Atomic Force Microscopy (AFM) is a powerful technique that has evolved from being a purely imaging tool to a one capable of providing multifunctional information, offering exciting new possibilities for nano-biotechnology. The project focuses on the use of the AFM in order to morphologically and mechanically characterise cells and biomaterials demonstrating how versatile this instrument can be. The project is divided in the following parts:Part 1: establishment of AFM protocols for the nano-scale morphological and mechanical characterisation of soft and hard macroscopic substrates and of objects such as adsorbed nanoparticles. In particular, these techniques were tested on:Hyaluronic acid (HA)/poly(ethylene glycol) (PEG)-based hydrogels, which provide an artificial model for the mechanical behaviour of some biological tissues and organs. The elastic modulus, measured via AFM nanoindentation, of these hydrogels increased by decreasing the concentration and the molecular weight (MW) of HA in the hydrogels. We have then verified a clear relation between the mechanical properties of the hydrogels and the proliferation of cells cultured on them. Chitosan nanoparticle (popular carriers for the delivery of negatively charged macromolecular payloads, e.g. nucleic acids) cross-linked with triphosphate (TPP) and then coated with HA. We focussed on the influence of chitosan molecular weight (Mw) on nanoparticle properties. HA was able to penetrate into the more porous nanoparticles (high MW chitosan), whereas it formed a corona around the more cross-linked ones (low MW chitosan). AFM imaging was used to highlight the presence of this corona and also to estimate its apparent thickness to about 20-30 nm (in dry state).Silicone substrates modified with amphiphilic triblock copolymer (Sil-GMMA) layers. Extensive AFM (imaging and nanoindentation) provided evidence that silicone substrates are prevalently coated with Sil-GMMA thin layers that exhibit negligible hydrophobic recovery during drying and change the surface from more to less cell-adhesive. Part 2: AFM mechanical characterisation of fibroblast-to-myofibroblast differentiation process. Fibroblasts were stimulated to differentiate into myofibroblasts by Transforming Grow Factor β1 (TGFβ1) on hard substrate. AFM force maps performed both on fibroblasts (untreated cells) and myofibroblasts (TGFβ1-treated cells) revealed a significant increase in the elastic modulus in treated cells. Part 3: preparation and AFM characterisation of poly(ethylene glycol) diacrylate/acrylate (PEGDA/A) hydrogels. Since the mechanical properties of the substrate plays a pivotal role in fibroblast-to-myofibroblast differentiation process, hydrogels were prepared and characterised at the macro/nanoscale with AFM indentation, providing us with cell-adhesive substrates that cover a wide range of elastic modulus. These substrates are optimal candidates for future investigations to better understand and possibly control the differentiation process.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:603144 |
| Date | January 2014 |
| Creators | Donno, Roberto |
| Contributors | Freemont, Anthony; Sherratt, Michael; Tirelli, Nicola |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/nanomechanical-characterisation-of-cells-and-biocompatible-substrates(f4d8bf94-035f-4798-ad64-02d319756974).html |
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