Infrared Spectroscopic Quantification of Methacrylation of Hyaluronic Acid: A Scaffold for Tissue Engineering Applications.

Methacrylated hyaluronic acid (MeHA) has been used extensively in tissue engineering and drug delivery applications. The degree of methacrylation (DM) of HA impacts hydrogel crosslinking, which is of pivotal importance for cell interactions. The methacrylation reaction occurs over several hours, and DM is currently assessed post reaction and after dialysis of the solution, using nuclear magnetic resonance (1 H NMR) data. Thus, there is little control over exact DM in a specific reaction. Here, infrared (IR) spectroscopy in attenuated total reflection (ATR) mode was investigated as an alternate modality for assessment of the DM of HA hydrogels, including during the reaction progression. Attenuated total reflection is a low-cost technique that is widely available in research and industry labs that can be used online during the reaction process. Strong correlations were achieved with IR-derived peak heights from dialyzed and lyophilized samples at 1708 cm-1 (from the methacrylic ester carbonyl vibration), and 1 H NMR values ( R = 0.92, P = 6.56E-11). Additional IR peaks of importance were identified using principal component analysis and resulted in significant correlations with the 1 H NMR DM parameter: 1454 cm-1 ( R = 0.85, P = 2.81E-8), 1300 cm-1 ( R = 0.95, P = 4.50E-14), 950 ( R = 0.85, P = 3.55E-8), 856 cm-1 ( R = 0.94, P = 1.20E-12), and 809 cm-1 ( R = 0.93, P = 3.54E-12). A multiple linear regression model to predict 1 H NMR-derived DM using the 1708, 1300, and 1200 cm-1 peak heights as independent variables resulted in prediction with an error of 3.2% using dialyzed and lyophilized samples ( P < 0.001). Additionally, a multilinear regression model to predict the DM in undialyzed liquid MeHA samples obtained during the reaction process using similar peak height positions as independent variables resulted in a prediction error of 0.81% ( P < 0.05). Thus, IR spectroscopy can be utilized as an alternate modality to 1 H NMR for quantification of the DM of MeHA while sampling either on-line during the methacrylation reaction as well as in post-lyophilized products. This could greatly simplify workflow for tissue engineering and other applications.