Development of self-assembled hydrogels for stem cell based tissue engineering applications

Abstract

a-cyclodextrin molecules (a-CDs) and poly(ethylene oxide)-poly(propylene oxide)- poly(ethylene oxide) (PEO-PPO-PEO, Pluronic) block copolymers can self-assemble to form structures known as pseudo-polyrotaxanes (PPRs) or polyrotaxane (PRs) if end-capped, also known as qmolecular necklacesq. In these complexes, the a-CDs are threaded onto the PEO polymer backbone and can rotate and slide along the polymer axis. These properties, when combined with the ability of PRs to self-assemble into hydrogels under specific conditions make them a very attractive system for tissue engineering applications. To this date, the use of PPR and PR hydrogels in tissue engineering has been limited and little is known about their potential as a biomaterial, especially for use with stem cells. Moreover, the mechanical and chemical properties of these hydrogels need to be finely tuned to re-create the complex microenvironment required to direct stem cells to form specific biological tissues. This thesis aimed to investigate the properties of a new range of PR based hydrogels and to design PR systems that may be used for stem cell based tissue engineering, both in 2D and 3D. Initially, the conditions for PPR hydrogel formation from Pluronic a-CD were investigated and the influence of the type and concentration of Pluronic, concentration of a-CD and temperature on the hydrogel properties determined. It was found that PPR hydrogels can be formed between a- CD and Pluronic F68 and F127 at concentrations of 10 % (w/v) and 20 % (w/v) when a sufficient amount of a-CD is present. The hydrogels obtained by this method were highly tunable in terms of gelation kinetics (a few minutes to a few hours) and mechanical properties (elastic modulus ranging between 1 kPa to 7 MPa). Such high values of mechanical stiffness had not been reported previously for this type of system. The microscopic features of the PPR hydrogels were investigated using Dynamic Light Scattering (DLS) and Small-Angle X-Ray Scattering (SAXS) and revealed a complex multi-levelled self-assembled structure, allowing the structure-function-property relationship between the various components of the hydrogels and the resultant materials to be elucidated. Building upon these new insights into these self-assembled PPR systems, an enzymatically mediated covalent crosslinking function and branched 8-arm poly(ethylene) glycol (PEG) were introduced into the hydrogel to overcome the inherent property of the self-assembled PPR hydrogels to dissociate when immersed in a liquid that contains lower concentrations or no Pluronic or a-CD, due to differences in chemical potential. The crosslinking mechanism was based on coupling of phenol groups using horseradish peroxidase (HRP) and hydrogen peroxide. The phenol groups were added onto the Pluronic backbone of the PPRs and onto the 8-arm PEG in a two-step reaction that introduced a hydrolytically cleavable ester bond into the hydrogel network. The 8-arm PEG also acted as an end-capping group, thus leading to the formation of PRs. The covalent FRONT MATTER iii crosslinking successfully extended the lifetime of the hydrogels from a few hours to several days and led to the formation of highly tunable hydrogels with an elastic modulus between 20 kPa and 410 kPa and a viscous modulus between 150 Pa and 22 kPa, by varying the concentrations of a-CD and 8-arm PEG. The resulting hydrogels showed potential in drug delivery and the viability of mouse fibroblasts encapsulated in the hydrogels for 24 hours was maintained. Subsequently, the stability of the PR hydrogels was further improved to make them suitable for long term tissue engineering applications by changing the chemistry of the phenol functionalisation process: the ester bonds introduced in the network during the functionalisation of the polymers with phenol groups were modified to carbamate bonds. This simple modification led to hydrogels that were stable for at least 90 days while still having highly tunable mechanical properties in the range of 70 to 190 kPa and that could maintain viability of encapsulated hMSCs for at least 7 days. Finally, the adhesion peptide RGD was successfully introduced to enhance interactions between hMSCs and the hydrogels: attachment of hMSCs was observed on the RGDfunctionalised hydrogels and the hydrogel surfaces were permissive of osteogenic differentiation under osteogenic conditions. The hydrogels developed in this thesis present unique features that make them an attractive system to add to the panoply of hydrogels available to the biomaterials community. These promising properties could assist in our quest to understand the impacts of hierarchical structures within hydrogels on stem cell behaviours and eventually lead to the development of novel regenerative therapeutic solutions.