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- ItemPoly(2-alkyl-2-oxazoline)-Heparin Hydrogels—Expanding the Physicochemical Parameter Space of Biohybrid Materials(Weinheim : Wiley-VCH, 2021) Hahn, Dominik; Sonntag, Jannick M.; Lück, Steffen; Maitz, Manfred F.; Freudenberg, Uwe; Jordan, Rainer; Werner, CarstenPoly(ethylene glycol) (PEG)-glycosaminoglycan (GAG) hydrogel networks are established as very versatile biomaterials. Herein, the synthetic gel component of the biohybrid materials is systematically varied by combining different poly(2-alkyl-2-oxazolines) (POx) with heparin applying a Michael-type addition crosslinking scheme: POx of gradated hydrophilicity and temperature-responsiveness provides polymer networks of distinctly different stiffness and swelling. Adjusting the mechanical properties and the GAG concentration of the gels to similar values allows for modulating the release of GAG-binding growth factors (VEGF165 and PDGF-BB) by the choice of the POx and its temperature-dependent conformation. Adsorption of fibronectin, growth of fibroblasts, and bacterial adhesion scale with the hydrophobicity of the gel-incorporated POx. In vitro hemocompatibility tests with freshly drawn human whole blood show advantages of POx-based gels compared to the PEG-based reference materials. Biohybrid POx hydrogels can therefore enable biomedical technologies requiring GAG-based materials with customized and switchable physicochemical characteristics. © 2021 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.
- ItemBio-responsive polymer hydrogels homeostatically regulate blood coagulation(London : Nature Publishing Group, 2013) Maitz, Manfred F.; Freudenberg, U.; Tsurkan, M.V.; Fischer, M.; Beyrich, T.; Werner, C.Bio-responsive polymer architectures can empower medical therapies by engaging molecular feedback-response mechanisms resembling the homeostatic adaptation of living tissues to varying environmental constraints. Here we show that a blood coagulation-responsive hydrogel system can deliver heparin in amounts triggered by the environmental levels of thrombin, the key enzyme of the coagulation cascade, which - in turn - becomes inactivated due to released heparin. The bio-responsive hydrogel quantitatively quenches blood coagulation over several hours in the presence of pro-coagulant stimuli and during repeated incubation with fresh, non-anticoagulated blood. These features enable the introduced material to provide sustainable, autoregulated anticoagulation, addressing a key challenge of many medical therapies. Beyond that, the explored concept may facilitate the development of materials that allow the effective and controlled application of drugs and biomolecules.
- ItemGeometry-Driven Cell Organization Determines Tissue Growths in Scaffold Pores: Consequences for Fibronectin Organization(San Francisco, CA : Public Library of Science, 2013) Joly, P.; Duda, G.N.; Schöne, M.; Welzel, P.B.; Freudenberg, U.; Werner, C.; Petersen, A.To heal tissue defects, cells have to bridge gaps and generate new extracellular matrix (ECM). Macroporous scaffolds are frequently used to support the process of defect filling and thus foster tissue regeneration. Such biomaterials contain micro-voids (pores) that the cells fill with their own ECM over time. There is only limited knowledge on how pore geometry influences cell organization and matrix production, even though it is highly relevant for scaffold design. This study hypothesized that 1) a simple geometric description predicts cellular organization during pore filling at the cell level and that 2) pore closure results in a reorganization of ECM. Scaffolds with a broad distribution of pore sizes (macroporous starPEG-heparin cryogel) were used as a model system and seeded with primary fibroblasts. The strategies of cells to fill pores could be explained by a simple geometrical model considering cells as tensioned chords. The model matched qualitatively as well as quantitatively by means of cell number vs. open cross-sectional area for all pore sizes. The correlation between ECM location and cell position was higher when the pores were not filled with tissue (Pearson's coefficient ρ = 0.45±0.01) and reduced once the pores were closed (ρ = 0.26±0.04) indicating a reorganization of the cell/ECM network. Scaffold pore size directed the time required for pore closure and furthermore impacted the organization of the fibronectin matrix. Understanding how cells fill micro-voids will help to design biomaterial scaffolds that support the endogenous healing process and thus allow a fast filling of tissue defects.
- ItemHeparin-based, injectable microcarriers for controlled delivery of interleukin-13 to the brain(Cambridge : Royal Soc. of Chemistry, 2020) Schirmer, Lucas; Hoornaert, Chloé; Le Blon, Debbie; Eigel, Dimitri; Neto, Catia; Gumbleton, Mark; Welzel, Petra B.; Rosser, Anne E.; Werner, Carsten; Ponsaerts, Peter; Newland, BenInterleukin-13 (IL-13) drives cells of myeloid origin towards a more anti-inflammatory phenotype, but delivery to the brain remains problematic. Herein, we show that heparin-based cryogel microcarriers load high amounts of IL-13, releasing it slowly. Intra-striatal injection of loaded microcarriers caused local up-regulation of ARG1 in myeloid cells for pro-regenerative immunomodulation in the brain. © 2020 The Royal Society of Chemistry.
- ItemTechniques for RNA extraction from cells cultured in starPEG-heparin hydrogels(London : Royal Society Publishing, 2021) Jaeschke, Anna; Harvey, Nicholas R.; Tsurkan, Mikhail; Werner, Carsten; Griffiths, Lyn R.; Haupt, Larisa M.; Bray, Laura J.Three-dimensional (3D) cell culture models that provide a biologically relevant microenvironment are imperative to investigate cell–cell and cell–matrix interactions in vitro. Semi-synthetic star-shaped poly(ethylene glycol) (starPEG)–heparin hydrogels are widely used for 3D cell culture due to their highly tuneable biochemical and biomechanical properties. Changes in gene expression levels are commonly used as a measure of cellular responses. However, the isolation of high-quality RNA presents a challenge as contamination of the RNA with hydrogel residue, such as polymer or glycosaminoglycan fragments, can impact template quality and quantity, limiting effective gene expression analyses. Here, we compare two protocols for the extraction of high-quality RNA from starPEG–heparin hydrogels and assess three subsequent purification techniques. Removal of hydrogel residue by centrifugation was found to be essential for obtaining high-quality RNA in both isolation methods. However, purification of the RNA did not result in further improvements in RNA quality. Furthermore, we show the suitability of the extracted RNA for cDNA synthesis of three endogenous control genes confirmed via quantitative polymerase chain reaction (qPCR). The methods and techniques shown can be tailored for other hydrogel models based on natural or semi-synthetic materials to provide robust templates for all gene expression analyses.
- ItemGlycosaminoglycan-based hydrogels to modulate heterocellular communication in in vitro angiogenesis models(London : Nature Publishing Group, 2014) Chwalek, K.; Tsurkan, M.V.; Freudenberg, U.; Werner, C.Angiogenesis, the outgrowth of blood vessels, is crucial in development, disease and regeneration. Studying angiogenesis in vitro remains challenging because the capillary morphogenesis of endothelial cells (ECs) is controlled by multiple exogenous signals. Therefore, a set of in situ-forming starPEG-heparin hydrogels was used to identify matrix parameters and cellular interactions that best support EC morphogenesis. We showed that a particular type of soft, matrix metalloproteinase-degradable hydrogel containing covalently bound integrin ligands and reversibly conjugated pro-angiogenic growth factors could boost the development of highly branched, interconnected, and lumenized endothelial capillary networks. Using these effective matrix conditions, 3D heterocellular interactions of ECs with different mural cells were demonstrated that enabled EC network modulation and maintenance of stable vascular capillaries over periods of about one month in vitro. The approach was also shown to permit in vitro tumor vascularization experiments with unprecedented levels of control over both ECs and tumor cells. In total, the introduced 3D hydrogel co-culture system could offer unique options for dissecting and adjusting biochemical, biophysical, and cell-cell triggers in tissue-related vascularization models.