Refine
Has Fulltext
- yes (21)
Is part of the Bibliography
- yes (21)
Year of publication
Document Type
- Doctoral Thesis (21) (remove)
Keywords
- Hydrogel (21) (remove)
Institute
- Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde (7)
- Institut für Funktionsmaterialien und Biofabrikation (3)
- Institut für Organische Chemie (3)
- Klinik und Poliklinik für Unfall-, Hand-, Plastische und Wiederherstellungschirurgie (Chirurgische Klinik II) (3)
- Lehrstuhl für Orthopädie (3)
- Graduate School of Life Sciences (2)
- Graduate School of Science and Technology (2)
- Theodor-Boveri-Institut für Biowissenschaften (2)
- Fakultät für Chemie und Pharmazie (1)
- Institut für Physikalische und Theoretische Chemie (1)
Hydrogele stehen als Material für den 3D-Biodruck zunehmend im Fokus aktueller Forschung, da sie aufgrund ihrer wasserhaltigen Struktur optimale Voraussetzungen für Anwendungen der Zellkultur aufweisen. Durch die Verarbeitung solcher Biotinten mittels additiver Fertigungstechniken der Biofabrikation erhofft man sich beschädigtes oder krankes Gewebe zu heilen oder zu ersetzen. Allerdings wird der Fortschritt in diesem Bereich durch einen Mangel an geeigneten Materialien gebremst, weshalb die Entwicklung neuer Biotinten von zentraler Bedeutung ist. Das Polymer GelAGE ist ein am Lehrstuhl für Funktionswerkstoffe der Medizin und Zahnheilkunde der Universität Würzburg synthetisiertes Hydrogelsystem. Zu diesem über eine Thiol-En Reaktion vernetzenden Material stehen systematische Untersuchungen der für die in vitro Zellkultur relevanten Eigenschaften noch aus. Das Ziel dieser Arbeit war daher die biologische Evaluation von GelAGE und der Vergleich mit der Biotinte Alginat-Gelatine.
Zu diesem Zweck wurden L929-Zellen für 7 Tage in verschiedenen Hydrogelzusammensetzungen in vitro kultiviert. Um die zytokompatiblen Eigenschaften in den verschiedenen Versuchsgruppen zu untersuchen, wurden die Proben mittels der in vitro Testverfahren Live/Dead Färbung, DNA-Assay, CCK-8-Assay und Phalloidin-Färbung analysiert.
Im Rahmen dieser Arbeit konnte ein Herstellungsprotokoll für das Material GelAGE etabliert werden, welches eine Grundlage für die Durchführung weiterer biologischer Experimente bietet. Das Resultat der biologischen Untersuchungen war, dass das Polymer GelAGE als zytokompatibel bewertet werden kann, es jedoch nicht die Qualität des Alginat-Gelatine Hydrogelsystems aufweist. Allerdings konnten die Eigenschaften der GelAGE Proben teilweise durch eine Modifikation mit Humanem Plättchenlysat verbessert werden. Des Weiteren konnten deutliche Unterschiede in der Zell-Material- Interaktion zwischen den verschiedenen GelAGE Varianten nachgewiesen werden.
The aim of the work was the development of thiol-ene cross-linked hydrogels based on functionalized poly(glycidol)s (PG) and hyaluronic acid (HA) for extrusion based 3D bioprinting. Additionally, the functionalization of the synthesized PG with peptides and the suitability of these polymers for physically cross-linked gels were investigated, in a proof of principle study in order to demonstrate the versatile use of PG polymers in hydrogel development.
First, the precursor polymers of the different hydrogel systems were synthesized. For thiol-ene cross-linked hydogels, linear allyl-functionalized PG (P(AGE-co-G)) and three different thiol-(SH-)functionalized polymers, ester-containing PG-SH (PG SHec), ester-free PG-SH (PG-SHef) and HA-SH were synthesized and analysed, The degree of functionalization of these polymers was adjustable.
For physically cross-linked hydrogels, peptide-functionalized PG (P(peptide-co-G)), was synthesized through polymer analogue thiol-ene modification of P(AGE-co-G).
Subsequently, thiol-ene cross-linked hydrogels were prepared with the synthesized thiol- and allyl-functionalized polymers. Depending on the origin of the used polymers, two different systems were obtained: on the one hand synthetic hydrogels consisting of PG-SHec/ef and P(AGE-co-G) and on the other hand hybrid gels, consisting of HA-SH and P(AGE-co-G). In synthetic gels, the degradability of the gels was determined by the applied PG-SH. The use of PG-SHec resulted in hydrolytically degradable hydrogels, whereas the cross-linking with PG-SHef resulted in non-degradable gels.
The physical properties of these different hydrogel systems were determined by swelling, mechanical and diffusion studies and subsequently compared among each other. In swelling studies the differences of degradable and non-degradable synthetic hydrogels as well as the differences of synthetic compared to hybrid hydrogels were demonstrated.
Next, the stiffness and the swelling ratios (SR) of the established hydrogel systems were examined in dependency of different parameters, such as incubation time, polymer concentration and UV irradiation. In general, these measurements revealed the same trends for synthetic and hybrid hydrogels: an increased polymer concentration as well as prolonged UV irradiation led to an increased network density. Moreover, it was demonstrated that the incorporation of additional non-bound HMW HA hampered the hydrogel cross-linking resulting in gels with decreased stiffness and increased SR. This effect was strongly dependent on the amount of additional HMW HA.
The diffusion of different molecular weight fluorescein isothiocyanate-dextran (FITC-dextran) through hybrid hydrogels (with/without HMW HA) gave information about the mesh size of these gels. The smallest FITC-dextran (4 kDa) completely diffused through both hydrogel systems within the first week, whereas only 55 % of 40 kDa and 5-10 % HMW FITC-dextrans (500 kDa and 2 MDa) could diffuse through the networks.
The applicability of synthetic and hybrid hydrogels for cartilage regeneration purpose was investigated through by biological examinations. It was proven that both gels support the survival of embedded human mesenchymal stromal cells (hMSCs) (21/28 d in vitro culture), however, the chondrogenic differentiation was significantly improved in hybrid hydrogels compared to synthetic gels. The addition of non-bound HMW HA resulted in a slightly less distinct chondrogenesis.
Lastly the printability of the established hydrogel systems was examined. Therefore, the viscoelastic properties of the hydrogel solutions were adjusted by incorporation of non-bound HMW HA. Both systems could be successfully printed with high resolution and high shape fidelity.
The introduction of the double printing approach with reinforcing PCL allowed printing of hydrogel solutions with lower viscosities. As a consequence, the amount of additional HMW HA necessary for printing could be reduced allowing successful printing of hybrid hydrogel solutions with embedded cells. It was demonstrated that the integrated cells survived the printing process with high viability measured after 21 d. Moreover, by this reinforcing technique, robust hydrogel-containing constructs were fabricated.
In addition to thiol-ene cross-linked hydrogels, hydrogel cross-linking via ionic interactions was investigated with a hybrid hydrogel based on HMW HA and peptide-functionalized PG. Rheological measurements revealed an increase in the viscosity of a 2 wt.% HMW HA solution by the addition of peptide-functionalized PG. The increase in viscosity could be attributed to the ionic interactions between the positively charge PG and the negatively charge HMW HA.
In conclusion, throughout this thesis thiol-ene chemistry and PG were introduced as promising cross-linking reaction and polymer precursor for the field of biofabrication. Furthermore, the differences of hybrid and synthetic hydrogels as well as chemically and physically cross-linked hydrogels were demonstrated.
Moreover, the double printing approach was demonstrated to be a promising tool for the fabrication of robust hydrogel-containing constructs. It opens the possibility of printing hydrogels that were not printable yet, due to too low viscosities.
In the field of biofabrication, biopolymer-based hydrogels are often used as bulk materials with defined structures or as bioinks. Despite their excellent biocompatibility, biopolymers need chemical modification to fulfill mechanical stability.
In this thesis, the primary alcohol of hyaluronic acid was oxidized using TEMPO/TCC oxidation to generate aldehyde groups without ring-opening mechanism of glycol cleavage using sodium periodate. For crosslinking reaction of the aldehyde groups, adipic acid dihydrazide was used as bivalent crosslinker for Schiff Base chemistry. This hydrogel system with fast and reversible crosslinking mechanism was used successfully as bulk hydrogel for chondrogenic differentiation with human mesenchymal stem cells (hMSC).
Gelatin was modified with pentenoic acid for crosslinking reaction via light controllable thiol-ene reaction, using thiolated 4-arm sPEG as multivalent crosslinker. Due to preservation of the thermo responsive property of gelatin by avoiding chain degradation during modification reaction, this gelatin-based hydrogel system was successfully processed via 3D printing with low polymer concentration. Good cell viability was achieved using hMSC in various concentrations after 3D bioprinting and chondrogenic differentiation showed promising results.
Structure-property relationships in poly(2-oxazoline)/poly(2-oxazine) based drug formulations
(2020)
According to estimates, more than 40% of all new chemical entities developed in pharmaceutical industry are practically insoluble in water. Naturally, the demand for excipients which increase the water solubility and thus, the bioavailability of such hydrophobic drugs is enormous. Poly(2-oxazoline)s (POx) are currently intensively discussed as highly versatile class of biomaterials. Although selected POx based micellar drug formulations exhibit extraordinarily high drug loadings > 50 wt.% enabling high anti-tumor efficacies in vivo, the formulation of other hydrophobic compounds has failed. This casts doubt on the general understanding in which a hydrophobic active pharmaceutical ingredient is dissolved rather unspecifically in the hydrophobic core of the micelles following the fundamental concept of “like dissolves like”. Therefore, a closer look at the interactions between all components within a formulation becomes increasingly important. To do so, a large vehicle platform was synthesized, loaded with various hydrophobic drugs of different structure, and the formulations subsequently characterized with conventional and less conventional techniques. The obtained in-depth insights helped to develop a more thorough understanding about the interaction of polymer and incorporated API finally revealing morphologies deviating from a classical core/shell structure. During these studies, the scarcely investigated polymer class of poly(2-oxazine)s (POzi) was found as promising drug-delivery vehicle for hydrophobic drugs. Apart from this fundamental research, the anti-tumor efficacy of the two APIs curcumin and atorvastatin has been studied in more detail. To increase the scope of POx and POzi based formulations designed for intravenous administration, a curcumin loaded hydrogel was developed as injectable drug-depot.
Der Kehlkopf ist ein stimmerzeugendes knorpelhaltiges Organ und spielt eine wichtige Rolle in der Atemfunktion und beim aspirationsfreien Schluckakt. Funktionsstörungen des Kehlkopfs wie Stimmbandlähmungen werden durch Schädigungen des Kehlkopfnervs nach operativen Eingriffen und Halsverletzungen hervorgerufen. Des Weiteren führen durch Traumen, Teil- und komplette Resektionen verursachte Substanzdefekte des Kehlkopfs zu Funktionsverlusten. Die hierfür notwendigen und komplexen Rekonstruktionen werden durch das schlechte Regenerationspotential von Knorpelgewebe eingeschränkt und können nur bedingt durch synthetische Ersatzmaterialen oder körpereigenes Ersatzgewebe bewerkstelligt werden. Ist es möglich, mit Hilfe des Tissue Engineerings aus körpereigenen Stammzellen und biokompatiblen Trägermaterialien implantierbares Knorpelersatzgewebe herzustellen, welches zur dauerhaften Wiederherstellung der Kehlkopffunktionen eingesetzt werden kann? Die zusätzliche Markierung von Stammzellen mit superparamagnetischen Eisenoxidnanopartikeln (VSOP) als Zellmarker bietet die Möglichkeit der Detektion und der Verfolgung der Zellen mittels nicht-invasiver Nachweismethoden nach deren Implantation. Ist die Verwendung dieser Nanopartikel ohne negative Folgen für die Stammzellen möglich und sind diese für den Einsatz in der Laryngologie geeignet?
Fettgewebsstammzellen (ASC) wurden aus humanem Liposuktionsmaterial und Kaninchen-Nackenfett isoliert und expandiert. Die Zellen wurden in Hydrogelkombinationen aus Kollagen Typ-I, Agarose, Fibrin und Hyaluronsäure eingebettet und mit den chondrogenen Wachstumsfaktoren TGF-β3, BMP-6 und IGF-I über 14 Tage differenziert. Anschließend wurden diese Zell-Hydrogelkonstrukte bezüglich Morphologie, extrazellulärer Matrixanreicherung und knorpelspezifischer Genexpression histologisch, immunhistochemisch und molekularbiologisch analysiert. In einem weiteren Schritt wurden die Integration der Zell-Hydrogelkonstrukte in natives Knorpelgewebe sowie die Defektdeckung in einem in vitro- und einem in vivo-Knorpeldefektmodell mit vor- und nicht-vordifferenzierten Zell-Hydrogelkonstrukten untersucht. Die Analyse möglicher zyto- und genotoxischer Effekte von VSOP sowie des Einflusses der Markierung von ASC mit VSOP auf die Proliferation, Migration und das Multidifferenzierungspotential erfolgte nach der Markierung der Zellen mit unterschiedlichen VSOP-Konzentrationen. Außerdem wurden VSOP-markierte ASC in Kaninchenstimmlippen injiziert und die Nachweisbarkeit dieser Zellen im Injektionsareal histologisch und mittels Magnetresonanztomographie (MRT) untersucht.
Nach 14-tägiger chondrogener Differenzierung wurde in den Zell-Hydrogelkonstrukten eine knorpelähnliche Morphologie, die Anreicherung knorpelspezifischer Matrixproteine und die Expression chondrogener Markergene nachgewiesen. Die Kombination der chondrogenen Wachstumsfaktoren zeigte keinen verstärkenden Einfluss auf die Chondrogenese von ASC. Hydrogele aus Kollagen Typ I und Hyaluronsäure wiesen die stärkste extrazelluläre Matrixanreicherung auf. Bei den agarosefreien Hydrogelen war eine ausgeprägte Gelschrumpfung auffällig. In den beiden Knorpeldefektmodellen konnte weder eine Integration der Zell-Hydrogelkonstrukte in den Nativknorpel noch eine vollständige Defektdeckung nachgewiesen werden. Nach der Markierung von ASC mit VSOP zeigte sich bei der höchsten Konzentration von 1,5 mM eine genotoxische Wirkung. Zytotoxische Effekte sowie Einflüsse der Markierung auf die Proliferation, Migration und das Multidifferenzierungspotential von ASC waren nicht nachweisbar. VSOP-markierte ASC konnten nach deren Injektion in Kaninchenstimmlippen im Injektionsareal nur vereinzelt mittels MRT und histologisch nachgewiesen werden.
Es ist möglich, mit Hilfe des Tissue Engineerings aus körpereigenen Stammzellen und biokompatiblen Trägermaterialien implantierbares knorpelähnliches Gewebe herzustellen. Dabei begünstigen agarosefreie Trägermaterialien die chondrogene Differenzierung von ASC. Diese könnte durch die jeweilige Erhöhung der Zelldichte und Wachstumsfaktorkonzentrationen sowie die Verlängerung der Induktionszeit verstärkt werden. Eine mögliche klinische Anwendung dieser knorpelähnlichen Gewebe in der Laryngologie ist jedoch durch deren Schrumpfung wie auch mangelnde Integration und Defektdeckung noch weit entfernt. Aufgrund ihrer genotoxischen Wirkung kann eine Verwendung von VSOP als Zellmarker auch unterhalb von 1,5 mM ohne negative Folgen für den Organismus nicht sicher ausgeschlossen werden. Der inhomogene Gewebekontrast im Kehlkopf, die schlechte Auflösung im MRT und die geringe Größe von VSOP erschweren die Nachweisbarkeit und Verfolgung markierter Zellen mittels MRT. Daher sind andere nicht-invasive Nachweismethoden für die Verwendung von VSOP im Kehlkopf zu evaluieren. Der möglichen Anwendung dieser knorpelähnlichen Gewebe und VSOP in der rekonstruktiven Laryngologie muss eine erfolgreiche Optimierung und ausführliche positive Validierung in klinischen Tests vorausgehen.
The aim of the thesis was to develop water soluble poly(2-oxazoline) (POx) copolymers with new side group functionalities, which can be used for the formation of hydrogels in biomedical applications and for the development of peptide-polymer conjugates.
First, random copolymers of the monomer MeOx or EtOx with ButEnOx and EtOx with DecEnOx were synthesized and characterized. The vinyl functionality brought into the copolymer by the monomers ButEnOx and DecEnOx would later serve for post-polymerization functionalization. The synthesized copolymers were further functionalized with thiols via post-polymerization functionalization using a newly developed synthesis protocol or with a protected catechol molecule for hydrogel formation. For the formation of peptide-polymer conjugates, a cyclic thioester, namely thiolactone acrylamide and an azlactone precursor, whose synthesis was newly developed, were attached to the side chain of P(EtOx-co-ButEnOx) copolymers.
The application of the functionalized thiol copolymers as hydrogels using thiol-ene chemistry for cross-linking was demonstrated. The swelling behavior and mechanical properties were characterized. The hydrophilicity of the network as well as the cross-linking density strongly influenced the swelling behavior and the mechanical strength of the hydrogels. All hydrogels showed good cell viability results.
The hydrogel networks based on MeOx and EtOx were loaded with two dyes, fluorescein and methylene blue. It was observed that the uptake of the more hydrophilic dye fluorescein depended more on the ability of the hydrogel to swell. In contrast, the uptake of the more hydrophobic dye methylene blue was less dependent on the swelling degree, but much more on the hydrophilicity of the network.
For the potential application as cartilage glue, (biohybrid) hydrogels were synthesized based on the catechol-functionalized copolymers, with and without additional fibrinogen, using sodium periodate as the oxidizing agent. The system allowed for degradation due to the incorporated ester linkages at the cross-linking points. The swelling behavior as well as the mechanical properties were characterized. As expected, hydrogels with higher degrees of cross-linking showed less swelling and higher elastic modulus. The addition of fibrinogen however increased the elasticity of the network, which can be favorable for the intended application as a cartilage glue. Biological evaluation clearly demonstrated the advantage of degradable ester links in the hydrogel network, where chondrocytes were able to bridge the artificial gap in contrast to hydrogels without any ester motifs.
Lastly, different ways to form peptide-polymer conjugates were presented. Peptides were attached with the thiol of the terminal cysteine group to the vinyl side chain of P(EtOx-co-ButEnOx) copolymers by radical thiol-ene chemistry. Another approach was to use a cyclic thioester, thiolactone, or an azlactone functionality to bind a model peptide via native chemical ligation. The two latter named strategies to bind peptides to POx side chains are especially interesting as one and in the case of thiolactone two free thiols are still present at the binding site after the reaction, which can, for example, be used for further thiol-ene cross-linking to form POx hydrogels.
In summary, side functional poly(oxazoline) copolymers show great potential for numerous biomedical applications. The various side chain functionalities can be introduced by an appropriate monomer or by post-polymerization functionalization, as demonstrated. By their multi-functionality, hydrogel characteristics, such as cross-linking degree and mechanical strength, can be fine-tuned and adjusted depending on the application in the human body. In addition, the presented chemoselective and orthogonal reaction strategies can be used in the future to synthesize polymer conjugates, which can, for example, be used in drug delivery or in tissue regeneration.
Für die Rekonstruktion von Gelenkknorpeldefekten des Kniegelenkes in Folge eines Traumas oder einer Osteochondrosis dissecans (OD) stehen verschiedene operative Verfahren zur Verfügung. Die Autologe Chondrozytentransplantation (ACT) hat sich als zuverlässiges Rekonstruktionsverfahren erwiesen. In der vorliegenden Arbeit wurde eine prospektive Fallseriestudie für eine neue Form der ACT mit einem Kollagen I Hydrogel (CaReS-Technologie) durchgeführt. Die Vorteile der Technologie liegen zum Einen darin, dass sich die Zellen homogen im Gel verteilen und zum Anderen, dass die Zellen unmittelbar nach dem Herauslösen aus dem Gelenkknorpel in das Gel eingebracht werden und dadurch eine geringere Dedifferenzierung der Chondrozyten stattfindet. Von März 2003 bis Ende 2006 wurden 29 Patienten in die Studie eingeschlossen. Die Ein- und Ausschlusskriterien erfüllten die Kriterien der Arbeitsgruppe ACT und Tissue Engineering der Deutschen Gesellschaft für Orthopädie und Unfallchirurgie. Die Eingangs- und Nachuntersuchungsbögen wurden an die IKDC Form 2000 angelehnt. Insgesamt zeigte sich ein signifikanter Anstieg des IKDC Scores im mittleren follow-up von 30,7 Monaten von 47,3 auf 74,9 bei den 29 Patienten. Bei Aufschlüsselung der Patienten bzgl. Diagnose, Defektgröße, Lokalisation und Defektanzahl zeigte sich bei den Behandlungsgruppen OD, Trauma/degenerativ, > 4 cm2, mediale Femurkondyle und Einzeldefekte eine signifikante Zunahme des IKDC Scores im zeitlichen Verlauf. Der postoperative Schmerz zeigte einhergehend mit dem Anstieg des IKDC Scores eine signifikante Abnahme der Schmerzintensität in den Behandlungsgruppen OD, Trauma/degenerativ, > 4 cm2, mediale Femurkondyle und Einzeldefekte. Nachgewiesen wurde ebenfalls ein Anstieg des SF36 Scores, der den gegenwärtigen Gesundheitszustand sowohl körperlich als auch psychisch beurteilt. Zusammen mit einer globalen Patientenzufriedenheit von 80% und einem IKDC Funktionsstatus von I und II bei 77% der Patienten spiegeln die gewonnenen Daten die Ergebnisse der klassischen ACT bzw. anderer matrixgekoppelten Verfahren wieder. Die CaReS-Technologie stellt somit ein gleichwertiges Verfahren zu den bisher auf dem Markt befindlichen Techniken der ACT dar.
Motivated by the great potential which is offered by the combination of additive manufacturing and tissue engineering, a novel polymeric bioink platform based on poly(2 oxazoline)s was developed which might help to further advance the young and upcoming field of biofabrication. In the present thesis, the synthesis as well as the characteristics of several diblock copolymers consisting of POx and POzi have been investigated with a special focus on their suitability as bioinks.
In general, the copolymerization of 2-oxazolines and 2-oxazines bearing different alkyl side chains was demonstrated to yield polymers in good agreement with the degree of polymerization aimed for and moderate to low dispersities.
For every diblock copolymer synthesized during the present study, a more or less pronounced dependency of the dynamic viscosity on temperature could be demonstrated. Diblock copolymers comprising a hydrophilic PMeOx block and a thermoresponsive PnPrOzi block showed temperature induced gelation above a degree of polymerization of 50 and a polymer concentration of 20 wt%. Such a behavior has never been described before for copolymers solely consisting of poly(cyclic imino ether)s.
Physically cross linked hydrogels based on POx b POzi copolymers exhibit reverse thermal gelation properties like described for solutions of PNiPAAm and Pluronic F127. However, by applying SANS, DLS, and SLS it could be demonstrated that the underlying gel formation mechanism is different for POx b POzi based hydrogels. It appears that polymersomes with low polydispersity are formed already at very low polymer concentrations of 6 mg/L. Increasing the polymer concentration resulted in the formation of a bicontinuous sponge like structure which might be formed due to the merger of several vesicles. For longer polymer chains a phase transition into a gyroid structure was postulated and corresponds well with the observed rheological data.
Stable hydrogels with an unusually high mechanical strength (G’ ~ 4 kPa) have been formed above TGel which could be adjusted over a range of 20 °C by changing the degree of polymerization if maintaining the symmetric polymer architecture. Variations of the chain ends revealed only a minor influence on TGel whereas the influence of the solvent should not be neglected as shown by a comparison of cell culture medium and MilliQ water.
Rotationally as well as oscillatory rheological measurements revealed a high suitability for printing as POx b POzi based hydrogels exhibit strong shear thinning behavior in combination with outstanding recovery properties after high shear stress.
Cell viability assays (WST-1) of PMeOx b PnPrOzi copolymers against NIH 3T3 fibroblasts and HaCat cells indicated that the polymers were well tolerated by the cells as no dose-dependent cytotoxicity could be observed after 24 h at non-gelling concentrations up to 100 g/L.
In summary, copolymers consisting of POx and POzi significantly increased the accessible range of properties of POx based materials. In particular thermogelation of aqueous solutions of diblock copolymers comprising PMeOx and PnPrOzi was never described before for any copolymer consisting solely of POx or POzi. In combination with other characteristics, e.g. very good cytocompatibility at high polymer concentrations and comparably high mechanical strength, the formed hydrogels could be successfully used for 3D bioprinting. Although the results appear promising and the developed hydrogel is a serious bioink candidate, competition is tough and it remains an open question which system or systems will be used in the future.
Improved treatment options for the degenerative joint disease osteoarthritis (OA) are of major interest, since OA is one of the main sources of disability, pain, and socioeconomic burden worldwide [202]. According to epidemiological data, already 27 million people suffer from OA in the US [23]. Moreover, the WHO expects OA to be the fourth most common cause of disability in 2020 [203], illustrating the need for effective and long-lasting therapy options of severe cartilage defects. Despite numerous clinically available products for the treatment of cartilage defects [62], the development of more cartilage-specific materials is still at the beginning.
Hyaluronic acid (HA) is a major component of the cartilaginous extracellular matrix (ECM) and inherently creates a cell-friendly niche by providing cell attachment and migration sites. Furthermore, it is known that the functional groups of HA are well suited for chemical modification. These characteristics render HA an attractive material for hydrogel-based tissue engineering approaches. Poly(glycidol) (PG) as chemical crosslinker basically features similar chemical characteristics as the widely used poly(ethylene glycol) (PEG), but provides additional side groups at each repeating unit that can be further chemically functionalized. With the introduction of PG as multifunctional crosslinker for HA gels, a higher cross-linking density and, accordingly, a greater potential for biomimetic functionalization may be achieved. However, despite the mentioned potential benefits, PG has not been used for cartilage regeneration approaches so far.
The initial aim of the study was to set up and optimize a HA-based hydrogel for the chondrogenic differentiation of mesenchymal stromal cells (MSCs), using different amounts and variations of cross-linkers. Therefore, the hydrogel composition was optimized by the utilization of different PEG diacrylate (PEGDA) concentrations to cross-link thiol-modified HA (Glycosil, HA-SH) via Michael addition. We aimed to generate volumestable scaffolds that simultaneously enable a maximum of ECM deposition. Histological and biochemical analysis showed 0.4% PEGDA as the most suitable concentration for these requirements (Section 5.1.2).
In order to evaluate the impact of a differently designed cross-linker on MSC chondrogenesis, HA-SH was cross-linked with PEGTA (0.6%) and compared to PEGDA (0.4%) in a next step. Following this, acrylated PG (PG-Acr) as multifunctional cross-linker alternative to acrylated PEG was evaluated. It provides around five times more functional groups when utilized in PG-Acr (0.6%) HA-SH hydrogels compared to PEGTA (0.6%) HA-SH hydrogels, thus enabling higher degrees of biomimetic functionalization. Determination of cartilage-specific ECM components showed no substantial differences between both cross-linkers while the deposition of cartilaginous matrix appeared more homogeneous in HA-SH PG-Acr gels. Taken together, we were able to successfully increase the possibilities for biomimetic functionalization in the developed HA-SH hydrogel system by the introduction of PG-Acr as cross-linker without negatively affecting MSC chondrogenesis (Section 5.1.3).
The next part of this thesis focused extensively on the biomimetic functionalization of PG-Acr (0.6%) cross-linked HA-SH hydrogels. Here, either biomimetic peptides or a chondrogenic growth factor were covalently bound into the hydrogels.
Interestingly, the incorporation of a N-cadherin mimetic (HAV), a collagen type II binding (KLER), or a cell adhesion-mediating peptide (RGD) yielded no improvement of MSC chondrogenesis. For instance, the covalent binding of 2.5mM HAV changed morphology of cell nuclei and reduced GAG production while the incorporation of 1.0mM RGD impaired collagen production. These findings may be attributed to the already supportive conditions of the employed HA-based hydrogels for chondrogenic differentiation. Most of the previous studies reporting positive peptide effects on chondrogenesis have been carried out in less supportive PEG hydrogels or in significantly stiffer MeHA-based hydrogels [99, 101, 160]. Thus, the incorporation of peptides may be more important under unfavorable conditions while inert gel systems may be useful for studying single peptide effects (Section 5.2.1).
The chondrogenic factor transforming growth factor beta 1 (TGF-b1) served as an example for growth factor binding to PG-Acr. The utilization of covalently bound TGF-b1 may thereby help overcome the need for repeated administration of TGF-b1 in in vivo applications, which may be an advantage for potential clinical application. Thus, the effect of covalently incorporated TGF-b1 was compared to the effect of the same amount of TGF-b1 without covalent binding (100nM TGF-b1) on MSC chondrogenesis. It was successfully demonstrated that covalent incorporation of TGF-b1 had a significant positive effect in a dose-dependent manner. Chondrogenesis of MSCs in hydrogels with covalently bound TGF-b1 showed enhanced levels of chondrogenesis compared to hydrogels into which TGF-b1 was merely mixed, as shown by stronger staining for GAGs, total collagen, aggrecan and collagen type II. Biochemical evaluation of GAG and collagen amounts, as well as Western blot analysis confirmed the histological results. Furthermore, the positive effect of covalently bound TGF-b1 was shown by increased expression of chondrogenic marker genes COL2A1, ACAN and SOX9. In summary, covalent growth factor incorporation utilizing PG-Acr as cross-linker demonstrated significant positive effects on chondrogenic differentiation of MSCs (Section 5.2.2).
In general, PG-Acr cross-linked HA hydrogels generated by Michael addition represent a versatile hydrogel platform due to their high degree of acrylate functionality. These hydrogels may further offer the opportunity to combine several biological modifications, such as the incorporation of biomimetic peptides together with growth factors, within one cell carrier.
A proof-of-principle experiment demonstrated the suitability of pure PG gels for studying single peptide effects. Here, the hydrogels were generated by the utilization of thiol-ene-click reaction. In this setting, without the supportive background of hyaluronic acid, MSCs showed enhanced chondrogenic differentiation in response to the incorporation of 1.0mM HAV. This was demonstrated by staining for GAGs, the cartilage-specific ECM molecules aggrecan and type II collagen, and by increased GAG and total collagen amounts shown by biochemical analysis. Thus, pure PG gels exhibit the potential to study the effects and interplay of peptides and growth factors in a highly modifiable, bioinert hydrogel environment.
The last section of the thesis was carried out as part of the EU project HydroZONES that aims to develop and generate zonal constructs. The importance of zonal organization has attracted increased attention in the last years [127, 128], however, it is still underrepresented in tissue engineering approaches so far. Thus, the feasibility of zonal distribution of cells in a scaffold combining two differently composed hydrogels was investigated. A HA-SH(FMZ) containing bottom layer was generated and a pure PG top layer was subsequently cast on top of it, utilizing both times thiol-ene-click reaction. Indeed, stable, hierarchical constructs were generated that allowed encapsulated MSCs to differentiate chondrogenically in both zones as shown by staining for GAGs and collagen type II, and by quantification of GAG amount. Thus, the feasibility of differently composed zonal hydrogels utilizing PG as a main component was successfully demonstrated (Section 5.4).
With the first-time utilization and evaluation of PG-Acr as versatile multifunctional cross-linker for the preparation of Michael addition-generated HA-SH hydrogels in the context of cartilage tissue engineering, a highly modifiable HA-based hydrogel system was introduced. It may be used in future studies as an easily applicable and versatile toolbox for the generation of biomimetically functionalized hydrogels for cell-based cartilage regeneration. The introduction of reinforcement structures to enhance mechanical resistance may thereby further increase the potential of this system for clinical applications.
Additionally, it was also demonstrated that thiol-ene clickable hydrogels can be used for the generation of cell-laden, pure PG gels or for the generation of more complex, coherent zonal constructs. Furthermore, thiol-ene clickable PG hydrogels have already been further modified and successfully been used in 3D bioprinting experiments [204]. 3D bioprinting, as part of the evolving biofabrication field [205], offers the possibilities to generate complex and hierarchical structures, and to exactly position defined layers, yet at the same time alters the requirements for the utilized hydrogels [159, 206–209]. Since a robust chondrogenesis of MSCs was demonstrated in the thiol-ene clickable hydrogel systems, they may serve as a basis for the development of hydrogels as so called bioinks which may be utilized in more sophisticated biofabrication processes.
The objective of this thesis was the synthesis and characterisation of two linear multifunctional PEG-alternatives for bioconjugation and hydrogel formation: i) Hydrophilic acrylate based copolymers containing peptide binding units and ii) hydrophilic polyether based copolymers containing different functional groups for a physical crosslinking.
In section 3.1 the successful synthesis of water soluble and linear acrylate based polymers containing oligo(ethylene glycol) methyl ether acrylate with either linear thioester functional 2-hydroxyethyl acrylate, thiolactone acrylamide, or vinyl azlactone via the living radical polymerisation technique Reversible Addition Fragmentation Chain Transfer (RAFT) and via free-radical polymerisation is described. The obtained polymers were characterized via GPC, 1H NMR, IR and RAMAN spectroscopy.
The RAFT end group was found to be difficult to remove from these short polymer chains and accordingly underwent the undesired side reaction aminolysis with the peptide during the conjugation studies. Besides that, polymers without RAFT end groups did not show any binding of the peptide at the thioester groups, which can be improved in future by using higher reactant concentrations and higher amount of binding units at the polymer. Polymers containing the highly reactive azlactone group showed a peptide binding of 19 %, but unfortunately this function also underwent spontaneous hydrolysis before the peptide could even be bound. In all cases, oligo(ethylene glycol) methyl ether acrylate was used with a relatively high molecular weight (Mn = 480 Da) was used, which eventually was efficiently shielding the introduced binding units from the added peptide. In future, a shorter monomer with Mn = 300 Da or less or hydrophilic N,N’-dialkyl acrylamide based polymers with less steric hindrance could be used to improve this bioconjugation system. Additionally, the amount of monomers containing peptide binding units in the polymer can be increased and have an additional spacer to achieve higher loading efficiency.
The water soluble, linear and short polyether based polymers, so called polyglycidols, were successfully synthesized and modified as described in section 3.2. The obtained polymers were characterized using GPC, 1H NMR, 31P{1H} NMR, IR, and RAMAN spectroscopy. The allyl groups which were present up to 20 % were used for radical induced thiol-ene chemistry for the introduction of functional groups intended for the formation of the physically crosslinking hydrogels. For the positively charged polymers, first a chloride group had to be introduced for the subsequent nucleophilic substitution with the imidazolium compound. There, degrees of modifications were found in the range 40-97 % due to the repulsion forces of the charges, decreased concentration of active chloride groups, and limiting solution concentrations of the polymer for this reaction. For the negatively charged polymers, first a protected phosphonamide moiety was introduced with a deprotection step afterwards showing 100 % conversion for all reactions. Preliminary hydrogel tests did not show a formation of a three-dimensional network of the polymer chains which was attributed to the short backbone length of the used polymers, but the gained knowledge about the synthetic routes for the modification of the polymer was successfully transferred to longer linear polyglycidols. The same applies to the introduction of electron rich and electron poor compounds showing π-π stacking interactions by UV-vis spectroscopy.
Finally, long linear polyglycidyl ethers were synthesised successfully up to molecular weights of Mn ~ 30 kDa in section 3.3, which was also proven by GPC, 1H NMR, IR and RAMAN spectroscopy. This applies to the homopolymerisation of ethoxyethyl glycidyl ether, allyl glycidyl ether and their copolymerisation with an amount of the allyl compound ~ 10 %. Attempts for higher molecular weights up to 100 kDa showed an uncontrolled polymerisation behaviour and eventually can be improved in future by choosing a lower initiation temperature. Also, the allyl side groups were modified via radical induced thiol-ene chemistry to obtain positively charged functionalities via imidazolium moieties (85 %) and negatively charged functionalities via phosphonamide moieties (100 %) with quantitative degree of modifications. Hydrogel tests have still shown a remaining solution by using long linear polyglycidols carrying negative charges with long/short linear polyglycidols carrying positive charges. The addition of calcium chloride led to a precipitate of the polymer instead of a three-dimensional network formation representing a too high concentration of ions and therefore shielding water molecules with prevention from dissolving the polymer. These systems can be improved by tuning the polymers structure like longer polymer chains, longer spacer between polymer backbone and charge, and higher amount of functional groups.
The objective of the thesis was partly reached containing detailed investigated synthetic routes for the design and characterisation of functional polymers which could be used in future with improvements for bioconjugation and hydrogel formation tests.