HA was modified with methacrylate groups to allow for free radical-initiated covalent crosslinking as described in [3]. To synthesize 35% modified MeHA, a 1% w/v solution of sodium hyaluronate (NaHy, 64 kDa, Lifecore) in dI H2O was dissolved at 4°C prior to addition of 2.2 mL methacrylic anhydride (Sigma) (Figure 4.1A). The solution was allowed to react 24 hours on ice within a pH range of 7.5-9. After the 24-hour period, another 1.1 mL of methacrylic anhydride was added to the reaction, and the pH was kept between 7.5-9 for another 5 hours. For synthesis of 100% modified MeHA, the volume of methacrylic anhydride was doubled at both steps, and the
Figure 4.1 (A) Reaction scheme of MeHA synthesis and (B) representative 1H NMR spectra of MeHA. Labeled peaks in (B) correspond to labels on MeHA chemical structure in (A).
pH was kept within a range of 8.5-9.5. After the second reaction period, the solution was dialyzed against dI H2O (6-8 kDa molecular weight cut off, SpectrumLabs) for 5-6 days and then freeze- dried. The extent of modification was characterized through 1H NMR (Figure 4.1B).
4.2.2 HeMA-HA Synthesis
HA was modified with HeMA groups to allow for radical-initiated covalent crosslinking, as well as hydrolytic degradation through ester group hydrolysis as described in [15]. First, the hydroxyl group in HeMA was converted to a carboxylic acid (i.e. HeMA-COOH) via an esterification reaction with succinic anhydride in the presence of N-methylimidazole. Next, a
Figure 4.2 (A) Reaction scheme of HeMA-HA synthesis and (B) representative 1H NMR spectra of HeMA-HA. Labeled peaks in (B) correspond to labels on HeMA-HA chemical structure in (A).
tetrabutylammonium salt of HA (HA-TBA) was formed by mixing NaHy with a Dowex ion exchange resin (Sigma) for 5-6 hours at room temperature, vacuum filtering out the resin, and then titrating the resulting solution with tetrabutylammonium hydroxide (Sigma) to a pH of 7.02- 7.05. To synthesize HeMA-HA, a 2% w/v solution of HA-TBA in anhydrous dimethyl sulfoxide was reacted with HeMA-COOH in the presence of 4-dimethylaminopyridine and di-tert-butyl dicarbonate under anhydrous conditions for 20 hours at 45°C (Figure 4.2A). The resulting HeMA- HA solution was then purified through dialysis (6-8 kDa molecular weight cut off, SpectrumLabs) against dI H2O for 2-3 weeks at 4°C. After freeze-drying, the extent of modification was characterized using 1H NMR (Figure 4.2B).
4.2.3 Fabrication of Fibrous HA Scaffolds
Multiple HA solutions for electrospinning were investigated, consisting of HA macromer content ranging from 1 to 4% w/v, poly(ethylene oxide) (PEO, Sigma, 900 kDa) content ranging from 1 to 3% w/v, and 0.05% w/v Irgacure 2959 dissolved in dI H2O. Electrospinning solutions were dissolved over a period of 24-48 hours to ensure the polymers were completely dissolved. To electrospin, a syringe was connected to a 12”-long 18G blunt-ended stainless steel needle positioned 12-18 cm away from the grounded aluminum mandrel (Figure 4.3A). The flow rate and applied voltage were kept between 1.0 to 2.0 mL/hr and 18 to 30 kV, respectively. After electrospinning, samples were purged with N2 in an airtight container and then crosslinked with 10 mW/cm2 UV light (320-390 nm collimated, Omnicure S1000 UV Spot Cure Systems). Crosslinking time depended on the relative thickness of the fibrous scaffold; thin samples (i.e. less than 200-300 µm) were polymerized for 10 minutes, and thick scaffolds (i.e. greater than 200-300 µm) were polymerized for 15 minutes on either side for a total of 30 minutes. After crosslinking, samples were stored with desiccant either at room temperature or -20°C (for HeMA- HA-containing scaffolds). Due to the hydrophilicity of HA, swelling of these fibrous HA scaffolds results in a “fibrous hydrogel” scaffold, where the macroscopic fibers are themselves water- swollen networks (Figure 4.3B).
4.2.4 Imaging of Fibrous Scaffolds
Dry fibrous scaffolds were imaged using scanning electron microscopy (SEM, JEOL 7500F HRSEM, Penn Regional Nanotechnology Facility). To visualize swollen scaffolds, methacrylated rhodamine dye (MeRho, Polysciences) was incorporated into electrospinning solutions prior to scaffold fabrication. Fibrous samples with MeRho were swollen in PBS at 37°C for 48 hours prior to imaging with confocal microscopy (Zeiss Axioobserver Inverted microscope, Penn CDB Microscopy Core).
4.2.5 Encapsulation and Release of FITC-BSA from HA Fibers
To measure the release of encapsulated proteins from HA fibers, fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA, Sigma) was dissolved at 0.5% w/v into the HA electrospinning solution. The resulting solution was then electrospun and crosslinked as described previously, and the mat was then cut into 0.5 x 0.5 cm squares. Samples were placed into 1.5 mL Eppendorf tubes, swollen with 1 mL of PBS, and incubated at 37°C. Releasate was collected and refreshed at desired timepoints. Samples were stored at -20°C until the final timepoint, at which point relative FITC-BSA levels were quantified using a microplate reader (Tecan infinite m200) with 480 and 520 nm for excitation and emission wavelengths, respectively. Figure 4.3 Schematic of (A) HA electrospinning system and post-fabrication processing and (B) “gel-within-a-gel” structure of HA fibers (adapted from [16]).
4.2.6 Fabrication of Depth-dependently Aligned HA Scaffolds
To alter the degree of fiber alignment, the rotational speed of the collecting mandrel was varied between 0.2 m/s for unaligned scaffolds, 5 m/s for scaffolds partial alignment, and 10 m/s for fully aligned scaffolds. These scaffolds were then imaged dry with SEM, and the degree of fiber alignment was quantified in ImageJ (NIH) by measuring the angle between each fiber and an arbitrary line drawn over the image. For fabrication of scaffolds with depth-dependent alignment, the mandrel rotation speed was set at 0.2 m/s for the first 4 hours and then switched to 10 m/s during the remaining 12 hours of electrospinning. Samples were then crosslinked and imaged as described previously.