2.3. Degree of Crosslinking

The most common chemical HA crosslinking method uses ether formation by reaction of HA with 1,4-butanediol diglycidyl ether (BDDE) [24]. Under alkaline conditions, epoxides of BDDE react with the HA hydroxyl groups to form derivatives of 1,4-dibutanediol dipropan-2,3-diolyl ether (BDPE). Some of the BDPE form “true cross-links”, binding to HA at both ends, while others only bind HA at one end (“mono-linked”). To describe these HA filler modifications, Kenne and Wende and colleagues proposed terms that can be applied to characterize HA hydrogels crosslinked with BDDE [25,26]:

The degree of modification (MoD) is the stoichiometric ratio between the sum of mono- and double-linked BDPE residues and HA disaccharide units. The more crosslink modifications seen when compared with the acetyl group, the higher the MoD%.

The crosslinker ratio (CrR) indicates the fraction of double-linked crosslinker residues compared to all linked crosslinkers and this represents the measure of crosslinker efficiency.

The degree of substitution (DS) is the proportion of the HA disaccharides that are substituted.

The degree of crosslinking (CrD) is the stoichiometric ratio between BDPE residues that are double-linked and HA disaccharide units.

The degree of crosslinking (DC) is the number of HA disaccharides involved in crosslinking in relation to the total number of HA disaccharides.

Kenne states that MoD and CrD quantify the total amount of linked BDPE and double-linked BDPE, respectively, in comparison to the total amount of HA. DS and DC are equivalent to the amount of the substituted HA and crosslinked HA, respectively, in comparison to the total amount of HA [26]. The relevance of MoD and DS is in describing the total change in the gel after modification. For example, a polymer with low MoD or DS is structurally similar with the original, intact gel. CrD and DC are more important in terms of describing the physical properties of the gel. A higher value of CrD for a gel reveals that it is a stronger gel (i.e., more crosslinked) and would swell less than a weaker gel with a lower CrD.

In this study, the determination of the degree of crosslinking of the nominated HA-based fillers was performed using the nuclear magnetic resonance (NMR)-based approach described by Wende and colleagues [25]. HA is hydrolyzed using an acidic medium, and then, the degradation products are analyzed using one-dimensional NMR. First, the samples were diluted to 4 mg/mL with acid water (HCl 0.1 M, pH 1.5), heated and gently mixed at 75 °C overnight. Once cooled, these samples were buffered at pH 7.0 with NaOH 1 M and 0.1 M, frozen and then lyophilized. After, the samples were dissolved in 0.5 mL of deuterated water (D₂O) and then subjected to NMR Bruker 400 mHz analysis for the determination of MoD%, CrR and CrD. With the ¹H-NMR set up, 64 scans and a recycle delay (D1) of 10 s were used. For the ¹³C-NMR set up, 8192 scans and a D1 of 10 s were used.

The three main parameters were then measured using the following formulae: (1) MoD (%) = (I ^δH1.5/4)/(I ^δH1.9/3) × 100, (2) CrR = 1-I^δC62.7/(^Iδ25.2/2) and (3) CrD (%) = (CrR × MoD) × 100.