Self-assembling silk hydrogels for mesenchymal stem cell and drug delivery
Silk fibroin has been commonly used as sutures and surgical meshes in humans. Additionally, silk fibroin had been used for a wide range of biomedical uses including as a coating for polymer scaffolds produced for tissue engineering and silk fibroin hydrogels containing silk nanoparticles have previously been studied for the controlled delivery of model drugs. The aims of this thesis was to characterise silk hydrogels by both FT-IR analysis and using compression testing. Next, the effect of loading drugs into silk hydrogels with different physicochemical properties. The final aim of the project was to investigate the trophic factors released from MSCs cultured on top (i.e. 2D) and in (i.e. 3D) a silk hydrogel. From the sol-gel transition experiments the ideal silk hydrogel to be used for incorporated MSCs is a 5% or 4% w/v 60 minute degummed silk hydrogel diluted with PBS due to a fast sol-gel transition allowing enough time for the addition of MSCs ensuring they were homogenously distributed throughout the silk hydrogel. Protease XIV increases the degradation in the 3% w/v silk hydrogel compared to the 5% w/v silk hydrogel. This is likely caused by the higher the silk concentration the higher the cross-linking density. This results in a stronger hydrogel structure. SDS-PAGE analysis showed that the 60 minute degummed silk hydrogel supernatants had smearing in the range 70 to 130 kDa. The 60 minute degummed silk hydrogel supernatants have a smaller average molecular weight. Mechanical testing of silk hydrogels revealed that the stress required to reach the yield point in hydrogels in their natural state and suspended in DMEM for 24 hours is ~35 KPa and ~32KPa respectively. MSCs grown in 2D secrete higher concentrations of cytokines. For example, there is a 2.3 fold higher concentration of VEGF in 2D in comparison to 3D environment. VEGF improves cardiac performance by enhancing angiogenesis. The obtained excretory profiles of MSCs would be beneficial in regenerative medicine and are promising for future in vitro studies. Overall, this thesis demonstrates that silk hydrogels can be used in MSC delivery in vitro and the nature of cell culture can affect the secretion of cytokines. This is promising for future studies using silk hydrogels to deliver MSCs where excretory profiles should be evaluated more rigorously using 3 independent replicates to validate the findings in this thesis. In addition, this thesis has shown that the degradation rate of silk hydrogels can be tuned in vivo by altering the silk content. This is an encouraging prospect for the use of silk hydrogels in drug and cell delivery which provides a foundation for further in vitro and in vivo studies.
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