Electrospun Fibres with Hyaluronic Acid-Chitosan Nanoparticles Produced by a Portable Device

Electrospinning is a versatile technique to produce nano/microscale fibrous scaffolds for tissue engineering and drug delivery applications. This research aims to demonstrate that hyaluronic acid-chitosan (HA-CS) nanoparticles can be electrospun together with polycaprolactone (PCL) and gelatine (Ge) fibres using a portable device to create scaffolds for tissue repair. A range of polymer solutions of PCL-gelatine at different weight/volume concentrations and ratios were electrospun and characterised. Fibre–cell interaction (F11 cells) was evaluated based on cell viability and proliferation and, from here, a few polymer blends were electrospun into random or aligned fibre arrangements. HA-CS nanoparticles were synthesised, characterised, and used to functionalise electrospun fibres (8% w/v at 70 PCL:30 Ge), which were chosen based on cell viability. Different concentrations of HA-CS nanoparticles were tested to determine cytotoxicity. A single dosage (1 × 10−2 mg/mL) was associated with higher cell proliferation compared with the cell-only control. This nanoparticle concentration was embedded into the electrospun fibres as either surface modification or blend. Fibres with blended NPs delivered a higher cell viability than unmodified fibres, while NP-coated fibres resulted in a higher cell proliferation (72 h) than the NP-blended ones. These biocompatible scaffolds allow cell attachment, maintain fibre arrangement, promote directional growth and yield higher cell viability.


Materials and Methods
Fibres were electrospun for 60 s and carefully placed in a 24-well plate (Corning Incorporated -Life Sciences, Oneonta, NY, US) in a unidirectional arrangement (i.e., parallel fibres). A press was placed overnight to secure the fibres to the bottom and, after it was removed, fibres were sterilised.
Three fibre coatings were explored: hyaluronic acid (HA; 0.1%), chitosan (CS; 1%) and neural growth factor (NGF; 50 ng/mL). Half a millilitre of each coating solution was added to each well (already containing the PCL-Ge fibres) and left for 3 hours at room temperature in order to ensure full fibre coating. After the excess was removed, samples were washed with half a millilitre of Phosphate-Buffered Saline (PBS; Gibco Life Technologies, Bleiswijk, the Netherlands).
Cells were seeded (50,000 cells/well) on top of the fibres and co-cultured for 72 h with 0.5 mL of media. Three fibre coatings (HA, CS and NGF) as well as controls of uncoated fibres and cell-only samples were used (n = 3). Cell viability was determined with the aid of a live/dead assay and a microplate reader, as previously outlined.

Hyaluronic Acid (HA)
A 0.1% solution of HA (Sigma-Aldrich, Prague, Czech Republic) in double distilled water was dissolved by magnetic stirring at room temperature until achieving homogeneity.

Chitosan (CS)
Chitosan (Sigma-Aldrich Life Science, Reykjavík, Iceland) was dissolved at 1% in acetic acid (Sigma-Aldrich, Saint Louis, MO, US) by magnetic stirring at room temperature for 3 h. Then, three washes with a 0.1 M NaOH solution and deionised water were performed.

Nerve Growth Factor (NGF)
NGF (PeproTech, Cranbury, NJ, US) at a concentration of 50 ng/mL was dissolved in PBS and left to solubilise for 30 min, vortexing occasionally.
In comparison with the unmodified fibres control, both hyaluronic acid and NGF yielded a statistically significant higher viability. On the other hand, when comparing the experimental fibres with the cells-only control, only chitosan was associated with a statistically significant lower viability. The HA and NGF coatings performed similar to the cells-only wells; however, it is worth noting that the advantage of using either of the coated electrospun fibres relies on their ability to direct cell growth by providing preferential attachment points.
When comparing between coatings, both HA and NGF delivered a statistically significant higher viability than chitosan. This could potentially be attributed to the solution in which CS was dissolved in (acetic acid).