we recorded spontaneous activity in dopaminergic neurons encapsulated within these 3D peptide scaffolds for more than 1 month without a decrease in signal intensity. In addition, we demonstrate a 3D bioprinted model of dopaminergic neurons inspired by the mouse brain using an extrusion-based 3D robotic bioprinting technology. We used our 3D in vitro neuronal models to study the effect of both gabapentin and pregabalin on the development of dopaminergic neurons. Pregabalin and gabapentin are frequently regarded as first-line therapies for a variety of neuropathic pain syndromes, regardless of the underlying cause. Our results showed that both drugs can interfere with the neurogenesis and morphogenesis of ventral midbrain dopaminergic neurons during early brain development. Finally, to gain a better understanding of the influence of cell-cell and cell- matrix interactions on cellular behavior and function in 3D cultured cells within our peptide-based scaffolds compared to the ones cultured in 2D, we studied the metabolic and transcriptomic profiles of 2D and 3D cultured cells. 2D cultured cells exhibited distinct metabolic and transcriptomic profiles compared to the 3D cultured cells. Advancements in the fields of 3D in vitro modeling, 3D bioprinting, and biomaterials are of extreme value for the development of efficient models suitable for investigating disease-specific pathways, aiding the discovery of novel treatments, and promoting tissue regeneration.

Osteoarthritis (OA) is a progressive cartilage degeneration disease with a complex pathologic mechanism. Although OA has devastating effects on patient quality of life and places a significant burden on the healthcare system, no disease-modifying drugs have been found, and surgical treatment options are often unsustainable. 3D bioprinting is a novel field within tissue engineering that focuses on developing biocompatible constructs that can be implanted to replace an organ or tissue. Such constructs have a great potential to become treatments for OA. Understanding cell mobility within hydrogels could play a vital role in advancing the development of biocompatible constructs. However, due to the novelty of bioprinting, limited research on cell mobility within hydrogels is available. Therefore, this project aims to fill the gap in existing research regarding cell mobility within bioprinted constructs with varying mechanical properties. To achieve this goal, green fluorescent protein-tagged mesenchymal stromal cells (MSCs) were developed to assess progenitor cell mobility in bioprinted hydrogel constructs. Constructs were printed with three zones: hydrogel with embedded chondrocytes or MSCs; hydrogel spacer; and chemoattractant. Designed constructs were bioprinted (BioAssemblyBot, Advanced Solutions) using GelMA:HAMA bioinks containing photoinitiator with varying bioink percentages. Cell viability and directional mobility within constructs were assessed by fluorescence viability assay and time-lapse fluorescence microscopy. The protocol to evaluate cell mobility in bioprinted constructs and optimized bioprinting settings for GelMA:HAMA bioinks were gained through this project. Overall, this project allowed us to fill the gap in existing knowledge regarding MSC and chondrocyte mobility in hydrogels and contribute to developing a novel treatment method for OA.

Cette thèse a pour objectif de développer une méthode de bioimpression 3D de tissus vivants. Ce nouveau champ disciplinaire a pour but la fabrication de tissus grâce à une bioimprimante en s’appuyant sur les principes fondamentaux de l’ingénierie tissulaire. Pour mener à bien ces travaux, une bio-encre spécifique a été formulée à l’aide de biomatériaux naturels afin de répondre aux critères de biocompatibilité, de maintien de la viabilité cellulaire et de support pour la formation d’un réseau cellulaire en trois dimensions. Plusieurs caractérisations ont ainsi pu être réalisées afin de démontrer l’innocuité du procédé de bioimpression 3D sur les cellules utilisées.L’évolution technologique de la bioimprimante utilisée est ensuite présentée en partant d’une technologie open-source pour arriver à l’utilisation d’un bras robotique 6 axes. L’exigence du cahier des charges de cette bioimprimante a évolué au fil des différents prototypes utilisés.La dernière partie de ce travail de thèse présente les résultats de bioimpression de tissus obtenus grâce à de multiples collaborations. Plusieurs tissus seront étudiés et caractérisés : le derme et sa maturation vers une peau totale, le cartilage et la bioimpression de cellules souches mésenchymateuses, un tissu microvascularisé grâce à l’incorporation de cellules endothéliales et pour finir un tissu perfusable en utilisant une approche de culture dynamique en bioréacteur / This thesis focus on the development of a 3D bioprinting process for living tissue. This new field of research, 3D bioprinting, aims to fabricate tissues using a bioprinter based on the tissue engineering fundamentals.To carry out this work, a specific bioink was formulated using natural biomaterials to meet the requirement of biocompatibility, cell viability and support of a three-dimensional cellular network. Several characterizations have been used to demonstrate the cells viability during the 3D bioprinting process.The bioprinter technological evolution is then presented, starting from an open-source technology and ending with the use of a 6-axis robotic arm. The specifications of this bioprinter evolved through different prototypes.The last part of this thesis concerns tissue bioprinting results obtained through multiple collaborations. Several tissues will be studied and characterized: the dermis and its maturation towards a total skin, the cartilage and the mesenchymal stem cells bioprinting, a microvascularized tissue thanks to the incorporation of endothelial cells and finally a perfusable tissue by using a dynamic culture approach in bioreactor

Cartilage tissue engineering has been emerging as a promising therapeutic approach, where engineered constructs or scaffolds are used as temporary supports to promote regeneration of functional cartilage tissue. Hybrid constructs fabricated from cells, hydrogels, and solid polymeric materials show the most potential for their enhanced biological and mechanical properties. However, fabrication of customized hybrid constructs with impregnated cells is still in its infancy and many issues related to their structural integrity and the cell functions need to be addressed by research. Meanwhile, it is noticed that nowadays monitoring the success of tissue engineered constructs must rely on animal models, which have to be sacrificed for subsequent examination based on histological techniques. This becomes a critical issue as tissue engineering advances from animal to human studies, thus raising a great need for non-invasive assessments of engineered constructs in situ. To address the aforementioned issues, this research is aimed to (1) develop novel fabrication processes to fabricate hybrid constructs incorporating living cells (hereafter referred as “construct biofabrication”) for cartilage tissue regeneration and (2) develop non-invasive monitoring methods based on synchrotron X-ray imaging techniques for examining cartilage tissue constructs in situ. Based on three-dimensional (3D) printing techniques, novel biofabrication processes were developed to create constructs from synthetic polycaprolactone (PCL) polymer framework and cell-impregnated alginate hydrogel, so as to provide both structural and biological properties as desired in cartilage tissue engineering. To ensure the structural integrity of the constructs, the influence of both PCL polymer and alginate was examined, thus forming a basis to prepare materials for subsequent construct biofabrication. To ensure the biological properties, three types of cells, i.e., two primary cell populations from embryonic chick sternum and an established chondrocyte cell line of ATDC5 were chosen to be incorporated in the construct biofabrication. The biological performance of the cells in the construct were examined along with the influence of the polymer melting temperature on them. The promising results of cell viability and proliferation as well as cartilage matrix production demonstrate that the developed processes are appropriate for fabricating hybrid constructs for cartilage tissue engineering. To develop non-invasive in situ assessment methods for cartilage and other soft tissue engineering applications, synchrotron phase-based X-ray imaging techniques of diffraction enhanced imaging (DEI), analyzer based imaging (ABI), and inline phase contrast imaging (PCI) were investigated, respectively, with samples prepared from pig knees implanted with low density scaffolds. The results from the computed-tomography (CT)-DEI, CT-ABI, and extended-distance CT-PCI showed the scaffold implanted in pig knee cartilage in situ with structural properties more clearly than conventional PCI and clinical MRI, thus providing information and means for tracking the success of scaffolds in tissue repair and remodeling. To optimize the methods for live animal and eventually for human patients, strategies with the aim to reduce the radiation dose during the imaging process were developed by reducing the number of CT projections, region of imaging, and imaging resolution. The results of the developed strategies illustrate that effective dose for CT-DEI, CT-ABI, and extended-distance CT-PCI could be reduced to 0.3-10 mSv, comparable to the dose for clinical X-ray scans, without compromising the image quality. Taken together, synchrotron X-ray imaging techniques were illustrated promising for developing non-invasive monitoring methods for examining cartilage tissue constructs in live animals and eventually in human patients.

In this diploma thesis we are discussing about using 3D bioprinting in tissue engineering. We are discribing using biomaterials for construction scaffolder and aplication stem cells in 3D bioprinting. Last section of theoretical part deals with very often used techniques of 3D bioprinting and we are focused on extrusion technique. In the practical part we propose a method for print vasculars structures. We realized prototype of print head, her design and 3D printing of individual parts. To mechanical part we create a control system for printing control. At the end we visualize the organization of the cells using program modules.

Articular cartilage is a highly specialized, hierarchically organized tissue covering the articular surfaces of diarthrodial joints that absorbs and distributes forces upon mechanical loading and enables low-friction movement between opposing bone ends. Despite a strong resilience towards mechanical stress, once damaged cartilage is generally not regenerated due to a limited repair potential of the residing cells (chondrocytes) and the local absence of vascularized blood vessels and nerves. Eventually, this may lead to osteoarthritis, a chronic degenerative disorder of the synovial joints which has a strongly growing prevalence worldwide. Modern regenerative therapies that aim to rebuild cartilage tissue in vivo and in vitro using chondrocyte- and stem cell-based methods are still not able to produce tissue constructs with desired biomechanical properties and organization for long-term repair. Therefore, cartilage tissue engineering seeks for new ways to solve these problems. In this regard, the application of hydrogel-based scaffolding materials as artificial matrix environments to support the chondrogenesis of embedded cells and the implementation of appropriate biofabrication techniques that help to reconstitute the zonal structure of articular cartilage are considered as promising strategies for sophisticated cartilage regeneration approaches. In this thesis, a modular starPEG-heparin hydrogel platform as cell-instructive hydrogel scaffold was used in combination with a custom-designed 3D inkjet bioprinting method with the intention to develop a printable 3D in vitro culture system that promotes the chondrogenic differentiation of human mesenchymal stromal cells (hMSC) in printed cell-laden hydrogels with layered architectures in order to fabricate cartilage-like tissue constructs with hierarchical organization. Firstly, the successful bioprinting of horizontally and vertically structured, cell-free and -laden hydrogel scaffolds that exhibit layer thicknesses in the range of the superficial zone, the thinnest articular cartilage layer is demonstrated. The long-term integrity of the printed constructs and the cellular functionality of the plotted cells that generally had a high viability after the printing process are shown by a successful PDGF-BB-mediated hMSC migration assay in a printed multilayered hydrogel construct over a culture period of 4 weeks. Secondly, when the established printing procedures were applied for the chondrogenic differentiation of hMSCs, it was found that the printed cell-laden constructs showed a limited potential for in vitro chondrogenesis as indicated by a weaker immunostaining for cartilage-specific markers compared to casted hydrogel controls. In order to increase the post-printing cell density to tackle the limited printable cell concentration which was regarded as the primary reason for the impaired performance of the printed scaffolds, different conditions with varying culture medium and hydrogel compositions were tested to stimulate 3D cell proliferation. However, a significant 3D cell number increase could not be achieved which ultimately resulted in shifting the further focus to casted hMSC-laden starPEG-heparin hydrogels. Thirdly, the chondrogenic differentiation of hMSCs in casted hydrogels proved to be successful which was indicated by a uniform deposition of cartilage-specific ECM molecules comparable with the outcomes of scaffold-free MSC micromass cultures used as reference system. However, the quantitative analysis of biochemical and physical properties of the engineered hydrogel constructs yielded still significant lower values in relation to native articular cartilage tissue. Fourthly, in order to improve these properties and to enhance the chondrogenesis in starPEGheparin hydrogels, a dualistic strategy was followed. In the first part, specific externally supplied stimulatory cues including a triple growth factor supply strategy and macromolecular crowding were applied. As second part, intrinsic properties of the modular hydrogel system such as the crosslinking degree, the enzymatic degradability and the heparin content were systematically and independently altered. It was found that while the external cues showed no supportive benefits for the chondrogenic differentiation, the reduction of the heparin content in the hydrogel proved to be a key trigger that resulted in a significantly increased cartilage-like ECM deposition and gel stiffness of engineered constructs with low and no heparin content. In conclusion, this work yielded important experiences with regards to the application of inkjet bioprinting for hMSC-based cartilage tissue engineering approaches. Furthermore, the obtained data provided valuable insights into the interaction of MSCs and a surrounding hydrogel-based microenvironment that can be used for the further development of chondrosupportive scaffolding materials which may facilitate the fabrication of cartilage-like tissue constructs.

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.

[pt] A tecnologia de bioimpressão 3D se tornou um importante recurso em pesquisas médicas no campo da engenharia de tecidos. Entretanto, apesar de essa tecnologia possuir grande convergência e demanda de competências do Design, o profissional designer ainda se encontra distante de tais trabalhos. Esta tese promove a investigação do papel que o Design pode desempenhar em pesquisas envolvendo o uso da bioimpressão 3D. A partir da revisão bibliográfica inicial e da compreensão acerca dos temas da engenharia de tecidos e de biomateriais, foram estabelecidas parcerias visando à execução de uma série de experimentos práticos. A partir da construção de uma bioimpressora desktop e posterior aquisição de uma bioimpressora profissional, foram realizados testes de configuração, calibragem e impressão de diferentes materiais viscosos, para uma melhor compreensão do processo de impressão dos biomateriais. Em parceria com o doutor Ronaldo Andrade, paralelamente se buscou solucionar a questão de simulação prática em treinamentos médicos, visando gerar modelos físicos semelhantes aos tecidos vivos. Por meio de uma parceria com o Grupo de Estudos do Fígado/UFRJ, e com o laboratório do Programa de Engenharia Nuclear, da COPPE/UFRJ, utilizou-se a microtomografia para gerar modelos tridimensionais virtuais que serviram de base para um software de visualização e para a impressão 3D de um fígado de rato. Da parceria com as pesquisadoras Sara Gemini e Jéssica Dornelas, foi possível realizar a impressão de construtos com esferóides celulares e a posterior análise via microscopia. Em nova rodada de experimentos, a biotinta desenvolvida foi aperfeiçoada e testada em diferentes formulações. Além de buscar avanços nos campos investigados, a presente tese buscar mostrar o papel interdisciplinar do Design e seu potencial agregador em pesquisas na área da Ciências Médicas e Biológicas, fugindo do lugar-comum em que o profissional muitas vezes é colocado. / [en] 3D bioprinting technology has become an important resource in medical research, in the field of tissue engineering. However, although this technology has great convergence and demand for skills that Design has, the designer is far from such works. Thisthesis promotesthe investigation of the role that Design can play in researches on the use of 3D bioprinting. From the preliminary bibliographic review and understanding of tissue engineering and biomaterials topics, partnerships were formed in order to develop a series of practical experiments. From the construction of a desktop bioprinter and subsequent acquisition of a professional bioprinter, configuration, calibration and printing tests of different viscous materials were carried out for a better understanding of the printing process of biomaterials. In partnership with Dr. Ronaldo Andrade, an attempt was also made to solve the issue of practical simulation in medical training, with a view to generating physical models similar to living tissue. Through a partnership with the Grupo de Estudos do Fígado/UFRJ, and the laboratory of the Nuclear Engineering Program at COPPE/UFRJ, microtomography was used to generate virtual three-dimensional models that served as the basis for a software for visualization and 3D printing of a mouse liver. In partnership with researchers Sara Gemini and Jéssica Dornelas, it was possible to print constructs with cellular spheroids and subsequently analyze them via microscopy. In a new round of experiments, the developed bioink was improved and tested in different formulations. In addition to seeking advances in the investigated fields, this thesis seeks to show the interdisciplinary role of Design and its aggregating potential in research in the Medical and Biological Sciences areas, escaping the commonplace in which the professional is often placed.