Calcium Peroxide Embedded in Dopamine-Grafted Hyaluronic Acid Hydrogel with Tunable Physicochemical Properties and Biocompatibility for Soft Tissue Regeneration

Adhesive hydrogels are pivotal biomaterials in tissue engineering. Recently, calcium peroxide (CaO₂) has gained attention for its oxygen-releasing capacity to promote healing, holding potential for diverse applications. However, the uncontrolled release of H₂O₂ from CaO₂ hydrolysis poses cytotoxicity challenges. This study developed a biocompatible adhesive hydrogel system, DaHC, comprising dopamine-grafted hyaluronic acid and a low concentration of CaO₂ (0.2 mg/mL), with horseradish peroxidase (HRP) enzyme to mitigate H₂O₂ toxicity, accelerate gelation, and enhance the biomedical performance of the adhesive hydrogel. The presence of HRP enzyme significantly accelerated the gelation time (from 2022 ± 150 s to 69 ± 32 s for the enzyme concentration of 0.8 U/mL) and improved rheological properties, with DaHC-0.4 (with 0.4 U/mL HRP) exhibiting the highest storage modulus (110.1 ± 8.4 Pa). HRP-containing hydrogels (0.02 to 0.6 U/mL) showed enhanced compressive strength and adhesion (∼300 Pa). The DaHC hydrogels exhibited controlled release of H₂O₂ and sustained release of Ca²⁺ ions over 7 days. All DaHC samples demonstrated hemocompatibility (hemolysis rate <5%) and significantly accelerated blood clotting time (<500 s). Cytotoxicity tests confirmed non-cytotoxicity (>95% cell viability) via extracts and improved cell viability with HRP inclusion in direct contact. In vivo experimental models underscore the DaHC hydrogel's potential with short-term biocompatibility (subcutaneous implantation) and hemostasis properties (reducing blood loss in a liver puncture model to approximately 30-40 mg). These findings suggest that the introduced material may serve as a promising candidate for soft tissue, surgical hemostats, and wound dressings.