1D NMR methods for determination of degree of cross-linking and BDDE substitution positions in HA hydrogels

Hyaluronic acid polymers cross-linked with BDDE are today among the most used hydrogels for biomedical applications. The physical properties of the hydrogels depend, among other parameters, on the degree of cross-linking of HA. Another parameter likely to affect the physical properties is the substitution position of the linker on the HA functional groups. A NMR-based method for the determination of these parameters in hyaluronic acid hydrogels is presented. The method is based on the degradation of HA cross linked hydrogels by chondroitinase ABC followed by one-dimensional 1H and 13C NMR analysis. The necessary structural information to obtain both the degree of cross-linking and the substitution positions can be obtained from the same NMR sample and no chromatographic separation step is required prior to NMR analysis.