15 research outputs found
Functionalization of the polysaccharide hydrogel gellan gum for tissue engineering applications
Hydrogels have for long been a promising class of materials for tissue engineering applications. Essentially, hydrogels form a scaffold-like support structure for cells and provide an aqueous environment. This artificial environment mimics natural tissues and allows for the research on cells and the effect of factors onto cells. Furthermore, hydrogels can be used in regenerative medicine for cell delivery to damaged tissue. The herein studied hydrogel material is the polysaccharide gellan gum, which has been developed as a food additive, but has recently been proposed as a suitable tissue engineering material.
Although most hydrogel materials are biocompatible and do not negatively affect cell growth, they are also biologically relatively inert. To combat this situation, various approaches have been described in the literature to functionalize hydrogels with an abundance of different bioactive molecules through the means of various chemical strategies. Likewise, gellan gum hydrogels have been used successfully in cell culture, but satisfying cell adhesion and response have not been achieved.
This thesis work describes the chemical functionalization of gellan gum and the covalent binding of the protein avidin to the gellan gum. Avidin is a tetrameric protein which binds biotin with high specificity and affinity. This allows for the convenient and flexible modification of the gellan gum network with biotin-labelled compounds, notably biotinylated ligands for cell attachment and signaling. Therefore, sodium purified gellan gum was successfully functionalized with avidin over carbodiimide coupling. Self-supporting gel samples could be created from the functionalized gellan gum. Commercial gellan gum was purified with an established method and its elemental composition was analyzed with atomic absorption spectroscopy. The covalent coupling of avidin was verified with gel electrophoresis, while its functionality was determined with fluorescence spectroscopy. Hydrogel samples were formed with calcium and bioamines and the mechanical properties of the gels were examined with compression testing.
The results verify that the presented approach offers a mild functionalization that does not disturb hydrogel gelation or the avidin-biotin binding. Further work is required to improve the cross-linking and gel sample production, in order to achieve consistent results of parallel samples with good gel structure and desired suitable mechanical behavior. The next steps will be to discern a suitable biotinylated bioactive cue, such as biotinylated RGD, and test the ability of the functionalized gellan gum to serve as a cell culture matrix
Design Strategies for Polysaccharide Hydrogels Used in Soft Tissue Engineering : Modification, Testing and Applications of Gellan Gum
Hydrogels are water-swollen polymer networks which provide an aqueous, three- dimensional environment and can mimic the biological cell environment and tissue architecture. Therefore, hydrogels are a valuable class of biomaterials for tissue engineering purposes that can be modified to support a specific application, such as the encapsulation of cells or as implantable device. Gellan gum is a microbial polysaccharide that readily forms self-supporting hydrogels in the presence of ions, and that has been investigated for medical applications due to its biocompatibility. However, due to its lack of innate cell recognition sites in its structure, gellan gum is highly inert and does not elicit any cell response required for in vitro cell culture or in vivo tissue integration.
Here, the possibilities to chemically modify gellan gum and render it bioactive for cell culture purposes are explored. The investigated modifications include purification, oxidation, reductive scissoring, as well as blending and chemical crosslinking, and are initially reviewed for their biocompatibility and ability to form hydrogels. The modified materials were assessed for their mechanical and viscoelastic properties, and basic cell response using the human fibroblast line WI-38. The cells were seeded either 2D on the surface of a gelated sample or encapsulated in the 3D hydrogel. Similarly, more advanced cell lines, such as human adipose stem cells, bone marrow-derived stem cells and a vascular co-culture model, were investigated using some of the formulations, and evaluated using different microscopic techniques. Furthermore, extrusion bioprinting was investigated as biofabrication method, and tissue response in vivo of cell-free hydrogels was ascertained by subcutaneous implantation.
In conclusion, the aim of thesis was to examine different modification approaches for the hydrogel gellan gum, but also to present a wholistic assessment protocol of modified hydrogel. Gellan gum acts as model polymer with the intent of projecting the design strategies and evaluation insights onto other polysaccharides and hydrogels. It has proven to be a suitable base polymer to create a material library with various mechanical and bioactive properties
Monitoring the gelation of gellan gum with torsion rheometry and brightness-mode ultrasound
publishedVersionPeer reviewe
Bioactivated gellan gum hydrogels affect cellular rearrangement and cell response in vascular co-culture and subcutaneous implant models
Hydrogels are suitable soft tissue mimics and capable of creating pre-vascularized tissues, that are useful for in vitro tissue engineering and in vivo regenerative medicine. The polysaccharide gellan gum (GG) offers an intriguing matrix material but requires bioactivation in order to support cell attachment and transfer of biomechanical cues. Here, four versatile modifications were investigated: Purified NaGG; avidin-modified NaGG combined with biotinylated fibronectin (NaGG-avd); oxidized GG (GGox) covalently modified with carbohydrazide-modified gelatin (gelaCDH) or adipic hydrazide-modified gelatin (gelaADH). All materials were subjected to rheological analysis to assess their viscoelastic properties, using a time sweep for gelation analysis, and subsequent amplitude sweep of the formed hydrogels. The sweeps show that NaGG and NaGG-avd are rather brittle, while gelatin-based hydrogels are more elastic. The degradation of preformed hydrogels in cell culture medium was analyzed with an amplitude sweep and show that gelatin-containing hydrogels degrade more dramatically. A co-culture of GFP-tagged HUVEC and hASC was performed to induce vascular network formation in 3D for up to 14 days. Immunofluorescence staining of the αSMA+ network showed increased cell response to gelatin-GG networks, while the NaGG-based hydrogels did not allow for the elongation of cells. Preformed, 3D hydrogels disks were implanted to subcutaneous rat skin pockets to evaluate biological in vivo response. As visible from the hematoxylin and eosin-stained tissue slices, all materials are biocompatible, however gelatin-GG hydrogels produced a stronger host response. This work indicates, that besides the biochemical cues added to the GG hydrogels, also their viscoelasticity greatly influences the biological response.publishedVersionPeer reviewe
Functionalization of the polysaccharide hydrogel gellan gum for tissue engineering applications
Hydrogels have for long been a promising class of materials for tissue engineering applications. Essentially, hydrogels form a scaffold-like support structure for cells and provide an aqueous environment. This artificial environment mimics natural tissues and allows for the research on cells and the effect of factors onto cells. Furthermore, hydrogels can be used in regenerative medicine for cell delivery to damaged tissue. The herein studied hydrogel material is the polysaccharide gellan gum, which has been developed as a food additive, but has recently been proposed as a suitable tissue engineering material.
Although most hydrogel materials are biocompatible and do not negatively affect cell growth, they are also biologically relatively inert. To combat this situation, various approaches have been described in the literature to functionalize hydrogels with an abundance of different bioactive molecules through the means of various chemical strategies. Likewise, gellan gum hydrogels have been used successfully in cell culture, but satisfying cell adhesion and response have not been achieved.
This thesis work describes the chemical functionalization of gellan gum and the covalent binding of the protein avidin to the gellan gum. Avidin is a tetrameric protein which binds biotin with high specificity and affinity. This allows for the convenient and flexible modification of the gellan gum network with biotin-labelled compounds, notably biotinylated ligands for cell attachment and signaling. Therefore, sodium purified gellan gum was successfully functionalized with avidin over carbodiimide coupling. Self-supporting gel samples could be created from the functionalized gellan gum. Commercial gellan gum was purified with an established method and its elemental composition was analyzed with atomic absorption spectroscopy. The covalent coupling of avidin was verified with gel electrophoresis, while its functionality was determined with fluorescence spectroscopy. Hydrogel samples were formed with calcium and bioamines and the mechanical properties of the gels were examined with compression testing.
The results verify that the presented approach offers a mild functionalization that does not disturb hydrogel gelation or the avidin-biotin binding. Further work is required to improve the cross-linking and gel sample production, in order to achieve consistent results of parallel samples with good gel structure and desired suitable mechanical behavior. The next steps will be to discern a suitable biotinylated bioactive cue, such as biotinylated RGD, and test the ability of the functionalized gellan gum to serve as a cell culture matrix
Acoustic parametrisation of gellan gum
National audienceGellan gum is a hydrogel with several applications in ultrasonic imaging, novel drug delivery, and tissue regeneration. As hydrogels are dynamic entities, they are viscocelastic and therefore their acoustic properties change over time, which is of interest to monitor. To determine the speed of sound from brightnessmode images, however, rather large quantities of hydrogel are needed. In this study, we investigated torsion rheometry as a means to determine acoustic properties. Perceived speeds of sound were derived and computed from torsion rheometry measurements of gelating gellan gum mixed with spermidine trihydrochloride crosslinker. For comparison, brightness-mode ultrasonic images were recorded of the same material inside a phantom well. The rheometry data converged to a speed of sound within a standard deviation of the speed of sound measured from the brightness-mode images. We have shown that dynamic acoustic properties of gelating gellan gum can be simulated and experimentally determined using torsion rheometry
Chemical modification strategies for viscosity-dependent processing of gellan gum
Recently, the hydrogel-forming polysaccharide gellan gum (GG) has gained popularity as a versatile biomaterial for tissue engineering purposes. Here, we examine the modification strategies suitable for GG to overcome processing-related limitations. We emphasize the thorough assessment of the viscoelastic and mechanical properties of both precursor solutions and final hydrogels. The investigated modification strategies include purification, oxidation, reductive chain scission, and blending. We correlate polymer flow and hydrogel forming capabilities to viscosity-dependent methods including casting, injection and printing. Native GG and purified NaGG are shear thinning and feasible for printing, being similar in gelation and compression behavior. Oxidized GGox possesses reduced viscosity, higher toughness, and aldehydes as functional groups, while scissored GGsciss has markedly lower molecular weight. To exemplify extrudability, select modification products are printed using an extrusion-based bioprinter utilizing a crosslinker bath. Our robust modification strategies have widened the processing capabilities of GG without affecting its ability to form hydrogels.publishedVersionPeer reviewe
Design of modular gellan gum hydrogel functionalized with avidin and biotinylated adhesive ligands for cell culture applications
This article proposes the coupling of the recombinant protein avidin to the polysaccharide gellan gum to create a modular hydrogel substrate for 3D cell culture and tissue engineering. Avidin is capable of binding biotin, and thus biotinylated compounds can be tethered to the polymer network to improve cell response. The avidin is successfully conjugated to gellan gum and remains functional as shown with fluorescence titration and electrophoresis (SDS-PAGE). Self-standing hydrogels were formed using bioamines and calcium chloride, yielding long-term stability and adequate stiffness for 3D cell culture, as confirmed with compression testing. Human fibroblasts were successfully cultured within the hydrogel treated with biotinylated RGD or biotinylated fibronectin. Moreover, human bone marrow stromal cells were cultured with hydrogel treated with biotinylated RGD over 3 weeks. We demonstrate a modular and inexpensive hydrogel scaffold for cell encapsulation that can be equipped with any desired biotinylated cell ligand to accommodate a wide range of cell types.publishedVersionPeer reviewe