Investigations of the Influence of the Topography and Surface Chemistry of Poly(hydroxyl ethyl methacrylate) based Hydrogels on Surface Properties and Cell Adhesion

Santander Borrego, Miriem (2013). Investigations of the Influence of the Topography and Surface Chemistry of Poly(hydroxyl ethyl methacrylate) based Hydrogels on Surface Properties and Cell Adhesion PhD Thesis, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland.

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Author Santander Borrego, Miriem
Thesis Title Investigations of the Influence of the Topography and Surface Chemistry of Poly(hydroxyl ethyl methacrylate) based Hydrogels on Surface Properties and Cell Adhesion
School, Centre or Institute Australian Institute for Bioengineering and Nanotechnology
Institution The University of Queensland
Publication date 2013
Thesis type PhD Thesis
Supervisor Idriss Blakey
Andrew Whittaker
Traian Chirila
Total pages 158
Language eng
Subjects 030307 Theory and Design of Materials
030503 Organic Chemical Synthesis
060199 Biochemistry and Cell Biology not elsewhere classified
030301 Chemical Characterisation of Materials
Formatted abstract
Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels have been widely used in ophthalmic applications, such as soft contact lenses and intraocular lenses. In porous forms this hydrophilic hydrogel has proved to be a suitable scaffold in artificial corneas and orbital implants. However, these biomedical applications are limited because PHEMA tends to resist cell attachment, spreading and growth, and lacks the protein absorption properties that are typically required for tissue engineering. The aim of this research was to investigate how the interaction between cells and PHEMA surfaces could be moderated by changes in chemistry and topography, and to understand the relative contributions of chemistry and topography to the surface properties.

Whilst modification of the pendant hydroxyl groups has been an effective method for altering surface chemistry, control of the surface coverage and the resulting surface properties requires significant optimisation of the reaction conditions. This work aimed to generate PHEMA gels with controlled degrees of azide functionalization to facilitate efficient modification via copper catalysed Huisgen 1, 3-dipolar cycloaddition. To achieve this, 2-hydroxyethyl methacrylate was copolymerised with specific amounts of glycidyl methacrylate to produce gels with epoxy groups, which were subsequently converted to azides via reaction with sodium azide. To test the accessibility of the functional groups, this hydrogel was first modified using small molecules as model compound. Finally, alkyne-GRGDS peptides were functionalised on azide-PHEMA surfaces in order to study effect of the ligand density on promoting cell adhesion. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) revealed the efficiency of P(HEMA-stat-GMA) functionalisation. The results of this work indicate that peptide functionalised PHEMA surfaces improved the cell adhesion of the human retinal pigment epithelial cell line (ARPE-19) when compared with unmodified PHEMA. More importantly, the peptide functionalised PHEMA membranes required an optimum peptide density for improving the cell adhesion, where the cell attachment decreased when the density of the immobilised RGD (Arginine-Glycine-Aspartic acid) peptide increased. The best cell attachment compared with the tissue culture plastic was observed for peptide densities of 3.2 pmol/cm2 at the surface of the gels.

Micro- and nano-structures on polymer surfaces also have been shown to have a significant influence on cell attachment, orientation, migration, and cytoskeletal organisation of cells. Different methods have been developed to change surface topography, however, often topographic changes are also accompanied by changes in surface chemistry, which makes it difficult to extract the exact contributions of surface chemistry and topography to cell adhesion. Inspired by the superhydrophobic properties of the lotus leaf, the second part of this research investigated replication of the topography of lotus leaves on PHEMA surfaces by the replica-mould technique. Scanning electron microscopy and atomic force microscopy observations demonstrated successful replication of micron-scale lotus leaf structures on PHEMA surfaces. Utilising the microstructured PHEMA surfaces, the effects of the topography on surface properties were evaluated. This study then explored the relationship between adhesion and surface roughness and scale dependence by AFM using different tip radii. By introducing microstructures on the PHEMA surfaces, the pull-off force was found to increase because of a decrease in the real area of contact between the AFM tip size of 10 nm and the moulded surface. In addition to the surface properties, wettability was studied on the flat and microstructured PHEMA. The water contact angles, measured using the sessile drop method, varied from 65o ± 3o on flat PHEMA to 86o ± 2o on microstructured PHEMA, which indicated that the hydrophobic properties increased due to the introduction of the lotus leaf topography. These experimental values were discussed with reference to the Wenzel and Cassie- Baxter models. These studies provide new insight in the development of polymer surfaces with controlled surface properties and the extent of their surface change effect on cell adhesion for tissue engineering applications.

In the final part of this research, the influences of chemistry and topography on the adhesion of cells to PHEMA surfaces were examined. The results indicate that peptide immobilisation on flat PHEMA surfaces and unfunctionalised PHEMA surfaces with lotus leaf topology enhanced the adhesion of human corneal epithelial cells (HCE-T). More importantly, the results showed that combining lotus leaf topography with RGD surface chemistry had a negative synergetic role on the adhesion of human corneal epithelial cells on PHEMA surfaces. Finally, an understanding of the factors that lead to an improvement of cell adhesion to PHEMA surfaces, which fulfils an essential criterion to advance development of artificial cornea substitutes. Consequently, future work should continue with studies of toxicity and biocompatibility in vitro and in vivo with these hydrogels, which could eventually be conducted in future clinical trials. 
Keyword Poly(2-hydroxyetly methacrylate)
Surface modification
Click chemistry
RGD peptide
Lotus leaf
Replica mould
Atomic force microscopy
Cell behaviour

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Created: Tue, 08 Apr 2014, 19:36:55 EST by Miriem Santander Borrego on behalf of Scholarly Communication and Digitisation Service