Fabrication and characterization of cross-linkable hydrogel particles based on hyaluronic acid: potential application in vocal fold regeneration.

There is a critical need to engineer hyaluronic acid (HA)-based hydrogels with prolonged in vivo residence time, temporal release of therapeutics and matching viscoelasticity for use in vocal fold tissue engineering. We have previously demonstrated the synthesis and characterization of HA-based soft hydrogel particles (HGP) and particle cross-linked networks as injectable materials to treat vocal fold scarring. In this paper, we report a more versatile technique for preparing cross-linkable HA HGP with reduced sizes. HA HGP were synthesized via chemical cross-linking with divinyl sulfone using a sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/isooctane reverse micelle system in the presence of 1-heptanol. These HGP were rendered cross-linkable by introducing aldehyde groups via sodium periodate oxidation (oxHGP). The presence of aldehyde groups was confirmed by multi-photon confocal microscope upon fluorescence staining using cascade blue hydrazide. The aldehyde groups were used as reactive handles for covalent cross-linking with HA that has been previously modified with adipic acid dihydrazide (HADH). The resulting doubly cross-linked networks (DXN) are highly pliable and do not break until approx. 200-300% strain. The measured elastic modulus of the DXN is around 500 Pa, while the dynamic viscosity decreases linearly with frequency in log- log scale. The mechanical characteristics of DXN are similar to that of vocal fold lamina propria. In vitro cell-proliferation assays showed that the cross-linkable HA HGP did not adversely affect the proliferation of the cultured fibroblasts as assessed by MTT assay. A low-molecular-weight model drug, rhodamine 6G (R6G), was loaded into oxHGP, and its release was monitored using UV-Vis spectroscopy. R6G-loaded oxHGP maintained their ability to form DXN when mixed with the HAADH solution. Approximately 84% of entrapped R6G was liberated from oxHGP at a rate of 0.24%/min in the first 6 h. When encapsulated in the DXN, R6G was released at a steady rate of 0.03%/min for over 3 days. These novel hydrogels are promising implant materials for vocal fold tissue regeneration.