The field of tissue engineering has grown immensely since its inception in the late 1980s. However, currently commercialized tissue engineered products are simple in structure. This is due to a pre-clinical bottleneck in which complex tissues are unable to be fabricated. 3D bioprinting has become a versatile tool in engineering complex tissues and offers a solution to this bottleneck. Characterizing the mechanical properties of engineered tissue constructs provides powerful insight into the viability of engineered tissues for their desired application. Current methods of mechanical characterization of soft hydrogel materials used in tissue engineering destroy the sample and ignore the effect of 3D bioprinting on the overall mechanical properties of a construct. Herein, this work reports on the novel use of a non-destructive method of viscoelastic analysis to demonstrate the influence of 3D bioprinting strategy on mechanical properties of hydrogel tissue scaffolds. 3D bioprinting is demonstrated as a versatile tool with the ability to control mechanical and physical properties. Structure-function relationships are developed for common 3D bioprinting parameters such as printed fiber size, printed scaffold pattern, and bioink formulation. Further studies include effective real-time monitoring of crosslinking, and mechanical characterization of multi-material scaffolds. We envision this method of characterization opening a new wave of understanding and strategy in tissue engineering. / Graduate

Bioprinting, defined as depositing cells, extracellular matrices and other biologically relevant materials in user-defined patterns to build tissue constructs de novo or to build upon pre-fabricated scaffolds, is among one of the most promising techniques in tissue engineering. Among the various technologies used for Bioprinting, pressure driven systems are most conducive to preserving cell viability. Herein, we explore the abilities of a novel bioprinter - Digilab, Inc.'s prototype cell printer. The prototype cell printer (Digilab Inc., Holliston, MA) is an automated liquid handling device capable of delivering cell suspension in user-defined patterns onto standard cell culture substrates or custom-designed scaffolds. In this work, the feasibility of using the cell printer to deliver cell suspensions to biological sutures was explored. Cell therapy using stem cells of various types shows promise to aid healing and regeneration in various ailments, including heart failure. Recent evidence suggests that delivering bone-marrow derived mesenchymal stem cells to the infarcted heart reduces infarct size and improves ventricular performance. Current cell delivery systems, however, have critical limitations such as inefficient cell retention, poor survival, and lack of targeted localization. Our laboratories have developed a method to produce discrete fibrin microthreads that can be bundled to form a suture and attached to a needle. These sutures can then be seeded with bone-marrow derived mesenchymal stem cells to deliver these cells to a precise location within the heart wall, both in terms of depth and surface localization. The efficiency of the process of seeding cells onto fibrin thread bundles (sutures) has previously been shown to be 11.8 ± 3.9 %, suggesting that 88% of the cells in suspension are not used. Considering that the proposed cell-therapy model for treatment of myocardial infarction contemplates use of autologous bone-marrow derived stem cells, an improvement in the efficiency of seeding cells onto the fibrin sutures is highly desirable. The feasibility of using Digilab's prototype cell printer to deliver concentrated cell suspension containing human mesenchymal stem cells (hMSCs) directly onto a fibrin thread bundle was explored in this work, in order to determine if this technology could be adapted to seed cells onto such biological sutures. First the effect of the printing process on the viability of hMSCs was assessed by comparing to cells dispensed manually using a hand-held pipette. The viability of hMSCs 24 hours post-dispensing using the cell printer was found to be 90.9 ± 4.0 % and by manual pipetting was 90.6 ± 8.2 % (p = ns). Thereafter a special bioreactor assembly composed of sterilizable Delrin plastic and stainless steel pins was designed to mount fibrin thread bundles onto the deck of the cell printer, to deliver a suspension containing hMSCs on the bundles. Highly targeted delivery of cell suspension directly onto fibrin thread bundles (average diameter 310 µm) was achieved with the bundle suspended in mid-air horizontally parallel to the printer's deck mounted on the bioreactor assembly. To compare seeding efficiency, fibrin thread bundles were simultaneously seeded with hMSCs using either the cell printer or the current method (tube-rotator method) and incubated for 24 hours. Seeded thread bundles were visualized using confocal microscopy and the number of cells per unit length of the bundle was determined for each group. The average seeding efficiency with the tube rotator method was 7.0 ± 0.03 % while the cell printer was 3.46 ± 2.24% (p = ns). In conclusion, the cell printer was found to handle cells as gently as manual pipetting, preserve their viability, with the added abilities to dispense cells in user-defined patterns in an automated manner. With further development, such as localized temperature, gas and humidity control on the cell printer's deck to aid cell survival, the seeding efficiency is likely to improve. The feasibility of using this automated liquid handling technology to deliver cells to biological scaffolds in specified patterns to develop vehicles for cell therapy was shown in this study. Seeding other cell types on other scaffolds along with selectively loading them with growth factors or multiple cell types can also be considered. In sum, the cell printer shows considerable potential to develop novel vehicles for cell therapy. It empowers researchers with a supervision-free, gentle, patterned cell dispensing technique while preserving cell viability and a sterile environment. Looking forward, de novo biofabrication of tissue replicates on a small scale using the cell printer to dispense cells, extracellular matrices, and growth factors in different combinations is a very realistic possibility.

Orientador: Joni Augusto Cirelli / Banca: Raquel Scarel Caminaga / Banca: Willian Fernando Zambuzzi / Resumo: Com a evolução no desenvolvimento de biomateriais, a utilização de matrizes tridimensionais (scaffolds), construídas a partir de impressão tridimensional (3DP), via SLS (selective laser sinterization) e por Extrusão de Filamentos, vêm ganhando bastante destaque no ramo da engenharia tecidual óssea. A possibilidade de impressão de modelos 3D, baseado em modelo virtual prévio, com forma, tamanho, e porosidade altamente controlados, assemelha o material ao osso perdido, favorecendo a reconstrução de defeitos ósseos em substituição aos autoenxentos, considerados "padrão ouro". O objetivo deste trabalho foi caracterizar físico-química e biologicamente scaffolds à base de Poli(3-hidroxibutirato) (PHB), confeccionados por impressão 3D via SLS, revestidos com celulose bacteriana (CB), funcionalizados ou não com apatitas (HA) e/ou peptídeo de crescimento osteogênico (osteogenic growth peptide - OGP) (Estudo 1), assim como matrizes de Poli(hidroxibutirato-co-valerato) (PHBV) e PHBV-apatita radiopaca dopada com Lantânio (PHBV-La20OAP) confeccionadas por Extrusão de Filamentos (Estudo 2), com finalidade de regeneração óssea. Os resultados de caracterização físico-química por meio da MEV/EDS demonstraram que os scaffolds produzidos apresentaram composição química e arquitetura (forma e porosidade) adequadas. A resistência mecânica nos scaffolds de PHB não sofreu alteração significativa após o revestimento com CB; porém, teve redução com a adição de apatita. O lantânio promoveu um aumento ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: With the evolution in the development of biomaterials, the use of three-dimensional matrices (scaffolds), constructed from three-dimensional printing (3DP) via SLS (selective laser sinterization) and Filament Extrusion are gaining a lot of attention in the field of bone tissue engineering. The possibility to print 3D models based on previous virtual models with highly controlled shape, size and porosity, makes the material similar to the lost bone and favours the reconstruction of bone defects in the replacement of autografts considered "gold standard". The aim of this study was to physicochemically and biologically characterize Poly (3-hydroxybutyrate) (PHB) scaffolds, made by 3D printing via SLS coated with bacterial cellulose, functionalized or not with apatite (HA) and / or osteogenic growth peptide- (OGP) (Study 1) and matrices of Poly (hydroxybutyrate-co-valerate) (PHBV) and PHBV-La containing radiopaque apatite (PHBV-La20OAP) made by filament extrusion (Study 2) for bone regeneration. Results from physicochemical characterization by SEM/EDS demonstrated that the all produced scaffolds presented appropriate chemical composition and architecture (shape and porosity). The mechanical strength of the PHB scaffolds was not significantly affected my CB coating but was reduced after apatite addition. The La allowed an increase in the elasticity modulus of PHBV matrices. Experiments for analysis of the biological behaviour of the scaffolds showed that they allowed the maintenan... (Complete abstract click electronic access below) / Mestre

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.

Tissue engineering, with the ultimate goal of engineering artificial tissues or organs to replace malfunctioning or diseased ones inside the human body, provides a substitute for organ transplantation. Driven by the growing, tremendous gap between the demand for and the supply of donated organs, tissue engineering has been advancing rapidly. There has been great success in engineering artificial organs such as skin, bone, cartilage and bladders because they have simple geometry, low cell oxygen consumption rates and little requirements for blood vessels. However, difficulties have been experienced with engineering thick, complex tissues or organs, such as hearts, livers or kidneys, primarily due to the lack of an efficient media exchange system for delivering nutrients and oxygen and removing waste. Very few types of cells can tolerate being more than 200 μm away from a blood vessel because of the limited oxygen diffusion rate. Without a vasculature system, three-dimensional (3D) engineered thick tissues or organs cannot get sufficient nutrients, gas exchange or waste removal, so nonhomogeneous cell distribution and limited cell activities result. Systems must be developed to transport nutrients, growth factors and oxygen to cells while extracting metabolic waste products such as lactic acid, carbon dioxide and hydrogen ions so the cells can grow, proliferate and make extracellular matrix (ECM), forming large-scale tissues and organs. However, available biomanufacturing technologies encounter difficulties in manufacturing and integrating vasculature networks into engineered constructs. This work proposed a novel 3D bioprinting technology that offers great potential for integration into thick tissue engineering. The presented system offered several advantages, including that it was perfusable, it could print conduits with smooth, uniform and well-defined walls and good biocompatibility, it had no post-fabrication procedure, and it enabled direct bioprinting of complex media exchange networks.

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.

Molecular self-assembly is the process of molecules able to associate into more ordered structures. Examples of self-assembling molecules is a class of ultrashort amphiphilic peptides with a distinct sequence motif, which consist of only three to seven amino acids. These peptides can self-assemble to form nanofibrous scaffolds, such as in form of hydrogels, organogels or aerogels, due to their amphiphilic structure which contains a dominant hydrophobic tail and a polar head group. Interestingly, these peptide scaffolds offer a remarkably similar fiber topography to that one found in collagen which is a dominant part of the extracellular matrix. The resemblance to collagen fibers brings a potential benefit in using these peptide scaffolds together with native human cells. Specifically, they can maintain high water content over 99 % weight per volume and are suitable for tissue engineering and regenerative medicine applications. Over the last decade, they have shown promising therapeutic potential in treating several diseases thanks to their high activity, target specificity, low toxicity, and minimal nonspecific and drug-drug interactions. This dissertation describes how to characterize and use ultrashort amphiphilic peptides for tissue engineering and biomedicine. The first chapter offers an overview of already reported self-assembling ultrashort peptides and their applications. As a proof-of-concept, ultrashort peptide scaffolds were used for osteogenic differentiation. Peptide nanoparticles were embedded into 5 peptide hydrogels with the goal to tune the stiffness of the peptide gels. Furthermore, the peptide scaffold was used for the generation of gold and silver nanoparticles after UV irradiation, which allowed the production of nanoparticles in the absence of any additional reducing agent. The mechanism of the generation of these nanoparticles was then investigated. The last chapter describes how tetrameric peptide solutions were utilized for 3D bioprinting applications. Compared to earlier reported self-assembling ultrashort peptide compounds, these tetrapeptides can form hydrogels at an extremely low concentration of 0.1% w/v in a relatively short time under physiological conditions. These promising findings suggest that the peptide solutions are promising bioinks for use in 3D bioprinting.

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Was vor einigen Jahren undenkbar erschien, könnte zukünftig möglich sein: Krankes Gewebe mit Gesundem ersetzen, das in vitro mit modernsten Biofabrikationstechniken hergestellt wird. Dabei werden bisherige Grenzen überschritten: Während lichtbasierte Biodruckverfahren wie die Zwei-Photonen-Polymerisation Auflösungen bis in den Nanometerbereich erzielen, ermöglicht der Volumetrische Biodruck (VB) den Druck zentimetergroßer Konstrukte in wenigen Sekunden. Diese Geschwindigkeiten erweisen sich unter Biodruckverfahren als konkurrenzlos und werden erreicht, da das Bioharz nicht konsekutiv, sondern zugleich vernetzt wird. Einschränkend gilt bislang nur der Mangel an geeigneten Bioharzen für den VB. Daher beschäftigt sich vorliegende Arbeit mit der Charakterisierung und Modifikation eines dafür geeigneten Bioharzes: Gelatine-Methacrylat (GelMA). Dank seiner Zusammensetzung ähnelt das etablierte Hydrogelsystem der Extratrazellularmatrix: Der Gelatine-Anteil ermöglicht Biokompatibilität und Bioaktivität durch zelladhäsive sowie degradierbare Aminosäure-Sequenzen. Zugleich können durch photovernetzbare Methacryloyl-Substituenten Konstrukte mit einer Formstabilität bei 37 °C erzeugt werden. Zunächst wurde das Bioharz zellbiologisch charakterisiert, indem mit der embryonalen Mausfibroblasten-Zelllinie NIH-3T3 beladene GelMA-Zylinder gegossen, photopolymerisiert und kultiviert wurden. Im Verlauf einer Woche wurde die Zytokompatibilität der Gele anhand der Proliferationsfähigkeit (PicoGreen-Assay), des Metabolismus (CCK-8-Assay) und der Vitalität (Live/Dead-Assay) der Zellen beurteilt. Dabei wurden Polymerkonzentrationen von 6 – 8 % sowie GelMA-Harze zweier verschiedener Molekulargewichte verglichen. Alle hergestellten Gele erwiesen sich als zytokompatibel, 6 % ige Gele ließen im Inneren jedoch zusätzlich eine beginnende Zellspreizung zu und ein niedriges GelMA-Molekulargewicht verstärkte die gemessene Proliferation. Die sich anschließende mechanische und physikalische Charakterisierung belegte, dass höher konzentrierte Gele einen größeren E-Modul aufwiesen und damit steifer waren. Eine Modifikation der Gele mit Fibronektin beeinflusste die Zellverträglichkeit weder positiv noch negativ und die Zugabe von Kollagen war wegen Entmischungseffekten nicht bewertbar. Es liegt die Vermutung nah, dass eine weitere Reduktion der Polymerkonzentration und damit Verringerung der Gelsteifigkeit der Schlüssel für mehr Zellspreizung und -wachstum ist. Da jedoch die Druckbarkeit des Bioharzes die weitere Senkung des GelMA-Gehalts limitiert, sollten zunächst Methoden entwickelt werden, welche die Netzwerkdichte des GelMAs anderweitig herabsetzen. / What seemed unthinkable a few years ago could be possible in the future: replacing diseased tissue with healthy tissue produced in vitro using the latest biofabrication techniques. Previous limits are being exceeded: While light-based bioprinting processes such as two-photon polymerization achieve resolutions down to the nanometer range, volumetric bioprinting (VB) makes it possible to print centimeter-sized constructs in just a few seconds. These speeds are unrivaled among bioprinting processes and are achieved because the bioresin is not cross-linked consecutively but simultaneously. The only limitation to date is the lack of suitable bioresins for VB. Therefore, the present work deals with the characterization and modification of a suitable bioresin: gelatine methacrylate (GelMA). Thanks to its composition, the established hydrogel system is similar to the extracellular matrix: The gelatine component enables biocompatibility and bioactivity through cell-adhesive as well as degradable amino acid sequences. At the same time, photo-crosslinkable methacryloyl substituents can be used to produce constructs with dimensional stability at 37 °C. First, the bioresin was characterized cell biologically by casting, photopolymerizing and culturing GelMA cylinders loaded with the embryonic mouse fibroblast cell line NIH-3T3. Over the course of a week, the cytocompatibility of the gels was assessed based on proliferation capacity (PicoGreen assay), metabolism (CCK-8 assay) and viability (Live/Dead assay) of the cells. Polymer concentrations of 6 - 8 % and GelMA resins of two different molecular weights were compared. All gels produced were found to be cytocompatible, however, 6 % gels additionally allowed incipient cell spreading inside and a low GelMA molecular weight increased the measured proliferation. The subsequent mechanical and physical characterization showed that gels with higher concentration had a higher modulus of elasticity and were therefore stiffer. Modifications of the gels with fibronectin had neither a positive nor negative effect on cell compatibility and the addition of collagen could not be evaluated due to segregation effects. It is reasonable to assume that further reduction in polymer concentration and thus a reduction in gel stiffness is the key to more cell spreading and growth. However, since the printability of the bioresin limits further reduction of the GelMA content, methods should first be developed to reduce the network density of the GelMA in other ways.