Academic literature on the topic 'Functionalized biopolymers'

In this review, we will discuss the current status of extracellular vesicle (EV) delivery via biopolymeric scaffolds for therapeutic applications and the challenges associated with the development of these functionalized scaffolds. EVs are cell-derived membranous structures and are involved in many physiological processes. Naïve and engineered EVs have much therapeutic potential, but proper delivery systems are required to prevent non-specific and off-target effects. Targeted and site-specific delivery using polymeric scaffolds can address these limitations. EV delivery with scaffolds has shown improvements in tissue remodeling, wound healing, bone healing, immunomodulation, and vascular performance. Thus, EV delivery via biopolymeric scaffolds is becoming an increasingly popular approach to tissue engineering. Although there are many types of natural and synthetic biopolymers, the overarching goal for many tissue engineers is to utilize biopolymers to restore defects and function as well as support host regeneration. Functionalizing biopolymers by incorporating EVs works toward this goal. Throughout this review, we will characterize extracellular vesicles, examine various biopolymers as a vehicle for EV delivery for therapeutic purposes, potential mechanisms by which EVs exert their effects, EV delivery for tissue repair and immunomodulation, and the challenges associated with the use of EVs in scaffolds.

A series of polymers, including chitosan (CS), carboxymethylcellulose (CMC) and a chitosan–gelatin (CS–GEL) hybrid polymer, were functionalized with ferulic acid (FA) derived from the enzymatic treatment of arabinoxylan through the synergistic action of two enzymes, namely, xylanase and feruloyl esterase. Subsequently, the ferulic acid served as the substrate for laccase from Agaricus bisporus (AbL) in order to enzymatically functionalize the above-mentioned polymers. The successful grafting of the oxidized ferulic acid products onto the different polymers was confirmed through ultraviolet–visible (UV–Vis) spectroscopy, attenuated total reflectance (ATR) spectroscopy, scanning electron microscopy (SEM) and nuclear magnetic resonance (NMR) spectroscopy. Additionally, an enhancement of the antioxidant properties of the functionalized polymers was observed according to the DDPH and ABTS protocols. Finally, the modified polymers exhibited strong antimicrobial activity against bacterial populations of Escherichia coli BL21DE3 strain, suggesting their potential application in pharmaceutical, cosmeceutical and food industries.

Although chitin is of the most available biopolymers on Earth its uses and applications are limited due to its low solubility. The deacetylation of chitin leads to chitosan. This biopolymer, composed of randomly distributed β-(1-4)-linked D-units, has better physicochemical properties due to the facts that it is possible to dissolve this biopolymer under acidic conditions, it can adopt several conformations or structures and it can be functionalized with a wide range of functional groups to modulate its superficial composition to a specific application. Chitosan is considered a highly biocompatible biopolymer due to its biodegradability, bioadhesivity and bioactivity in such a way this biopolymer displays a wide range of applications. Thus, chitosan is a promising biopolymer for numerous applications in the biomedical field (skin, bone, tissue engineering, artificial kidneys, nerves, livers, wound healing). This biopolymer is also employed to trap both organic compounds and dyes or for the selective separation of binary mixtures. In addition, chitosan can also be used as catalyst or can be used as starting molecule to obtain high added value products. Considering these premises, this review is focused on the structure and modification of chitosan as well as its uses and applications.

Supramolecular biopolymers (SBPs) are those polymeric units derived from macromolecules that can assemble with each other by noncovalent interactions. Macromolecular structures are commonly found in living systems such as proteins, DNA/RNA, and polysaccharides. Bioorganic chemistry allows the generation of sequence-specific supramolecular units like SBPs that can be tailored for novel applications in tissue engineering (TE). SBPs hold advantages over other conventional polymers previously used for TE; these materials can be easily functionalized; they are self-healing, biodegradable, stimuli-responsive, and nonimmunogenic. These characteristics are vital for the further development of current trends in TE, such as the use of pluripotent cells for organoid generation, cell-free scaffolds for tissue regeneration, patient-derived organ models, and controlled delivery systems of small molecules. In this review, we will analyse the 3 subtypes of SBPs: peptide-, nucleic acid-, and oligosaccharide-derived. Then, we will discuss the role that SBPs will be playing in TE as dynamic scaffolds, therapeutic scaffolds, and bioinks. Finally, we will describe possible outlooks of SBPs for TE.

Three-dimensional (3D) bioprinting technology is now one of the best ways to generate new biomaterial for potential biomedical applications. Significant progress in this field since two decades ago has pointed the way toward use of natural biopolymers such as polysaccharides. Generally, these biopolymers such as alginate possess specific reactive groups such as carboxylate able to be chemically or enzymatically functionalized to generate very interesting hydrogel structures with biomedical applications in cell generation. This present review gives an overview of the main natural anionic polysaccharides and focuses on the description of the 3D bioprinting concept with the recent development of bioprinting processes using alginate as polysaccharide.

Antibacterial resistance is a major worldwide threat due to the increasing number of infections caused by antibiotic-resistant bacteria with medical devices being a major source of these infections. This suggests the need for new antimicrobial biomaterial designs able to withstand the increasing pressure of antimicrobial resistance. Recombinant protein polymers (rPPs) are an emerging class of nature-inspired biopolymers with unique chemical, physical and biological properties. These polymers can be functionalized with antimicrobial molecules utilizing recombinant DNA technology and then produced in microbial cell factories. In this work, we report the functionalization of rPBPs based on elastin and silk-elastin with different antimicrobial peptides (AMPs). These polymers were produced in Escherichia coli, successfully purified by employing non-chromatographic processes, and used for the production of free-standing films. The antimicrobial activity of the materials was evaluated against Gram-positive and Gram-negative bacteria, and results showed that the polymers demonstrated antimicrobial activity, pointing out the potential of these biopolymers for the development of new advanced antimicrobial materials.

The work presented in this thesis focuses on the development of strategies and smart bioactive materials for the treatment of osteoporosis. High and low molecular weight soluble hyaluronic acid-bisphosphonate (HA-BP) derivatives were investigated for their ability to inhibit osteoclasts. Low molecular weight HA-BP (L-HA-BP) was most effective in inhibiting active resorption of both murine and human osteoclasts (without affecting osteoblasts) compared to free bisphosphonate (BP). Precursor monocytes were unaffected, suggesting the specificity of HA-BP towards osteoclasts. This new class of functionalized hyaluronic acid could lead to rapid development of tailor-made pro-drugs for targeted treatment of osteoporosis. Polyphosphoesters (PEP) have been widely studied for their pro-osteoblast effects, primarily due to their involvement in cellular energy production pathway leading to the formation of inorganic phosphates that contribute to mineralized bone. Given that the effect of PEP on human osteoclasts is little studied, this work on poly(ethylene sodium phosphate) (PEP.Na) explores the potential to use PEP.Na as an inhibitor of osteoclast activity for the first time. PEP.Na exposure led to a dose-dependent toxicity of osteoclasts with reduction in their capacity to form resorption pits over 24h. Currently, there is a dearth of in vitro cell-culture systems that can study osteoclast-related resorption and osteoblast-related mineralization in a single co-culture system, and to simultaneously quantify the effects of soluble factors on these processes. Described here, is the development of a novel and simple two-sided co-culture system that can overcome these limitations with reliable and quantifiable readouts. In comparison with traditional one-sided co-culture systems, the two-sided co-culture was able to generate similar readouts for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) markers. There is also the advantage of distinctly separate and quantifiable readouts for mineralization and resorption, which has been demonstrated using Pamidronate. Finally, HA-BP was synthesized with pre-determined amounts of BP groups. The BP groups attached to HA allowed the tunable incorporation of BMP-2 in hydrogels. The charge-based affinity of BMP-2 and BP allowed stable incorporation of specific amounts of BMP-2, which could be tuned by the ratio of BP groups. 125I-labelled BMP-2 was loaded into hydrogels and their release was studied. Radioactive measurements revealed the tunable sequestration and controlled release of protein over time. This result was corroborated by ALP measurements of cells exposed to released BMP-2. ALP production was found to be almost 5-fold higher in HA-BP hydrogels loaded with BMP-2 which suggested that the sequestered BMP-2 is not only available to cells but also remains highly potent, even in entrapped form, The release of BMP-2 is dependent upon the rate of diffusion, swelling in hydrogels and degradation pattern of the gels and may assist in the long-term and rapid regeneration of osteoblasts in vitro.

Les pathologies du cartilage articulaire constituent aujourd’hui un problème majeur de santé publique. À ce jour, il n’existe pas de traitement permettant la réparation du cartilage. Cependant, parmi les approches thérapeutiques en cours d’évaluation, l’ingénierie tissulaire, dont l’objectif est la formation de néo-tissus semble être une solution thérapeutique prometteuse.L’objectif final de ce projet de thèse concerne l’élaboration d’une matrice génétiquement activée (MGA) injectable, permettant de contrôler la différenciation chondrocytaire des cellules stromales mésenchymateuses (CSM). Pour mener à bien ce projet, le premier objectif a été de trouver un vecteur d’acides nucléiques adapté à l’élaboration d’une MGA et capable de transfecter efficacement les CSM. Nous avons donc développé un nouveau nanovecteur de siARN, nommé SELF (Solvent Exchange Lipoplexe Formulations), dont la taille est ajustable en contrôlant les paramètre de formulaion, qui est stable dans le temps dans des conditions de culture cellulaire et qui possède une grande efficacité pour transfecter les cellules stromales mésenchymateuses humaines primaires. Nous avons associé les SELF à des microsphères 3D poreuses de collagène et démontré que l'efficacité du chargement et la cinétique de libération sont corrélées à la taille des SELF. Ce type original et unique de MGA, avec une cinétique de libération adaptable, pourrait être intéressant pour des profils de transfection à long terme et/ou séquentielle de cellules souches en culture 3D. Ainsi, nous avons formé différentes MGA capables d’induire différents profils d’inhibition d’un gène spécifique sur au moins 21 jours. Enfin, nous avons étudié l’efficacité de la MGA sur un modèle in vitro de différenciation chondrocytaire de CSM humaines. Nous avons montré que la MGA induit une inhibition prolongée de l’expression du gène Runx2 pendant au moins 21 jours. En conditions de différenciation chondrocytaire, cette diminution d’expression de Runx2 semble diminuer l’expression de certains marqueurs de l’hypertrophie. Malgré ces résultats prometteurs, un effet inhibiteur de la MGA sur la différenciation des CSM reste à vérifier. En résumé, nos travaux ont montré l’intérêt de notre approche pour contrôler l’expression des marqueurs hypertrophiques d’un néocartilage. Un contrôle plus précis de la libération des vecteurs devrait permettre d’améliorer l’efficacité de la MGA pour des applications d’ingénierie tissulaire du cartilage
Articular cartilage pathologies are a major public health problem today. To date, there is no treatment that can repair the cartilage. However, among the therapeutic approaches currently being evaluated, tissue engineering, the objective of which is the formation of neo-tissues, seems to be a promising therapeutic solution.The final objective of this thesis project concerns the development of an injectable genetically activated matrix (MGA), making it possible to control the hypertrophic chondrocyte differentiation of mesenchymal stromal cells (MSCs). To carry out this project, the first objective was to find a nucleic acid vector suitable for the development of an MGA and capable of efficiently transfecting MSCs. We therefore designed a new siRNA nanovector, called solvent exchange lipoplexe formulation (SELF), which has a tunable size, is stable over time in cell culture conditions and possess a high efficiency to transfect primary human mesenchymal stromal cells. We associated SELF with porous collagen 3D microspheres and demonstrated that loading efficiency and release kinetics are correlated with SELF size. This original and unique type of gene activated matrix, with adaptable release kinetics, could be of interest for long-term and/or sequential transfection profiles of stem cells in 3D culture. Thus, we formed different MGAs capable of inducing different inhibition profiles of a specific gene over at least 21 days. Finally, we studied the efficiency of MGA on an in vitro model of chondrocyte differentiation of human MSCs. We have shown that MGA induces a prolonged inhibition of Runx2 gene expression for at least 21 days. Under chondrocyte differentiation conditions, this decrease in Runx2 expression seems to decrease the expression of certain markers of hypertrophy. Despite these promising results, an inhibitory effect of MGA on MSC differentiation remains to be verified. In summary, our work has shown the interest of our approach to control the expression of hypertrophic markers of a neocartilage. More precise control of vector release should improve the efficiency of MGA for cartilage tissue engineering applications

Cette thèse de doctorat a pour objectif le développement d'un catalyseur hétérogène très actif et sélectif pour la conversion d'huiles végétales usées en monomères pour le développement de bioplastiques via la réaction d’auto-métathèse. Le méthyltrioxorhénium supporté sur l’alumine mésoporeuse modifiée avec ZnCl2 s’est avéré un catalyseur actif pour l'auto-métathèse de l'oléate de méthyle, une molécule modèle pour les triglycérides des huiles. Les produits obtenus à partir de la métathèse de l'oléate de méthyle comprennent, un diester, le 1,18-octadecenedioate et un alcène, 9-octadécène, qui peut être utilisé comme monomère pour la synthèse des polymères lorsqu'il est associé à d'autres molécules. Les conditions de réaction telles que la température, le temps de réaction et le promoteur utilisé ont été optimisées pour obtenir un rendement élevé en produits de la métathèse de l'oléate de méthyle. Le catalyseur 3% MTO/ZnCl2-Al2O3-meso avec un rapport Al/Zn de 8 à la température de réaction de 45°C pendant 60 min a été trouvé actif pour la métathèse de l'oléate de méthyle. Il a été également observé que les autres halogénures métalliques et des chlorures n'ont pas favorisé la réaction comme le ZnCl2. L'amélioration de l'activité du catalyseur par l’addition de ZnCl2 à Al2O3-méso a été expliquée en raison de la nature acide de Lewis améliorée par le ZnCl2. Les études cinétiques ont montré que le catalyseur a été très actif permettant une conversion supérieure à 80% en 30 min de réaction entre 25°C et 60°C. L'activité catalytique à la température ambiante était aussi encourageante pour l'auto métathèse de l'oléate de méthyle. Les conditions opératoires optimisées pour la métathèse de l'oléate de méthyle ont par la suite été étendues à l'application de triglycérides, en l’occurrence la trioléïne qui est abondante dans l’huile de tournesol et autres huiles végétales. Le catalyseur a été trouvé actif avec la formation de l'alcène 9-octadécène ainsi que des dimères et des trimères de la trioléine. Le catalyseur à base de MTO a été actif pour la formation des produits de métathèse désirés et le produit non désiré de métathèse comme le polymère réticulé formé par la réaction à catalyse homogène a été évité.Les résultats prometteurs pour la métathèse des trioléïnes prouvent que le catalyseur 3% MTO/ZnCl2-Al2O3-meso peut être utilisé pour la métathèse des huiles végétales comme l'huile de tournesol riche en acide oléique qui contient la trioléïne comme sa principale composante.
This doctoral thesis aims at the development of highly active and selective heterogeneous catalyst for conversion of used vegetable oils into monomers for bioplastic development using self-metathesis reaction. The catalyst based on methyltrioxorhenium supported on mesoporous alumina modified with ZnCl2 was found to be an active catalyst for self-metathesis of methyl oleate, a model molecule for triglycerides of oils with high turnover number. The products obtained from the metathesis of methyl oleate include the diester, 1,18-Octadecenedioate and an alkene, 9-Octadecene which can be used as monomer for polymer synthesis when reacted with other molecules. The reaction conditions such as temperature, reaction time, the promoter used were optimized to obtain high yield of products from the metathesis of methyl oleate. The catalyst 3% MTO/ZnCl2-Al2O3-meso with Al/Zn ratio of 8 at reaction temperature of 45°C for 60 min was found to be active for metathesis of methyl oleate. It was also found that the other halides and metal chlorides did not promote the reaction similar to ZnCl2. The enhancement of activity by addition of ZnCl2 to Al2O3-meso was found to be due to the Lewis acidic nature enhanced by addition of ZnCl2. The kinetic studies showed that the catalyst was highly active resulting in conversion> 80% within 30 min of reaction between 25°C to 60°C. The catalytic activity at room temperature was also promising proving it to be a very efficient catalyst for self metathesis of methyl oleate. The conditions optimized for methyl oleate metathesis was extended for the application of triolein, the abundant triglyceride present in the high oleic sunflower oil and other vegetable oils. The catalyst was found to be active with formation of the alkene 9-Octadecene and the dimers and trimers of triolein as desired metathesis products. The MTO based catalyst was active in forming the desired metathesis product and the undesired product namely the cross-linked polymer formed with the homogeneously catalyzed reaction was avoided. The promising results for the metathesis of triolein prove that the catalyst 3% MTO/ZnCl2-Al2O3-meso can be used for the metathesis of vegetable oils such as high-oleic sunflower oil which contains triolein as its major component.

Hintergrund: Die Anzahl von Knochenfrakturen im Zusammenhang mit Traumata, sowie osteoporosebedingten Fragilitätsfrakturen oder auch Knochendefekten in Folge von Tumorresektionen steigt stetig an. Die Nutzung autologen, aber auch allogenen und xenogenen Spendermaterials ist limitiert. Eine vielversprechende Alternative sind Knochenkonstrukte, die über einen Tissue Engineering-Ansatz hergestellt werden. Dabei werden resorbierbare Biomaterialien mit biologisch aktiven Substanzen wie Wachstumsfaktoren oder Zellen kombiniert. Diese funktionalisierten Konstrukte regen nach einer Implantation in den Patienten die gesunde Knochensubstanz zur Heilung an und resorbieren idealerweise zugunsten des nachwachsenden, natürlichen Knochens. Eine neuartige Form des Tissue Engineerings ist der 3D-Biodruck („Bioprinting“), bei dem biologisch aktive Proteine und/oder Zellen mit Biomaterialien vermischt werden und anschließend durch ein additives Fertigungsverfahren zu Konstrukten verarbeitet werden. Dies hat einige Vorteile: Z.B. die Fertigung eines patientenspezifischen Konstrukts, welches direkt an den Defekt angepasst ist, aber auch eine gute Einstellbarkeit der Porosität des finalen Konstrukts, was vorteilhaft für die Nährstoffversorgung und Vaskularisierung sein kann. Vor allem erlaubt es eine ortsaufgelöste Verteilung, wodurch beispielsweise Zellen in einem Konstrukt so positioniert werden können, dass diese zu einem gewebeähnlichen Knochenkonstrukt reifen können. Fragestellung: Im letzten Jahrzehnt wurden einige technologische Fragestellungen im Bereich des Bioprintings gelöst. Für das Knochen-Tissue Engineering sind bisher allerdings nur wenige Ansätze präsentiert wurden. Dies liegt unter anderem daran, dass im Bioprinting vor allem Hydrogele verarbeitet werden. Diese sind allerdings sowohl chemisch, als auch mechanisch weit von natürlichem Knochengewebe entfernt und daher weniger als Knochenersatz geeignet. In dieser Arbeit wurde daher untersucht, ob (Bio-)printing eine für Knochen-Tissue Engineering-Strategien geeignete Methode ist. Dazu wurden zwei vielversprechende Ansätze verfolgt: (I) Mehrphasendruck von bioaktiven Calciumphosphatzementen in Kombination mit Zellen oder mit Wachstumsfaktoren funktionalisierten, biologisch aktiven Hydrogelen. (II) Entwicklung einer neuen Bioink, indem ein wachstumsfaktor- oder zellbeladenes Hydrogel mit einem bioaktiven Füllstoff geblendet wird. Die in der Doktorarbeit vorgestellten Studien sollen dabei insbesondere die Entwicklung dieser Ansätze darstellen, sowie deren Grenzen aufzeigen. Zusätzlich sollen grundlegende mechanische und biologische Eigenschaften der biogedruckten Knochenkonstrukte untersucht werden. Materialien und Methoden: Eine Technologie, die das Prinzip des Bioprintings ermöglicht, ist das sogenannte 3D-Plotten. Mit Hilfe eines Multikanal-Plotters können mehrphasige Konstrukte (Ansatz I), aber natürlich auch einphasige Konstrukte (Ansatz II) hergestellt werden. Für Ansatz I wurde ein klinisch zugelassener Calciumphosphatzement (CPC) als bioaktive Komponente verwendet. Für Ansatz II wurde ein bisher noch wenig erforschtes Nanomaterial namens Laponit verwendet, welches großes Potential für das Tissue Engineering besitzt. Die Biopoylmere Alginat und Methylcellulose bildeten die Grundlage für plottbare, wachstumsfaktor- und zellbeladene Pasten (Biomaterial-inks bzw. Bioinks). Zur Entwicklung einer spezifischen Bioink wurde humanes gefrorenes Frischplasma verwendet. Die rheologischen Eigenschaften neu entwickelter Biomaterial-inks und Bioinks, sowie die mechanischen Eigenschaften der geplotteten Hydrogele wurden charakterisiert. Weitere Untersuchungen schlossen die Quellung der Hydrogele und die Porosität der Konstrukte ein. Ein besonderes Augenmerk wurde auf die Formgenauigkeit der geplotteten Strukturen gelegt. Entsprechend der Untersuchungsansätze wurden verschiedene Zelltypen verwendet, insbesondere mesenchymale Stammzellen (MSC), die direkt mit der Paste verdruckt wurden. Als Modellwachstumsfaktor diente der angiogene vascular endothelial growth factor (VEGF). Dessen Freisetzung aus geplotteten Scaffolds wurde mittels ELISA überprüft; die biologische Aktivität wurde anhand des Wachstums von humanen Nabelschnurendothelzellen (HUVEC) untersucht. Ergebnisse: Zunächst wurde untersucht, ob Multikanal-Plotten geeignet ist, um CPC-Konstrukte patientenindividuell zu fertigen. Dies wurde mit Hilfe einer auflösbaren Methylcellulosepaste erreicht. Dieses Verfahren erlaubte die Herstellung von inneren Kavitäten, die mit anderen Herstellungsverfahren nicht möglich gewesen wären. Darüber hinaus konnte aus einem CT-Scan einer Hand ein Kahnbein extrahiert und virtuell modelliert werden, welches mit hoher Formgenauigkeit geplottet werden konnte. Es wurde gezeigt, dass dies auch auf biphasige Konstrukte aus CPC und einer Bioink anwendbar ist. Dies wurde durch die Entwicklung und Verarbeitung von Bioinks ermöglicht. Biogedruckte Zellen können in vitro und in vivo spezifische biologische Effekte bewirken. Dazu wurden innerhalb der Arbeit zwei Bioinks als plottbare Zellträgermaterialien entwickelt. Eine Bioink enthielt das Nanomaterial Laponit (Ansatz II), welches bereits in anderen Studien vorteilhafte Effekte für Knochen-Tissue Engineering-Ansätze gezeigt hat. Die neuentwickelte Laponit-haltige Bioink erlaubte die Fabrikation von Konstrukten mit hoher Formgenauigkeit. Darüber hinaus war die Zellviabilität, sowie die Zelldichteentwicklung erhöht im Vergleich zu einer Laponit-freien Kontrolle. Da Laponit eine heterogene Ladungsverteilung aufweist, wurde überprüft, inwieweit es ein geeignetes Freisetzungssystem für VEGF darstellt. Scaffolds, die aus einer VEGF-haltigen Paste hergestellt wurden, wiesen ein deutlich verändertes Freisetzungsprofil in Anwesenheit von Laponit auf, als Scaffolds ohne Laponit. So konnte eine initiale Freisetzung (Burstrelease) vermieden und gleichzeitig eine gleichmäßige Freisetzung beobachtet werden. VEGF war auch nach längerer Zeit im Scaffold noch biologisch aktiv. Die zweite Bioink wurde auf Basis gefrorenen, menschlichen Frischplasmas entwickelt. Blutplasma enthält Fibrinogen, das eine RGD-Sequenz für die Anheftung von MSC besitzt. Biogedruckte MSC, aber auch präosteoblastäre Zellen, zeigten eine hohe Neigung, sich in der Bioink aufzuspreizen, was für eingekapselte Zellen erschwert ist. Die plasmahaltige Bioink war dazu geeignet, zusammen mit CPC zu biphasigen Konstrukten (Ansatz I) verarbeitet zu werden. \par Dazu musste zunächst ein Postprozessierungsprotokoll für biphasige Konstrukte aus CPC und zellhaltigen Bioinks entwickelt werden. Aus vorherigen Studien ist bekannt, dass geplottete CPC-Konstrukte in wässrigen Lösungen Mikrorisse bilden, die die mechanischen Eigenschaften signifikant verschlechtern. Die Ausbildung der Mikrorisse kann durch eine Aushärtung in wasserdampfgesättigter Atmosphäre vermieden werden. In biphasigen Konstrukten mit Bioinks sollte diese Aushärtungsphase allerdings nur kurz sein, da eine lange Inkubation ohne wässrige Zellmedien zu einem Absterben der biogedruckten Zellen führen würde. Es konnte gezeigt werden, dass eine Inkubation für 20 min in wasserdampfgesättigter Atmosphäre ausreichend ist, um die Ausbildung von Mikrorissen im CPC zu vermeiden. Diese Zeitspanne konnte von den Zellen toleriert werden. In Kombination mit der plasmahaltigen Bioink wurde eine starke Proliferation und osteogene Reifung von biogedruckten präosteoblastären Vorläufern beobachtet. Schlussfolgerungen: In der vorliegenden Doktorarbeit wurde das Prinzip des extrusionsbasierten Biodrucks (3D-Plotten) verwendet, um biofunktionelle Konstrukte herzustellen. Dies erfolgte entweder durch die Beladung mit Wachstumsfaktoren oder mit Zellen vor der Fabrikation der Konstrukte. Bioaktive Materialien wurden entweder durch Multikanal-Plotten oder durch Supplementierung einer Bioink eingebracht. Beide Ansätze können prinzipiell sogar miteinander kombiniert werden. Die erzielten Ergebnisse belegen, dass Bioprinting eine geeignete Methode für das Knochen-Tissue Engineering darstellt. Patientenindividualisierte Konstrukte können mit dieser Technologie gefertigt werden. Auf diesen Ergebnissen aufbauend können weitere Untersuchungen in vivo die Wirksamkeit der vorgestellten Ansätze überprüfen und neue Therapieansätze für die Heilung von Knochendefekten entwickelt werden.:Abstract 9 Zusammenfassung 13 Index of Abbreviations 19 List of Figures 20 Preface 23 i generalis 1 introduction to the topic 29 1.1 Background 29 1.2 Terminology 29 1.3 Physiological Properties of Bone Tissue 31 1.3.1 Composition of Bone 31 1.3.2 Bone Cytology 33 1.3.3 Crosstalk 34 1.4 Bone Grafting 34 1.4.1 Biopolymers 35 1.4.2 Calcium Phosphates 38 1.4.3 Nanoclays 41 1.5 Additive Manufacturing in Medicine & Bioprinting 43 1.5.1 Additive Manufacturing in Tissue Engineering 43 1.5.2 Bioprinting Techniques 44 1.6 Bioinks & Biomaterial Inks 48 1.6.1 Rheology 48 1.6.2 Plottability & Shape Fidelity 49 1.6.3 Post-Processing 52 1.6.4 Biocompatiblity & Biodegradation 53 1.6.5 The Biofabrication Window 53 2 aim of the thesis 55 2.1 Preliminary Studies 55 2.2 Research Questions 57 ii specialis 3 A methylcellulose hydrogel as support for 3D plotting of complex shaped calcium phosphate scaffolds 61 4 Development of a clay based bioink for 3D cell printing for skeletal application 77 5 Bioprinting of mineralized constructs utilizing multichannel plotting of a self-setting calcium phosphate cement and a cell-laden bioink 97 6 A novel plasma-based bioink stimulates cell proliferation and differentiation in bioprinted, mineralized constructs 113 iii conclusio 7 Summary & Conclusion 133 7.1 Bioprinting of bone tissue constructs 133 7.2 Technological Improvements 134 7.3 Bioink Development 136 7.4 Limitations & Future Research Directions 138 Bibliography 140 Danke 155 Appendix Erklärungen zur Eröffnung des Promotionsverfahrens 165 Erklärung über die Einhaltung gesetzlicher Bestimmungen 166 Auszug aus dem Journal Citation Report 166 Conferences 167
Background: The number of trauma-related bone fractures, fragility fractures resulting from osteoporosis or bone defects after tumor resections is increasing. The usability of autologous, but also allogenous and xenogenous bone grafts is limited. Bone grafts being manufactured using a tissue engineering approach are a promising alternative. For this, resorbable biomaterials are combined with biological components such as cells and growth factors. These functionalized constructs stimulate the formation of novel bone tissue after implantation in the patient and resorb in favor of regrowing, native bone. A new form of tissue engineering is 3D bioprinting. Biologically active proteins and/or cells are mixed with biomaterials and get fabricated to constructs by a convenient additive manufacturing technology. This offers great advantages. For example, the patient-specific tissue engineered constructs can be manufactured fitting exactly to the respective defect. Further, it allows full control about the porosity of the final construct which is considered to be advantageous for nutrient supply and vascularization. Most crucial, it allows the spatial distribution of cells within the three-dimensional construct, which facilitate the maturation of the construct to the tissue-like graft. Research Questions: In the last decade some technological challenges in the field of bioprinting have been solved. Nevertheless, for bone tissue engineering only a small number of approaches had been developed. One of the reasons for this is that bioprinting technologies usually enable the processing of materials that are chemically and mechanically rather distant from the bone, particularly hydrogels. These materials are less suitable as bone substitutes. The aim of this work was to research new approaches of extrusion-based (bio-)printing for bone tissue engineering strategies. For this purpose two promising approaches were investigated: (I) Multichannel printing of bioactive calcium phosphate cements in combination with biologically active hydrogels which were loaded either with growth factors or cells. (II) Development of a new bioink by supplementation of growth factor- or cell-laden hydrogels with a bioactive filler material. The presented studies of this thesis demonstrate the feasibility of these approaches as well as their limits. In addition, fundamental mechanical and biological properties of the bioprinted bone constructs are investigated. Materials and Methods: A technology that makes the principle of bioprinting possible is the so-called 3D plotting. With the aid of a multichannel plotter, multiphasic constructs can be fabricated (approach I), but of course also monophasic constructs are possible (approach II). For approach I, a clinically certified calcium phosphate cement (CPC) was used as bioactive component. For approach II, a less investigated nanomaterial called Laponite was used which was shown before to hold great potential for tissue engineering applications. The biopolymers alginate and methylcellulose formed the basis for plottable, growth factor-laden (biomaterial inks) and cell-laden (bioinks) pastes. For the development of one specific bioink, human fresh frozen plasma was used. Rheological properties of the newly developed biomaterial inks and bioinks were characterized, additionally mechanical properties of plotted constructs were investigated. Further studies investigated the swelling of the hydrogels and the porosity of the constructs. Particular attention was payed to the shape fidelity of the plotted structures. Different cell types were used according to the aim of the subject of research; special attention was payed to the use of mesenchymal stem cells which were plotted directly in combination with the biomaterial, forming the bioink. The angiogenic vascular endothelial growth factor (VEGF) was used as model protein for release studies from bioprinted structures; its biological activity was investigated by proliferation studies of human umbilical vein endothelial cells (HUVEC). Results Firstly, it was investigated whether multichannel plotting is a suitable technology for the fabrication of patient-specific CPC constructs. This was achieved by plotting of a fugitive methylcellulose support ink. This procedure allowed the manufacturing of inner cavities which would not have been possible with other scaffold fabrication methods. Moreover, it was possible to extract a scaphoid bone from a CT scan of a human hand which was modeled virtually and fabricated subsequently with high shape fidelity. Later it was demonstrated that this procedure can be adapted to biphasic constructs consisting of CPC and cell-laden hydrogels. This was achieved by developing and processing bioinks. Bioprinted cells can evoke biological effects in vitro and in vivo. For this purpose two bioinks were developed within this work acting as cell carrier materials. The first bioink contained the nano material Laponite (approach II) which has demonstrated positive effects for bone tissue engineering before. The novel Laponite-based bioink enabled the fabrication of constructs with high shape fidelity. Furthermore, cell viability and cell density were increased compared to a Laponite-free control. Since Laponite offers a heterogeneous charge distribution, it was investigated whether it is a suitable delivery system for VEGF. Scaffolds with Laponite demonstrated a distinct different release profile compared to Laponite-free scaffolds. Thus an initial burst-like release could be avoided and at the same time a uniform release could be observed. The released VEGF was biologically active also after longer time in the scaffold. The second bioink was developed using fresh frozen human blood plasma. Plasma contains fibrinogen which holds a RGD motif for the attachment of MSC. Bioprinted MSC and preosteoblastic cells showed a high affinity to spread within the bioink, which is difficult to achieve for encapsulated cells. The plasma-based bioink was suitable for the combined fabrication of biphasic constructs with CPC (approach I). To achieve this, firstly a suitable post-processing for biphasic constructs consisting of CPC and cell-laden bioinks had to be developed. From previous studies it is known that plotted CPC constructs form microcracks in aqueous media during setting, which impair mechanical properties. The formation of the microcracks can be avoided by setting in water-saturated atmosphere. In biphasic constructs with bioinks this phase should only be short since a long incubation in absence of aqueous cell culture media would lead to cell death within the bioink. It could be shown that incubation for 20 min in water-saturated atmosphere is convenient to avoid the formation of microcracks in CPC strands. This time could be tolerated by the cells. In combination with the plasma-based bioink, a strong proliferation and osteogenic maturation of bioprinted preosteoblastic cells could be observed. Conclusion: In this thesis, the principle of extrusion-based bioprinting (3D plotting) was used to fabricate biofunctionalized constructs. This was achieved by loading cells or growth factors before manufacturing of the constructs. Bioactive materials could be embedded into the constructs by either multichannel plotting or by supplementation of a bioink with a bioactive filler material. In principle both approaches even could be combined with each other. The results obtained prove that bioprinting is a suitable method for bone tissue engineering. Patient-specific constructs can be fabricated by this technology. Based on these results, further studies should be performed in vivo to investigate the potency of the approaches for the development of new regenerative therapies to treat bone defects.:Abstract 9 Zusammenfassung 13 Index of Abbreviations 19 List of Figures 20 Preface 23 i generalis 1 introduction to the topic 29 1.1 Background 29 1.2 Terminology 29 1.3 Physiological Properties of Bone Tissue 31 1.3.1 Composition of Bone 31 1.3.2 Bone Cytology 33 1.3.3 Crosstalk 34 1.4 Bone Grafting 34 1.4.1 Biopolymers 35 1.4.2 Calcium Phosphates 38 1.4.3 Nanoclays 41 1.5 Additive Manufacturing in Medicine & Bioprinting 43 1.5.1 Additive Manufacturing in Tissue Engineering 43 1.5.2 Bioprinting Techniques 44 1.6 Bioinks & Biomaterial Inks 48 1.6.1 Rheology 48 1.6.2 Plottability & Shape Fidelity 49 1.6.3 Post-Processing 52 1.6.4 Biocompatiblity & Biodegradation 53 1.6.5 The Biofabrication Window 53 2 aim of the thesis 55 2.1 Preliminary Studies 55 2.2 Research Questions 57 ii specialis 3 A methylcellulose hydrogel as support for 3D plotting of complex shaped calcium phosphate scaffolds 61 4 Development of a clay based bioink for 3D cell printing for skeletal application 77 5 Bioprinting of mineralized constructs utilizing multichannel plotting of a self-setting calcium phosphate cement and a cell-laden bioink 97 6 A novel plasma-based bioink stimulates cell proliferation and differentiation in bioprinted, mineralized constructs 113 iii conclusio 7 Summary & Conclusion 133 7.1 Bioprinting of bone tissue constructs 133 7.2 Technological Improvements 134 7.3 Bioink Development 136 7.4 Limitations & Future Research Directions 138 Bibliography 140 Danke 155 Appendix Erklärungen zur Eröffnung des Promotionsverfahrens 165 Erklärung über die Einhaltung gesetzlicher Bestimmungen 166 Auszug aus dem Journal Citation Report 166 Conferences 167

Biopolymers have a great potential in biomedical engineering, having been used as scaffolds for hard and soft tissues, such as bone and blood vessels for many years. More recently biopolymers have also found applications in surgical fixation devices. Compared with conventional metal fixation devices, bone grafts and organ substitutes, biopolymer products have advantages of no long-term implant palpability or temperature sensitivity, predictable degradation to provide progressive bone loading and no stress shielding, all of which leads to a better bone healing, reduced patient trauma and cost, elimination of second surgery for implant removal, and fewer complications from infections. However lack of initial fixation strength and bioactivity are two major concerns which limited more widespread applications of biopolymers in orthopedic surgery. Nanodiamond is attractive for its use in reinforcement of composite materials due to their outstanding mechanical, chemical and biological properties. Nanotechnology shows us many innovations and it is generally accepted view that many could be further developed and applied in tissue engineering. In this work, we conduct poly(L-lactic acid) (PLLA) and octadecylamine functionalized nanodiamond (ND-ODA) composite research to optimize the polymer/ND interface, thus to reinforce the mechanical strength. Composites comprising PLLA matrix with embedded ND-ODA were prepared by mixing PLLA/chloroform solution with chloroform suspension of nanodiamonds at concentrations of 0–10 by weight percent. The dispersion of ND-ODA was observed by transmission electron microscopy (TEM). TEM micrographs show that ND-ODA can disperse uniformly in PLLA till 10% wt. Nanoindentation result shows the mechanical strength of ND-ODA/PLLA composites improving following increasing the concentration of ND-ODA in composites. The noncytotoxicity of ND-ODA was demonstrated on 7F2 Osteoblasts. To test the usefulness of ND-ODA/PLLA composites as scaffolds for supporting cell growth, 7F2 Osteoblasts were cultured on scaffolds for 6 days. The attachment and proliferation of 7F2 on all scaffolds were assessed by fluorescent nuclear staining with Hoechst 33258 and Alamar BlueTM assay. The results showed that the adding ND-ODA does small influence cell growth, which indicates the composites have good biocompatibility. The morphology of 7F2 cells growing on all ND-ODA/PLLA composite scaffolds was determined by SEM, which confirms the Osteoblasts spread on the scaffolds. All these results combined suggest that ND-ODA/PLLA might provide a novel composite suitable for surgical fixation devices.

Biopolymers are emerging materials with numerous capabilities of minimizing the environmental hazards caused by synthetic materials. The competitive mechanical properties of bio-based poly(lactic acid) (PLA) reinforced with cellulose nanocrystals (CNCs) have attracted a huge interest in improving the mechanical properties of the corresponding nanocomposites. To obtain optimal properties of PLA-CNC nanocomposites, the compatibility between PLA and CNCs needs to be improved through uniform dispersion of CNCs into PLA. The application of chemical surface functionalization technique is an essential step to improve the interaction between hydrophobic PLA and hydrophilic CNCs. In this study, a combination of a time-efficient esterification technique and masterbatch approach was used to improve the CNCs dispersibility in PLA. Nanocomposites reinforced by 1, 3, and 5 wt% functionalized CNCs were prepared using twin screw extrusion followed by injection molding process. The mechanical and dynamic mechanical properties of pure PLA and nanocomposites were studied through tensile, impact and dynamic mechanical analysis. The impact fractured surfaces were characterized using scanning electron microscopy. The mechanical test results exhibited that tensile strength and modulus of elasticity of nanocomposites improved by 70% and 11% upon addition of functionalized CNCs into pure PLA. The elongation at break and impact strength of nanocomposites exhibited 43% and 35% increase as compared to pure PLA. The rough and irregular fracture surface in nanocomposites confirmed the higher ductility in PLA nanocomposites as compared to pure PLA. The incorporation of functionalized CNCs into PLA resulted in an increase in storage modulus and a decrease in tan δ intensity which was more profound in nanocomposites reinforced with 3 wt% functionalized CNCs.

Nanodiamond (ND) is an attractive nanomaterial for reinforcement of biopolymers due to the ND’s superior mechanical and chemical properties, and low biotoxicity. A novel composite material has been produced for bone scaffolds utilizing the biodegradable polymer poly(L-lactic acid) (PLLA) and octadecylamine-functionalized nanodiamond (ND-ODA). Composites were prepared by admixing to a PLLA/chloroform solution chloroform suspension of ND-ODA in concentration range of 0–10% (w/w). The dispersion of ND-ODA evaluated by transmission electron microscopy (TEM) shows uniform distribution of ND-ODA in PLLA matrix. The composites were characterized by differential scanning calorimetry (DSC). DSC analysis of the composites showed no significant thermal behavior changes with the addition of ND-ODA into the polymer. Biomineralization test shows that ND-ODA can enhance the mineral deposition on scaffolds. Improved mechanical properties and good biocompatibility with enhanced biomineralization combined suggest that ND-ODA/PLLA might have potential applications for bone tissue engineering.