Abstract: BACKGROUND: Currently, the crosslinking process of gels can be characterized by the degree of crosslinking measured by nuclear magnetic resonance and other methods, or by the change of viscoelastic behavior monitored by the rheometer during the crosslinking solidification process. However, these methods are complicated to operate and will destroy the sample, so it is urgent to establish a simple method to monitor the crosslinking reaction process without special sample preparation and without sample damage. 
OBJECTIVE: To establish a method for characterizing the crosslinking reaction process of sodium hyaluronate. 
METHODS: 1,4-Butanediol diglycidyl ether was used as the cross-linking agent, and four factors were selected to prepare sodium hyaluronate gels, including different cross-linking degrees (1%, 2%, 3%, and 4%), different molecular weights of sodium hyaluronate (400, 700, 1 500, and 3 000 kD), different concentrations of sodium hydroxide solution (1%, 2%, and 3%), and different reaction temperatures (4, 25, 37, and 50 ℃). The compression stress of the crosslinked gel was measured by an electronic universal tensile testing machine when the cross-linked gel was pressed down and deformed for 1 mm. The influence of crosslinking parameters on the reaction process and adequacy sufficiency of the crosslinking reaction were characterized according to the compressive stress. 
RESULTS AND CONCLUSION: (1) With the extension of reaction time, the hardness of gel increased, and the compressive stress gradually increased. After reaching a certain value, the cross-linked macromolecular structure of sodium hyaluronate degraded under the action of strong alkali, and the gel hardness reduced while the compressive stress decreased. Therefore, the hardness of the gel could be characterized according to the compressive stress. The process of crosslinking reaction was characterized by the curve drawn by compressive stress and crosslinking time. According to compressive stress, the crosslinking reaction temperatures had significantly influenced the crosslinking reaction time and the gel hardness, whereas other factors such as crosslinking degree, alkali concentration, and molecular weight had negligible effects on the reaction time but exerted a substantial impact on the gel hardness. (2) According to CORREL, with the change of crosslinking time, the change trend of compressive stress was consistent with that of elastic modulus and dynamic viscosity, which could be used to characterize the crosslinking process of sodium hyaluronate gel. (3) Based on the compression force of cross-linked gel, not only the process of crosslinking reaction can be characterized, but also the gel hardness can be preliminarily determined. This method can be used for real-time and in-situ detection and has the advantages of simple operation and no damage to the sample, providing a solution and new ideas for monitoring the crosslinking reaction process of sodium hyaluronate or other substances.

Key words: sodium hyaluronate, compression stress, crosslinking reaction, tensile machine, crosslinked gel, process characterization, technologic parameter, in-situ detection