D gluconic acid δ lactone gdl

Sodium alginate (alginic acid sodium salt; Sigma-Aldrich, USA) in salt form, calcium carbonate (CaCO₃, Sigma-Aldrich, USA), ammonium fluoride (NH₄F, Sigma-Aldrich, USA), D-( +)-gluconic acid δ-lactone (GDL; Sigma-Aldrich, USA), and PDRN (PDRN; Genoss, South Korea) were prepared to fabricate the Alg-PDRN hydrogel according to literature^{51 (link)}. Briefly, mixture of 2–5 w/v% Alg with a calculated amount of CaCO₃ was prepared in distilled water (DW) under continuous stirring with a fixed Alg to calcium carbonate ratio of 1 w/v%:12.5 mM, as shown in Table 1. The amount of GDL was set to achieve a CaCO₃ to GDL molar ratio of 0.5 to attain a neutral pH^{51 (link)}. The samples were named according to the Alg concentration as 2%Alg-PDRN, 3%Alg-PDRN, 4%Alg-PDRN, and 5%Alg-PDRN; PDRN was loaded in each sample at a concentration of 100 μg/ml. CaCO₃ particles were homogeneously dispersed in the composite solution, and as soon as GDL was added, CaCO₃ started to dissolve and release Ca²⁺ ions to induce the crosslinking of the hydrogel. The gelling solution was continuously stirred for different times depending on the composition of the composite solution; then, each composite solution was pipetted into a 12-mm-diameter cylindrical mold. The gels were left at room temperature for 72 h to allow for complete gelation.

Conventional CMHs were prepared by adding 0.1 wt.% CaCO₃ nanoparticles (NPs, 10–80 nm, American Elements^®) and 28.14 mM solution of D-Gluconic acid δ-lactone (GDL, Sigma-Aldrich) to an aqueous SA solution^{60 (link)}. Upon dissolution in water and addition to the alginate mixture, GDL slowly hydrolyzes, reducing the pH and gradually ionizing the CaCO₃ NPs to release Ca²⁺ crosslinker ions^{61 (link)}. This is preferred over the addition of instantly soluble Ca salt (like CaCl₂) since it allows more homogeneous SA gel formation.