In vitro evaluation of the biodegradability of chitosan–genipin hydrogels

Abstract

Biomaterials intended for in vivo applications should ideally be biodegradable to prevent their retention in the body, while avoiding the need for surgical removal. This study investigates the in vitro lysozyme degradation of chitosan–genipin hydrogels using fluorescence formed during the crosslinking reaction between chitosan and genipin, resulting from highly conjugated heterocyclic structures. Fluorescence degradation studies showed that the supernatant of degraded hydrogels significantly fluoresced, suggesting that although the hydrogel structure was broken down, chitosan–genipin crosslinks prevail. Further studies employing FTIR showed that this is not entirely the case, and that one of the bifunctional crosslinks between chitosan and genipin is broken. Results suggest that lysozyme degrades the secondary amide linkage, whilst the tertiary aromatic amine linkage remains unbroken. Seeking to evaluate feasibility and likely mechanism of removal of degraded hydrogels in vivo, degraded particle size was measured. Results show existence of particles as small as 1.7 nm, which is below the threshold for renal filtration. At the same time clusters of larger particles with a mean diameter of 218.4 μm ± 17.8 were detected and shown to likely form via agglomeration, rather than incomplete degradation. Collectively, our findings show that lysozyme partially degrades chemical crosslinks in chitosan–genipin hydrogels, and that these hydrogels have potential to be eliminated from the body via urinary excretion.