2.2.2. Swelling Study in Phosphate Buffered Saline (PBS) with Different pH Values

The behavior of the swelling of the hydrogel samples was carried out in PBS at different pH values (pH 5, 7, 9). The desired basic and acidic pH values were obtained by pH adjustment using NaOH and HCl solutions, respectively. To measure the swelling, after autoclaving, samples of the excised hydrogel discs (diameter 3 mm) were dried at 80 ◦C for 1 h, then a dried sample with known weight was placed in 24-well plates and incubated in a solution (1:10). The swelling process took place at room temperature for up to 48 h. Swollen gels were periodically (1, 24, and 48 h) removed, blotted on dry filter paper to remove excess water and immediately weighed. Then, the mass increase (*MI*) was calculated as:

$$MI\ (\%) = ((Mt-Mo)/Mo) \times 100$$

where *Mt* is the weight of the hydrogel at a certain time, *Mo* is the initial hydrogel weight. All experiments were carried out with *n* = 6.
