Effect of Carboxymethylation on the Rheological Properties of Hyaluronan

<div><p>Chemical modifications made to hyaluronan to enable covalent crosslinking to form a hydrogel or to attach other molecules may alter the physical properties as well, which have physiological importance. Here we created carboxymethyl hyaluronan (CMHA) with varied degree of modification and investigated the effect on the viscosity of CMHA solutions. Viscosity decreased initially as modification increased, with a minimum viscosity for about 30–40% modification. This was followed by an increase in viscosity around 45–50% modification. The pH of the solution had a variable effect on viscosity, depending on the degree of carboxymethyl modification and buffer. The presence of phosphates in the buffer led to decreased viscosity. We also compared large-scale production lots of CMHA to lab-scale and found that large-scale required extended reaction times to achieve the same degree of modification. Finally, thiolated CMHA was disulfide crosslinked to create hydrogels with increased viscosity and shear-thinning aspects compared to CMHA solutions.</p></div

Effect of CMHA synthesis parameters on degree of modification.

<p>Effect of CMHA synthesis parameters on degree of modification.</p

Effect of thiolation and disulfide crosslinking on viscosity of CMHA.

<p>Steady-shear viscosity as a function of shear rate for solutions of CMHA (25 mg/ml) in PBS or the corresponding thiolated and crosslinked CMHA gels (xCMHA-S). Degree of thiolation and CMHA-S concentration in the gel for each gel type is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162849#pone.0162849.t005" target="_blank">Table 5</a>.</p

Effect of pH on steady-shear viscosity of CMHA solutions.

<p><b>A:</b> Steady-shear viscosity at a shear rate of 25 s^-1 for solutions of mock CMHA and CMHA (25 mg/ml) in 0.9% NaCl (saline). Mock CMHA has 0% CM modification; CMHA has 33% CM modification. <b>B:</b> Steady-shear viscosity at a shear rate of 25 s^-1 for solutions of CMHA (25 mg/ml) in 0.9% NaCl (saline) or DI water. CMHA has 25, 33, or 74% CM modification as indicated in the legend by the number following CMHA.</p

Effect of biopolymer concentration on viscosity.

<p>Steady-shear viscosity is shown as a function of shear rate for solutions of HA (10 or 25 mg/ml), mock CMHA (10 or 25 mg/ml), and CMHA (25, 30, 35, or 40 mg/ml) in PBS. Mock CMHA has 0% CM modification; CMHA has 33% CM modification.</p

General structure of hyaluronan.

<p>Modifications at the R group are indicated to generate CMHA and CMHA-S.</p

Effect of %CM modification on steady-shear viscosity.

<p>Viscosity was determined at a shear rate of 25 s^-1 for solutions of CMHA (25 mg/ml) in PBS. Various lots of CMHA are grouped by molecular weight: <280, 280–300, 300–310, 315–325, and 330–350 kDa.</p

Variability in large-scale production of CMHA.

<p>Variability in large-scale production of CMHA.</p