Advanced electrospun matrices based on polysaccharide derivatives for applications in regenerative medicine

The complete regeneration of damaged human tissues and organs is still a significant challenge. The integrative use of biomaterials, cells and bioactive factors in all-in-one devices exploits all current knowledge of materials science, nanotechnology and stem cell biology to best mimic the complex hierarchical architecture of native tissues. The artificial microenvironment design must be properly tuned to match the physicochemical features of the target, offering adequate nanoscale patterns and biological domains for cellular interactions. Scaffolds must promote and guide the regeneration route by mimicking host signalling pathways through the controlled release and retention of drugs or growth factors. For these purposes, polysaccharides have been often used and proposed to manufacture bioengineered scaffolds for regenerative medicine applications due to high biomimetic characteristics. The efforts made by researchers have highlighted how the cellular interactions with electrospun biomaterials offer excellent performances to achieve the desired differentiation and integration with the surrounding tissue. Alkyl derivatives of gellan gum (GG) and hyaluronic acid (HA) have been investigated as novel bioactive electrospun membranes for wound healing and periodontal regeneration. In this thesis, all the derivatives of these two polysaccharides have been produced via activation of the primary hydroxyl groups of β-glucose of gellan gum or N-acetyl-D-glucosamine of hyaluronic acid with bis(4-nitrophenyl) carbonate and the grafts of aliphatic chains at a different length. For hyaluronic derivatives, small moieties with free amino groups have been additionally inserted with the same chemistry. Membranes based on the octyl- and dodecyl-derivative of gellan gum (GG-C8 and GG-C12) have been produced by electrospinning and characterized in terms of fiber distribution and orientation verifying the improved processability of the polymers compared to native gellan gum. Rheological analyses have studied the influence of alkyl derivatization on the spinnability of blends. The feasibility of the process regarded the octyl-derivative one, so the swelling ability of such scaffold has been analyzed under physiological conditions after crosslinking with calcium chloride at different concentrations. To treat partial-thickness wounds, the membrane has been proposed to improve the cell homing at the damaged site, the adhesion of cells, and to encourage the regeneration of the extracellular matrix lost. Similar instrumental settings are used to incorporate the growth factor FGF-2 in the octyl-based membrane (GG-C8). The study has investigated physical interactions between the active and the polymer and how the ionotropic sensitivity of the GG-C8 could be exploited to assess a suitable FGF-2 releasing profile. The fabrication of the bilayer biodevice has involved a hydrophilic layer covered by a synthetic polyurethane layer loaded with ciprofloxacin. Considering the crosslinking degree of the membrane, the dissolution rate and the releases of FGF-2 and ciprofloxacin have been valued with Franz cells. The antibacterial effect of the bilayer has been investigated against the inhibition of units forming colonies of Staphylococcus aureus in the timeframe compatible with the complete regeneration of the tissue. The chemoattraction ability of the scaffold and the cytocompatibility have been tested using cultures of human fibroblasts (NIH3T3) by a specific migration assay. Electrospun membranes based on four chemical derivatives of hyaluronic acid at increasing hydrophobic character have been prepared and loaded with dexamethasone (a known osteoinductive drug) to induce osteogenic differentiation in pre-osteoblasts (MC3T3). The fibrillar supports have been characterized with a scanning electron microscope, and the hydrolytic and enzymatic degradation, as well as dexamethasone releases, have been tested after an autocrosslinking procedure. HA membranes have been designed as guided-bone regeneration barriers for periodontal regeneration; thus, wettability properties and biological performances have been investigated. The proliferation of cells above membranes is valued for up to seven days, and in vitro osteogenic induction is followed by quantifying the activity of alkaline phosphatases and evaluating the calcium content after one month of MC3T3 cultures. The final aim of the thesis is to fabricate a hyaluronan based membrane with high antibacterial properties for the healing of chronic wounds. The proposed membrane consists of incorporating graphene oxide in an electrospun membrane of a hydrophilic derivative of HA loaded with ciprofloxacin. According to an external laser stimulation in the near-infrared, the drug-releasing profile and hyperthermal features have been studied and related to the ability to eradicate bacterial infections and inhibit the generation of biofilm. The cytocompatibility has been valued culturing fibroblasts for up to three days, and the membrane was valued as a wound dressing system.