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Hyaluronan Based ECM-Mimetics with Tunable Charge Densities - Physico-Chemical Properties and Biological Implications

Hegger, Patricia

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The hydrated and highly charged extracellular matrix (ECM) plays a vital role in many biological processes. Therefore, mimicking this environment is of great interest in material science as well as biomedical research. In this thesis, an ECM mimetic is presented based on hyaluronic acid also called hyaluronan (HA) as one of the ECM’s abundant negatively charged components. Due to HA’s unique properties, this anionic biopolymer was chosen as biological "module" to design biocompatible charge defined hydrogels in a synthetic biology approach. By varying the degree of chemical modification on the carboxyl groups of the glucuronic acid moiety of HA in addition to differently charged but structurally similar heteroaromatic crosslinkers, the charge density of the hydrogels presented in this thesis can be adjusted. Using thiolation as chemical modification together with two armed crosslinkers carrying unsaturated end groups, the thiol-Michael addition click reaction can be employed for byproduct free crosslinking of HA chains. The composition of the presented hydrogels remains constant and hydrogels with the same degree of thiolation and differently charged heteroaromatic crosslinker cores even result in the same covalent connectivity of the network. I could show that size defined HA with an average molecular weight of 74 kDa is suitable for homogeneous and reproducible hydrogel formation at degrees of thiolation from 18% to 36%. Additionally, the presented hydrogels show a high stability over time, with constant mechanical stiffness over the course of at least four weeks. Using a set of hydrogels synthesized from 74 kDa HA with three different degrees of thiolation in the range of 18% to 33% and one neutral and one structurally similar, positively charged crosslinker, physico-chemical properties were monitored. Two general trends could be observed for mechanical stiffness in the form of Young’s moduli ranging from 0.44 kPa to 6.31 kPa, swelling ratios and mesh sizes between 53.54nm to 183.76nm. (1) With increasing degree of thiolation, mechanical stiffness increases whereas swelling ratios and respective mesh sizes decrease. (2) At each individual degree of thiolation, hydrogels with the charged crosslinker show twice as high Young’s moduli and roughly halved swelling ratios and mesh sizes, compared to the same hydrogels with uncharged crosslinkers. Furthermore, swelling ratios of the presented polyelectrolyte hydrogels are highly influenced by the ionic strength of the swelling solution but independent of the solution’s pH at physiological salt concentration. Biological properties, as for example enzymatic degradability, show the trends described for physico-chemical characterization. (1) Half-lives of hydrogels in hyaluronidase and hyaluronate lyase solutions are increasing with increasing degree of thiolation from 2.5 h to 11.7 h in hyaluronidase with the uncharged crosslinker and (2) for each individual degree of thiolation, half-lives for hydrogels with the charged crosslinkers are always larger than for respective hydrogels with the uncharged crosslinker. Furthermore, cell adhesion of fibroblasts, lymph endothelial cells and breast cancer cells is (1) increasing with increasing degree of thiolation. (2) It could also be observed, that cells adhere better on hydrogels with charged crosslinkers, compared to hydrogels with the same degree of thiolation with uncharged crosslinkers. For all of these trends in physico-chemical as well as biological properties, two influencing factors could clearly be determined in this thesis. Firstly, correlation of all presented properties with the negative network charge of each individual hydrogel could be observed: With decreasing negative network charge, hydrogels are becoming stiffer, show lower swelling capacity and smaller mesh sizes, degradation by hyaluronan degrading enzymes becomes slower, and more cells are able to adhere to their surface. Secondly, the aromatic core of the crosslinker seems to be another important factor for material properties. The naturally occurring pyridinium causes considerably stiffer hydrogels with smaller mesh sizes and lower swelling ratios compared to a structurally similar triazolium based crosslinker. To conclude, a charge defined hydrogel system could be presented, mimicking the hydrated ECM niche, in a minimal model adjusted for studying non-integrin mediated cell attachment. From results obtained by characterizing the material properties, I suggest a strong influence of secondary interactions on physico-chemical and biological properties of the ECM. Both electrostatic interactions, resulting from negative network charge of the hydrogels, as well as aromatic core interactions due to different heteroaromats used in crosslinker design are critically influencing hydrogel properties in a specific way. With the establishment of this defined system further studies on charge dependent ECM characteristics as well as application in three dimensional (3D) tissue engineering, drug delivery or regenerative medicine are possible, given the opportunity to specifically tune material properties by changing the secondary interactions within the network.

Item Type: Dissertation
Supervisor: Spatz, Prof. Dr. Joachim
Date of thesis defense: 29 November 2017
Date Deposited: 22 Jan 2018 07:18
Date: 2018
Faculties / Institutes: Fakultät für Chemie und Geowissenschaften > Institute of Physical Chemistry
Subjects: 540 Chemistry and allied sciences
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