Antibacterial, macroporous chitosan hydrogels for soft tissue engineering applications

Abstract

One of the goals of tissue engineering is to be able to develop functional tissue substitutes that can one day replace the function of an organ that is damaged or requires replacement. The development of macroporous scaffolds that can potentially replicate or mimic the functionality and architecture of native extracellular matrix (upon implantation at the site of defect) is one of the major focus in the field of tissue engineering. For a large number of tissue engineering strategies surgical implantation of the scaffold is necessary, yet it also increases the risk of infections. The growing of antimicrobial resistance has necessitated the development of new strategies to tackle drug-resistant bacteria.

The focus of this study was to develop porous hydrogel scaffolds for soft tissue engineering and antimicrobial wound healing. This research was first focused on the development of macroporous chitosan hydrogel scaffolds utlilising thermally induced phase separation (TIPS). Two different types of gelation (freeze neutralisation vs freeze crosslinking) were employed for the fabrication of porous chitosan scaffolds. Pore size and porosities are important scaffold parameters for neovascularisation, potentially improving the incorporation of the implant into the host tissue. Hence, various physical parameters such as temperature, chitosan concentration and crosslinking were varied to study their effects on the pore size distribution (60-150 µm) and porosity (65-80%) of the chitosan scaffolds. Mechanical properties of these scaffolds were tunable (compressive modulus: 1-450 kPa) via change of crosslinking and chitosan concentrations. The study was also able to demonstrate scaffold mechanical properties which were relatively similar to the that of the soft tissues (2.5-40 kPa). Mouse 3T3 fibroblasts (relevant cell line for dermis) were shown to adhere, proliferate and penetrate into the scaffolds in vitro.

A novel combined mechanical gas foaming and TIPS technique was also developed in order to better control the pore size and morphology of the scaffolds. To establish the range where mechanical foaming results in optimal entrainment of air (hence porous voids) the effects of surfactant (polyvinyl alcohol (PVA)) concentration, foaming speed, foaming time and chitosan concentration were probed and correlated to the foam generating potential. Upon establishing the physical constraints on mechanical foaming for this system, various parameters (PVA concentration, chitosan concentration, mixing speed) that could affect the pore size distribution of these chitosan/PVA scaffolds were studied. This combined technique yielded scaffolds with a more homogeneous spherical morphology. Although pore sizes were generally larger (average pore sizes 120-170 µm) compared to TIPS standalone method, however, pore size distributions were wide and highlighted the need for further improvement of pore size distribution. Scaffolds fabricated with this technique presented soft mechanics (elastic modulus: 2.5-25 kPa), which were similar to those of soft tissues. Experiments with mouse 3T3 fibroblasts revealed that these novel scaffolds supported cell proliferation, attachment without any observable cytotoxicity.

Wet chemical synthesis was utilised in order to incorporate selenium (Se) and silver (Ag) nanostructures (separately) into the novel foamed TIPS scaffold, to improve its antimicrobial properties. The morphology of the Ag and Se nanostructures formed in situ on chitosan/PVA scaffolds revealed stark differences which were attributed to the interaction between the chitosan backbone and selenite ions via a hydrogen bonding mechanism. This study also demonstrated the fine control of the loading of both the elements and measured its release in various culture media (complete DMEM, LB media and deionised water). There were significant differences in release of Ag species in all three types of media, indicating the possible role of O2, chloride ions and various organic molecules (e.g., glucose, proteins) that can affect Ag release. Se release was largely unaffected in DMEM and LB media. Extracts from chitosan/PVA scaffolds loaded with various doses of Se (0, 0.06, 0.25, 0.92, 3.67 w/w %) or Ag (0, 0.37, 2.33, 8.97, 17.79 w/w %) showed significant toxicity for Ag based scaffolds. However, no significant cytotoxicity was observed for Se-chitosan/PVA scaffolds. Antibacterial studies carried out on S.aureus, MRSA and E.coli via a Syto9/PI flow cytometric assay revealed significant membrane damage in presence of Se scaffolds and no membrane damage for Ag scaffolds. Colony forming unit (CFU) counts of all three bacterial species tested revealed that CS-Ag caused significant cell death to all bacterial species. However, CS-Se did not lead to significant reduction in bacterial cell numbers, which suggested that while CS-Se extracts caused membrane damage, it did not lead to cell death at the loadings tested.

Overall, this thesis focuses on the development of soft scaffolds with tunable pore sizes, morphology and porosities utilising TIPS and gas foaming methodologies. The results also indicate these scaffolds have soft mechanics, support mouse 3T3 fibroblast proliferation, attachment without cytotoxicity. This point towards the possible application of these scaffolds for wound healing. Furthermore, incorporation of Se nanostructures led to enhancement of the scaffold’s bacterial cell permeabilisation properties which could be useful as an adjuvant to enhance the potency of current antimicrobials against drug resistant bacteria.