TY - JOUR A1 - El Hajj, Nady A1 - Dittrich, Marcus A1 - Böck, Julia A1 - Kraus, Theo F. J. A1 - Nanda, Indrajit A1 - Müller, Tobias A1 - Seidmann, Larissa A1 - Tralau, Tim A1 - Galetzka, Danuta A1 - Schneider, Eberhard A1 - Haaf, Thomas T1 - Epigenetic dysregulation in the developing Down syndrome cortex JF - Epigenetics N2 - Using Illumina 450K arrays, 1.85% of all analyzed CpG sites were significantly hypermethylated and 0.31% hypomethylated in fetal Down syndrome (DS) cortex throughout the genome. The methylation changes on chromosome 21 appeared to be balanced between hypo- and hyper-methylation, whereas, consistent with prior reports, all other chromosomes showed 3-11times more hyper- than hypo-methylated sites. Reduced NRSF/REST expression due to upregulation of DYRK1A (on chromosome 21q22.13) and methylation of REST binding sites during early developmental stages may contribute to this genome-wide excess of hypermethylated sites. Upregulation of DNMT3L (on chromosome 21q22.4) could lead to de novo methylation in neuroprogenitors, which then persists in the fetal DS brain where DNMT3A and DNMT3B become downregulated. The vast majority of differentially methylated promoters and genes was hypermethylated in DS and located outside chromosome 21, including the protocadherin gamma (PCDHG) cluster on chromosome 5q31, which is crucial for neural circuit formation in the developing brain. Bisulfite pyrosequencing and targeted RNA sequencing showed that several genes of PCDHG subfamilies A and B are hypermethylated and transcriptionally downregulated in fetal DS cortex. Decreased PCDHG expression is expected to reduce dendrite arborization and growth in cortical neurons. Since constitutive hypermethylation of PCDHG and other genes affects multiple tissues, including blood, it may provide useful biomarkers for DS brain development and pharmacologic targets for therapeutic interventions. KW - trisomy 21 KW - DNA methylation KW - Down syndrome KW - fetal brain development KW - frontal cortex KW - protocadherin gamma cluster Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191239 VL - 11 IS - 8 ER - TY - THES A1 - Böck, Thomas T1 - Multifunctional Hyaluronic Acid / Poly(glycidol) Hydrogels for Cartilage Regeneration Using Mesenchymal Stromal Cells T1 - Multifunktionale Hyaluronsäure / Poly(glycidol) Hydrogele für die Knorpelregeneration mit Mesenchymalen Stromazellen N2 - 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. N2 - Es ist von großem Interesse die Therapieoptionen für die degenerative Gelenkerkrankung Osteoarthrose (OA) zu verbessern, da OA als eine der weltweit häufigsten Ursachen von Bewegungseinschränkungen und Schmerzen gilt und somit eine sozioökonomische Belastung darstellt [202]. Laut epidemiologischen Studien leiden bereits 27 Millionen Menschen in den USA an OA [23]. Darüber hinaus geht die WHO davon aus, dass OA bereits im Jahr 2020 die vierthäufigste Ursache von körperlichen Behinderungen sein wird [203], was die Notwendigkeit für effektive und langanhaltende Therapien von schweren Knorpeldefekten zeigt. Obwohl sich bereits eine Vielzahl von Therapien in klinischer Anwendung für die Behandlung von Knorpeldefekten befindet [62], ist die Entwicklung von knorpelspezifischen Produkten noch nicht weit fortgeschritten. Hyaluronsäure (HA), als Hauptbestandteil der Extrazellulären Matrix (ECM) von Knorpel, stellt eine generell zytokompatible Umgebung dar, die Zellen von Natur aus Bindungsstellen zur Adhäsion und Fortbewegung bietet. Zudem ist bekannt, dass die funktionellen Gruppen von HA besonders gut für chemische Modifikationen geeignet sind. Aufgrund dieser Eigenschaften wird HA häufig als Material für das hydrogelbasierte Tissue Engineering verwendet. Durch die Verwendung von Poly(glycidol) (PG) als Cross-linker stehen die gleichen chemischen Eigenschaften wie bei der Verwendung des gängigen Cross-linkers Poly(ethylene glycol) (PEG) zur Verfügung, allerdings bietet es zusätzliche Seitenketten an jeder Wiederholungseinheit. Durch die Einführung von PG als multifunktionalem Cross-linker zur Herstellung von HA-Gelen ergibt sich letztlich eine höhere Vernetzungsdichte und damit auch ein größeres Potenzial für biomimetische Funktionalisierungen. Trotz dieser genannten Vorteile wird PG bisher noch nicht im Bereich der Knorpelregeneration verwendet. Das erste Ziel dieser Arbeit beinhaltete die Etablierung und Optimierung eines HA-basierten Hydrogels für die chondrogene Differenzierung von Mesenchymalen Stromazellen (MSCs). Hierzu wurden verschiedene Mengen und Derivate von Cross-linkern eingesetzt. Zunächst wurde die Hydrogelzusammensetzung mithilfe von verschiedenen PEG-Diacrylat (PEGDA)-Konzentrationen zur Vernetzung von thiolmodifizierter HA (Glycosil, HASH) mittels Michael-Addition optimiert. Das Ziel war hierbei die Herstellung eines volumenstabilen Konstrukts, das gleichzeitig die größtmögliche Ablagerung von ECM erlaubt. Histologische und biochemische Analysen zeigten in Bezug darauf, dass eine Konzentration von 0,4% PEGDA die zuvor genannten Anforderungen am besten erfüllte (Abschnitt 5.1.2). Um im weiteren Verlauf den Einfluss von verschiedenen Cross-linkern auf die chondrogene Differenzierung von MSCs zu untersuchen, wurde die HA-SH vergleichend mit PEGTA (0,6%) und PEGDA (0,4%) vernetzt. Nachfolgend wurde acryliertes PG (PG-Acr) als eine Alternative zu acrylierten PEG-Derivaten evaluiert. Der Vorteil in der Verwendung von PG-Acr (0,6%) im Vergleich zu PEGTA (0,6%) liegt darin, dass es eine ca. fünfmal höhere Anzahl an funktionellen Gruppen bietet, was wiederum ein deutlich höheres Maß an biomimetischer Funktionalisierung ermöglicht. Hierbei zeigte die Untersuchung der knorpelspezifischen ECM-Bestandteile keine grundlegenden Unterschiede zwischen beiden Cross-linkern, wobei durch die Verwendung von PG-Acr eine gleichmäßigere Ablagerung von Knorpelmatrix in die entsprechenden Gele zu erkennen war. Zusammenfassend lässt sich feststellen, dass die Möglichkeiten für eine biomimetische Funktionalisierung durch die Verwendung von PG-Acr deutlich erhöht wurden, ohne dabei die Chondrogenese von MSCs negativ zu beeinträchtigen (Abschnitt 5.1.3). Der nächste Teil dieser Arbeit befasste sich mit der umfangreichen biomimetischen Funktionalisierung von mit PG-Acr (0,6%) vernetzten HA-SH Hydrogelen. Hierzu wurden entweder biomimetische Peptide oder ein chondrogener Wachstumsfaktor kovalent in das Hydrogel eingebunden. Interessanterweise führte weder das Einbringen des N-Cadherin-mimetischen (HAV), des Kollagen II-bindenden (KLER), noch des Zelladhäsions-vermittelnden (RGD) Peptids zu einer Verbesserung der chondrogenen Differenzierung der MSCs. Beispielsweise führte das kovalente Anbinden von 2,5mM HAV zu einer Veränderung der Zellkernmorphologie und einer Verringerung der Glykosaminoglykan (GAG)-Produktion, wohingegen das Einbringen von 1,0mM RGD die Kollagenproduktion hemmte. Diese Ergebnisse könnten möglicherweise darauf zurückzuführen sein, dass die hier verwendeten HA-SH-Hydrogele selbst bereits ausreichend effizient für die chondrogene Differenzierung von MSCs sind. Im Vergleich dazu wurden die vorherigen Studien, die positive Effekte von Peptiden nachweisen konnten, entweder in neutralen PEG-Hydrogelen oder in wesentlich festeren MeHA-Hydrogelen durchgeführt [99, 101, 160]. Daraus lässt sich folgern, dass die Verwendung von Peptiden gerade unter ungünstigen Bedingungen von Bedeutung sein könnte und ein neutrales Gelsystem für die Untersuchung von einzelnen Peptideffekten geeignet scheint (Abschnitt 5.2.1). Als nächstes wurde exemplarisch der chondrogene Wachstumsfaktor Transforming Growth Factor Beta 1 (TGF-b1) kovalent an PG-Acr angebunden. Durch die Verwendung von kovalent gebundenem TGF-b1 könnte somit die Notwendigkeit einer wiederholten Zugabe von TGF-b1 bei in vivo-Anwendungen vermieden werden, was wiederum bei einer potentiellen klinischen Anwendung von Vorteil sein könnte. Deshalb wurde der Einfluss von kovalent gebundenem TGF-b1 auf die Chondrogenese von MSCs mit der gleichen Menge ungebundenem TGF-b1 (100nM TGF-b1) verglichen. Hierbei wurde ein signifikant positiver, dosisabhängiger Effekt von kovalent gebundenem TGF-b1 erfolgreich nachgewiesen. Die Chondrogenese von MSCs in Hydrogelen mit kovalent gebundenem TGF-b1 war dabei der Chondrogenese von MSCs in Hydrogelen, in die TGF-b1 lediglich gemischt wurde, deutlich überlegen. Dies wurde anhand von stärkeren Färbungen für GAGs, Gesamtkollagen, Aggrecan und Kollagen II in den TGF-b1-modifizierten Gelen gezeigt. Darüber hinaus bestätigten sowohl biochemische Analysen des GAG- und Kollagengehalts, als auch Western Blot-Analysen die histologischen Daten. Zusätzlich wurde der positive Effekt von kovalent gebundenem TGF-b1 durch erhöhte Expressionsraten der chondrogenen Markergene COL2A1, ACAN und SOX9 nachgewiesen. Zusammenfassend konnte gezeigt werden, dass durch die kovalente Bindung des Wachstumsfaktors TGF-b1 ein signifikant positiver Effekt auf die chondrogene Differenzierung von MSCs entsteht (Abschnitt 5.2.2). Generell stellen die auf Basis von Michael-Addition hergestellten PG-Acr-HA-SH-Hydrogele aufgrund ihrer hohen Acrylat-Funktionalität eine vielseitige Hydrogelplattform dar. So bieten diese Hydrogele zahlreiche Möglichkeiten für das Einbringen von verschiedensten biologischen Modifikationen wie die kovalente Bindung von biomimetischen Peptiden zusammen mit Wachstumsfaktoren in ein und demselben Zellträger. Anhand eines Proof-of-principle-Experiments wurde die generelle Eignung von reinen PG-Hydrogelen für die Evaluation von einzelnen Peptideffekten demonstriert. Dazu wurden die Hydrogele unter Verwendung der Thiol-ene-click-Reaktion hergestellt. In diesem Hydrogelsystem, ohne den unterstützenden Effekt von HA, zeigten MSCs eine verstärkte chondrogene Differenzierung in Anwesenheit von 1,0mM HAV. Diese ließ sich anhand von stärkeren Färbungen für GAGs, Aggrecan und Kollagen II nachweisen. Außerdem waren die GAG- und Gesamtkollagen-Werte deutlich erhöht. Hiermit wurde gezeigt, dass sich die vielseitig modifizierbaren, reinen PG-Hydrogele für die Analyse von Peptideffekten und deren Interaktion mit Wachstumsfaktoren eignen (Abschnitt 5.3). Der letzte Teil dieser Arbeit wurde im Rahmen des EU-Projektes HydroZONES durchgeführt, welches an der Entwicklung und Herstellung von zonalen Konstrukten arbeitet. Der Aspekt der zonalen Organisation von Knorpel rückte in den letzten Jahren verstärkt in den Fokus [127, 128], jedoch findet er im Bereich des Tissue Engineering noch immer wenig Beachtung. Deshalb wurde im Folgenden die zonale Verteilung von Zellen innerhalb eines Zellträgers realisiert. Dazu wurden zwei unterschiedlich zusammengesetzte Hydrogele mithilfe der Thiol-ene-click-Reaktion hergestellt: eine aus HA-SH(FMZ) bestehende untere Lage und eine darauf liegende Lage aus reinem PG. Hierbei gelang es stabile, zonale Konstrukte herzustellen, in denen MSCs in beiden Zonen chondrogen differenzierten, was anhand von GAG- und Kollagen II-Färbungen, sowie durch die Quantifizierung des GAG-Gehalts bestätigt wurde. Hiermit konnte ein aus zwei verschiedenen Hydrogelen zusammengesetztes zonales Konstrukt erfolgreich hergestellt werden (Abschnitt 5.4). Durch den erstmaligen Einsatz des multifunktionalen Cross-linkers PG-Acr für das Tissue Engineering von Knorpel wurde ein auf Michael-Addition basierendes, vielseitiges HA-SH-Hydrogelsystem etabliert. Das hier vorgestellte Hydrogelsystem besitzt das Potenzial zukünftig als eine einfach anwendbare und vielseitige Toolbox zur Herstellung von biomimetischen Hydrogelen für die zellbasierte Knorpelregeneration verwendet zu werden. Vor allem könnte dabei der Einsatz von Stützstrukturen von entscheidender Bedeutung sein, um die mechanische Widerstandskraft der Zellträger zu erhöhen und somit das Potenzial für klinische Anwendungen zu vergrößern. Zusätzlich wurde gezeigt, dass Thiol-ene-click-Hydrogele sowohl zur Herstellung von zellbeladenen, reinen PG-Gelen, als auch zur Herstellung von deutlich komplexeren, zonalen Konstrukten geeignet sind. Diese Thiol-ene-click-Hydrogele wurden bereits erfolgreich weiterentwickelt und für 3D-Bioprinting-Prozesse verwendet [204]. 3D-Bioprinting ist eine Teildisziplin des sich immer weiter entwickelnden Feldes der Biofabrikation [205]. Die Verwendung in diesem Bereich verändert zwar die Anforderungen an die hierfür verwendeten Hydrogele, ermöglicht es aber gleichzeitig deutlich komplexere sowie hierarchische Strukturen herzustellen und kleinere Lagen noch exakter zu positionieren [159, 206–209]. Da in den hier vorgestellten Thiol-ene-click-Hydrogelen eine deutliche chondrogene Differenzierung von MSCs nachgewiesen wurde, ist es vorstellbar, dass sie als Basis für die Herstellung sogenannter Bioinks dienen, welche in zukünftigen, anspruchsvollen Biofabrikationsprozessen Anwendung finden sollen. KW - Hyaluronsäure KW - Hydrogel KW - Knorpel KW - Tissue Engineering KW - Hyaluronic acid KW - Poly(glycidol) KW - Hydrogel KW - Cartilage Regeneration KW - Mesenchymal Stromal Cells Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-155345 ER - TY - JOUR A1 - Schneider, Eberhard A1 - Dittrich, Marcus A1 - Böck, Julia A1 - Nanda, Indrajit A1 - Müller, Tobias A1 - Seidmann, Larissa A1 - Tralau, Tim A1 - Galetzka, Danuta A1 - El Hajj, Nady A1 - Haaf, Thomas T1 - CpG sites with continuously increasing or decreasing methylation from early to late human fetal brain development JF - Gene N2 - Normal human brain development is dependent on highly dynamic epigenetic processes for spatial and temporal gene regulation. Recent work identified wide-spread changes in DNA methylation during fetal brain development. We profiled CpG methylation in frontal cortex of 27 fetuses from gestational weeks 12-42, using Illumina 450K methylation arrays. Sites showing genome-wide significant correlation with gestational age were compared to a publicly available data set from gestational weeks 3-26. Altogether, we identified 2016 matching developmentally regulated differentially methylated positions (m-dDMPs): 1767 m-dDMPs were hypermethylated and 1149 hypomethylated during fetal development. M-dDMPs are underrepresented in CpG islands and gene promoters, and enriched in gene bodies. They appear to cluster in certain chromosome regions. M-dDMPs are significantly enriched in autism-associated genes and CpGs. Our results promote the idea that reduced methylation dynamics during fetal brain development may predispose to autism. In addition, m-dDMPs are enriched in genes with human-specific brain expression patterns and/or histone modifications. Collectively, we defined a subset of dDMPs exhibiting constant methylation changes from early to late pregnancy. The same epigenetic mechanisms involving methylation changes in cis-regulatory regions may have been adopted for human brain evolution and ontogeny. KW - Autism spectrum disorders KW - DNA methylation KW - Genome KW - Autism KW - Frontal cortex KW - Human prefrontal cortex KW - Gene-expression KW - Schizophrenia KW - Patterns KW - Transcription KW - Epigenetics KW - Environment KW - Fetal brain development KW - DNA methylation dynamics KW - Methylome Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-186936 VL - 592 IS - 1 ER - TY - JOUR A1 - Shan, Junwen A1 - Böck, Thomas A1 - Keller, Thorsten A1 - Forster, Leonard A1 - Blunk, Torsten A1 - Groll, Jürgen A1 - Teßmar, Jörg T1 - TEMPO/TCC as a Chemo Selective Alternative for the Oxidation of Hyaluronic Acid JF - Molecules N2 - Hyaluronic acid (HA)-based hydrogels are very commonly applied as cell carriers for different approaches in regenerative medicine. HA itself is a well-studied biomolecule that originates from the physiological extracellular matrix (ECM) of mammalians and, due to its acidic polysaccharide structure, offers many different possibilities for suitable chemical modifications which are necessary to control, for example, network formation. Most of these chemical modifications are performed using the free acid function of the polymer and, additionally, lead to an undesirable breakdown of the biopolymer’s backbone. An alternative modification of the vicinal diol of the glucuronic acid is oxidation with sodium periodate to generate dialdehydes via a ring opening mechanism that can subsequently be further modified or crosslinked via Schiff base chemistry. Since this oxidation causes a structural destruction of the polysaccharide backbone, it was our intention to study a novel synthesis protocol frequently applied to selectively oxidize the C6 hydroxyl group of saccharides. On the basis of this TEMPO/TCC oxidation, we studied an alternative hydrogel platform based on oxidized HA crosslinked using adipic acid dihydrazide as the crosslinker. KW - hyaluronic acid KW - oxidation KW - hydrogel formation KW - Schiff base chemistry Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248362 SN - 1420-3049 VL - 26 IS - 19 ER - TY - JOUR A1 - Jacobs, Graeme A1 - Bock, Stefanie A1 - Schuch, Anita A1 - Moschall, Rebecca A1 - Schrom, Eva-Maria A1 - Zahn, Juliane A1 - Reuter, Christian A1 - Preiser, Wolfgang A1 - Rethwilm, Axel A1 - Engelbrecht, Susan A1 - Krekau, Thomas A1 - Bodem, Jochen T1 - Construction of a high titer Infectious HIV-1 subtype C proviral clone from South Africa N2 - The Human Immunodeficiency Virus type 1 (HIV-1) subtype C is currently the predominant subtype worldwide. Cell culture studies of Sub-Saharan African subtype C proviral plasmids are hampered by the low replication capacity of the resulting viruses, although viral loads in subtype C infected patients are as high as those from patients with subtype B. Here, we describe the sequencing and construction of a new HIV-1 subtype C proviral clone (pZAC), replicating more than one order of magnitude better than the previous subtype C plasmids. We identify the env-region for being the determinant for the higher viral titers and the pZAC Env to be M-tropic. This higher replication capacity does not lead to a higher cytotoxicity compared to previously described subtype C viruses. In addition, the pZAC Vpu is also shown to be able to down-regulate CD4, but fails to fully counteract CD317. KW - HIV KW - HIV-1; subtype C; proviral plasmid; viral replication; resistance assays; Vpu; CD317; CD4 Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-76340 ER - TY - JOUR A1 - Paxton, Naomi A1 - Smolan, Willi A1 - Böck, Thomas A1 - Melchels, Ferry A1 - Groll, Jürgen A1 - Jungst, Tomasz T1 - Proposal to assess printability of bioinks for extrusion-based bioprinting and evaluation of rheological properties governing bioprintability JF - Biofabrication N2 - The development and formulation of printable inks for extrusion-based 3D bioprinting has been a major challenge in the field of biofabrication. Inks, often polymer solutions with the addition of crosslinking to form hydrogels, must not only display adequate mechanical properties for the chosen application but also show high biocompatibility as well as printability. Here we describe a reproducible two-step method for the assessment of the printability of inks for bioprinting, focussing firstly on screening ink formulations to assess fibre formation and the ability to form 3D constructs before presenting a method for the rheological evaluation of inks to characterise the yield point, shear thinning and recovery behaviour. In conjunction, a mathematical model was formulated to provide a theoretical understanding of the pressure-driven, shear thinning extrusion of inks through needles in a bioprinter. The assessment methods were trialled with a commercially available crème, poloxamer 407, alginate-based inks and an alginate-gelatine composite material. Yield stress was investigated by applying a stress ramp to a number of inks, which demonstrated the necessity of high yield for printable materials. The shear thinning behaviour of the inks was then characterised by quantifying the degree of shear thinning and using the mathematical model to predict the window of printer operating parameters in which the materials could be printed. Furthermore, the model predicted high shear conditions and high residence times for cells at the walls of the needle and effects on cytocompatibility at different printing conditions. Finally, the ability of the materials to recover to their original viscosity after extrusion was examined using rotational recovery rheological measurements. Taken together, these assessment techniques revealed significant insights into the requirements for printable inks and shear conditions present during the extrusion process and allow the rapid and reproducible characterisation of a wide variety of inks for bioprinting. KW - bioprinting KW - rheology KW - modelling KW - bioink Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-254061 VL - 9 IS - 4 ER - TY - JOUR A1 - Frischholz, Sebastian A1 - Berberich, Oliver A1 - Böck, Thomas A1 - Meffert, Rainer H. A1 - Blunk, Torsten T1 - Resveratrol counteracts IL‐1β‐mediated impairment of extracellular matrix deposition in 3D articular chondrocyte constructs JF - Journal of Tissue Engineering and Regenerative Medicine N2 - When aiming at cell‐based therapies in osteoarthritis (OA), proinflammatory conditions mediated by cytokines such as IL‐1β need to be considered. In recent studies, the phytoalexin resveratrol (RSV) has exhibited potent anti‐inflammatory properties. However, long‐term effects on 3D cartilaginous constructs under inflammatory conditions with regard to tissue quality, especially extracellular matrix (ECM) composition, have remained unexplored. Therefore, we employed long‐term model cultures for cell‐based therapies in an in vitro OA environment and evaluated effects of RSV. Pellet constructs made from expanded porcine articular chondrocytes were cultured with either IL‐1β (1–10 ng/ml) or RSV (50 μM) alone, or a cotreatment with both agents. Treatments were applied for 14 days, either directly after pellet formation or after a preculture period of 7 days. Culture with IL‐1β (10 ng/ml) decreased pellet size and DNA amount and severely compromised glycosaminoglycan (GAG) and collagen content. Cotreatment with RSV distinctly counteracted the proinflammatory catabolism and led to partial rescue of the ECM composition in both culture systems, with especially strong effects on GAG. Marked MMP13 expression was detected in IL‐1β‐treated pellets, but none upon RSV cotreatment. Expression of collagen type I was increased upon IL‐1β treatment and still observed when adding RSV, whereas collagen type X, indicating hypertrophy, was detected exclusively in pellets treated with RSV alone. In conclusion, RSV can counteract IL‐1β‐mediated degradation and distinctly improve cartilaginous ECM deposition in 3D long‐term inflammatory cultures. Nevertheless, potential hypertrophic effects should be taken into account when considering RSV as cotreatment for articular cartilage repair techniques. KW - articular chondrocytes KW - cartilage KW - cell‐based therapy KW - extracellular matrix KW - IL‐1β KW - inflammation KW - osteoarthritis KW - resveratrol Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215471 VL - 14 IS - 7 SP - 897 EP - 908 ER - TY - JOUR A1 - Haertle, Larissa A1 - Maierhofer, Anna A1 - Böck, Julia A1 - Lehnen, Harald A1 - Böttcher, Yvonne A1 - Blüher, Matthias A1 - Schorsch, Martin A1 - Potabattula, Ramya A1 - El Hajj, Nady A1 - Appenzeller, Silke A1 - Haaf, Thomas T1 - Hypermethylation of the non-imprinted maternal MEG3 and paternal MEST alleles is highly variable among normal individuals JF - PLoS ONE N2 - Imprinted genes show parent-specific activity (functional haploidy), which makes them particularly vulnerable to epigenetic dysregulation. Here we studied the methylation profiles of oppositely imprinted genes at single DNA molecule resolution by two independent parental allele-specific deep bisulfite sequencing (DBS) techniques. Using Roche (GSJunior) next generation sequencing technology, we analyzed the maternally imprinted MEST promoter and the paternally imprinted MEG3 intergenic (IG) differentially methylated region (DMR) in fetal cord blood, adult blood, and visceral adipose tissue. Epimutations were defined as paternal or maternal alleles with >50% aberrantly (de)methylated CpG sites, showing the wrong methylation imprint. The epimutation rates (range 2–66%) of the paternal MEST and the maternal MEG3 IG DMR allele, which should be completely unmethylated, were significantly higher than those (0–15%) of the maternal MEST and paternal MEG3 alleles, which are expected to be fully methylated. This hypermethylation of the non-imprinted allele (HNA) was independent of parental origin. Very low epimutation rates in sperm suggest that HNA occurred after fertilization. DBS with Illumina (MiSeq) technology confirmed HNA for the MEST promoter and the MEG3 IG DMR, and to a lesser extent, for the paternally imprinted secondary MEG3 promoter and the maternally imprinted PEG3 promoter. HNA leads to biallelic methylation of imprinted genes in a considerable proportion of normal body cells (somatic mosaicism) and is highly variable between individuals. We propose that during development and differentiation maintenance of differential methylation at most imprinting control regions may become to some extent redundant. The accumulation of stochastic and environmentally-induced methylation errors on the non-imprinted allele may increase epigenetic diversity between cells and individuals. KW - DNA methylation KW - genomic imprinting KW - polymerase chain reaction KW - blood KW - epigenetics KW - sequence alignment KW - sperm Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170433 VL - 12 IS - 8 ER - TY - JOUR A1 - Brand, Jessica S. A1 - Forster, Leonard A1 - Böck, Thomas A1 - Stahlhut, Philipp A1 - Teßmar, Jörg A1 - Groll, Jürgen A1 - Albrecht, Krystyna T1 - Covalently Cross-Linked Pig Gastric Mucin Hydrogels Prepared by Radical-Based Chain-Growth and Thiol-ene Mechanisms JF - Macromolecular Bioscience N2 - Mucin, a high molecular mass hydrophilic glycoprotein, is the main component of mucus that coats every wet epithelium in animals. It is thus intrinsically biocompatible, and with its protein backbone and the o-glycosidic bound oligosaccharides, it contains a plethora of functional groups which can be used for further chemical modifications. Here, chain-growth and step-growth (thiol-ene) free-radical cross-linked hydrogels prepared from commercially available pig gastric mucin (PGM) are introduced and compared as cost-efficient and easily accessible alternative to the more broadly applied bovine submaxillary gland mucin. For this, PGM is functionalized with photoreactive acrylate groups or allyl ether moieties, respectively. Whereas homopolymerization of acrylate-functionalized polymers is performed, for thiol-ene cross-linking, the allyl-ether-functionalized PGM is cross-linked with thiol-functionalized hyaluronic acid. Morphology, mechanical properties, and cell compatibility of both kinds of PGM hydrogels are characterized and compared. Furthermore, the biocompatibility of these hydrogels can be evaluated in cell culture experiments. KW - click chemistry KW - photopolymerization KW - hydrogels KW - mucin KW - thiol-ene Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318453 VL - 22 IS - 4 ER - TY - JOUR A1 - Lekszas, Caroline A1 - Nanda, Indrajit A1 - Vona, Barbara A1 - Böck, Julia A1 - Ashrafzadeh, Farah A1 - Donyadideh, Nahid A1 - Ebrahimzadeh, Farnoosh A1 - Ahangari, Najmeh A1 - Maroofian, Reza A1 - Karimiani, Ehsan Ghayoor A1 - Haaf, Thomas T1 - Unbalanced segregation of a paternal t(9;11)(p24.3;p15.4) translocation causing familial Beckwith-Wiedemann syndrome: a case report JF - BMC Medical Genomics N2 - Background The vast majority of cases with Beckwith-Wiedemann syndrome (BWS) are caused by a molecular defect in the imprinted chromosome region 11p15.5. The underlying mechanisms include epimutations, uniparental disomy, copy number variations, and structural rearrangements. In addition, maternal loss-of-function mutations in CDKN1C are found. Despite growing knowledge on BWS pathogenesis, up to 20% of patients with BWS phenotype remain without molecular diagnosis. Case presentation Herein, we report an Iranian family with two females affected with BWS in different generations. Bisulfite pyrosequencing revealed hypermethylation of the H19/IGF2: intergenic differentially methylated region (IG DMR), also known as imprinting center 1 (IC1) and hypomethylation of the KCNQ1OT1: transcriptional start site (TSS) DMR (IC2). Array CGH demonstrated an 8 Mb duplication on chromosome 11p15.5p15.4 (205,827-8,150,933) and a 1 Mb deletion on chromosome 9p24.3 (209,020-1,288,114). Chromosome painting revealed that this duplication-deficiency in both patients is due to unbalanced segregation of a paternal reciprocal t(9;11)(p24.3;p15.4) translocation. Conclusions This is the first report of a paternally inherited unbalanced translocation between the chromosome 9 and 11 short arms underlying familial BWS. Copy number variations involving the 11p15.5 region are detected by the consensus diagnostic algorithm. However, in complex cases which do not only affect the BWS region itself, characterization of submicroscopic chromosome rearrangements can assist to estimate the recurrence risk and possible phenotypic outcomes. KW - Familial Beckwith-Wiedemann syndrome KW - copy number variation KW - duplication-deficiency KW - genomic imprinting KW - submicroscopic chromosome rearrangement KW - reciprocal translocation Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-200422 VL - 12 ER -