IntroductionBiomedicine, tissue engineering, and pharmacology increasingly employ nanotechnology to develop innovative solutions for regenerative therapy and oxidative stress-related disorders. Among such materials, hydroxyapatite (HaP) is widely studied due to its excellent biocompatibility and osteoconductive properties, making it suitable for implantology, orthopedics, and dentistry1. HaP promotes bone cell adhesion and proliferation, which supports its use in bone grafts and implant coatings2,3,4,5. As a scaffold component, HaP is often combined with polymers to regulate pore size, cell proliferation, and tissue integration4,6. Its properties can be enhanced by the addition of titanium dioxide (TiO₂), which improves antimicrobial activity and may reduce inflammation-related oxidative stress7.Conventional biomaterials used in wound healing and tissue regeneration include natural polymers such as collagen, alginate, chitosan, hyaluronic acid, and fibrin, which mimic the extracellular matrix and support cell adhesion, migration, and matrix deposition8,9,10. Synthetic polymers like PLGA (poly(lactic-co-glycolic acid) offer customizable mechanical properties and degradation rates, while hybrid biomaterials combining natural and synthetic components improve moisture retention, infection control, and drug delivery8,11,12. However, several limitations restrict their broader use: some materials may induce prolonged inflammation, elicit toxic responses, or degrade inappropriately, while others are difficult to fabricate consistently or fail to prevent microbial infections9,11,13,14. These challenges underscore the need for novel scaffolds with enhanced biocompatibility, structural stability, and redox-regulating capacity—such as HaP-based composites.Although HaP is traditionally applied in bone-related fields, recent studies have extended its evaluation to soft tissues. Its interaction with fibroblasts—critical players in wound healing and tissue repair—is under growing scrutiny. Evidence suggests that HaP nanoparticles (HaP NPs) can induce oxidative stress by elevating reactive oxygen species (ROS) levels and decreasing antioxidant capacity in various cell types, including human blood cells and rat gastric tissues15,16,17. These findings point to the need for further investigation into the oxidative effects of HaP, especially in non-bone cellular environments7,18.Quercetin (Q), a bioactive flavonoid widely present in plant-derived foods, exhibits strong antioxidant, anti-inflammatory, and anticancer properties. It modulates ROS levels and regulates antioxidant enzymes, thereby protecting cells from oxidative damage19. Q also contributes to wound healing by enhancing fibroblast proliferation, angiogenesis, and collagen deposition, and by promoting inflammatory cell infiltration at injury sites20. Its ability to protect skin cells from UV-induced damage and reduce oxidative stress has been confirmed in several studies21, and Q-based hydrogels have shown efficacy in supporting muscle regeneration through macrophage modulation and oxidative stress attenuation22.Oxidative stress, caused by excessive ROS production, plays a key role in cellular damage and impaired tissue regeneration23,24. Studies have shown that HaP NPs can suppress the activity of key antioxidant enzymes such as glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT), while increasing lipid peroxidation and nitric oxide generation16,17. In contrast, Q has been shown to protect against oxidative stress by lowering intracellular ROS and preserving antioxidant enzyme activity25,26. Combining Q with HaP may thus enhance the antioxidant profile and biocompatibility of HaP-based materials.Fibroblast migration, proliferation, and extracellular matrix remodeling are essential for wound healing. The scratch assay is a widely used in vitro model to evaluate fibroblast migration. Both HaP and Q have been shown to influence fibroblast activity and modulate key cellular signaling pathways involved in wound repair27. HaP-containing composite membranes enhance fibroblast proliferation and migration, supporting their use in regenerative applications28,29. Likewise, Q accelerates wound closure in scratch assays and improves healing outcomes in both in vitro and in vivo models by stimulating fibroblast migration30,31.Therefore, this study aimed to evaluate the biocompatibility and redox-modulating effects of a novel composite material consisting of HaP and Q, using both in vitro (human fibroblast culture) and in ovo (Gallus gallus) models. We hypothesized that the combination of HaP and Q would exert synergistic effects, reducing intracellular oxidative stress, enhancing antioxidant enzyme activity, and stimulating fibroblast migration more effectively than either compound alone. Furthermore, we assumed that this composite would not negatively affect embryonic development, confirming its biocompatibility and potential applicability in tissue engineering. To test this hypothesis, the study assessed redox balance, antioxidant enzyme activity, ROS levels, cell viability, and wound closure capacity in vitro, as well as embryotoxicity and liver oxidative stress markers in the in ovo model.ResultsPhysicochemical analysis of hap, Q, and their complexesHaP, Q, and their complexes QHaP were characterized at concentrations of 10 ppm and 100 ppm (Fig. 1). At 10 ppm, the Z-average particle size of HaP was 1455 nm, with a low polydispersity index (PdI) of 0.153, indicating moderate size uniformity and minimal aggregation. However, at 100 ppm, the Z-average particle size increased to 1700 nm, with a higher PdI of 0.367, suggesting greater aggregation due to increased particle interactions and reduced electrostatic stabilization. Q showed distinct behavior compared to HaP. At 10 ppm, the Z-average particle size of Q was below 500 nm, with a very low PdI, reflecting excellent dispersibility and size uniformity. This stability persisted at 100 ppm, as no significant increase in particle size or aggregation was observed, highlighting the stable molecular properties of Q in solution. For the QHaP complexes, particle size and aggregation behavior were influenced by both the concentration of HaP and its interaction with Q. At 10 ppm, the Z-average particle size of QHaP complexes ranged from 1335 to 1455 nm, representing a dominant particle population (96.8–100% intensity) with minimal secondary aggregation (~ 1.7% contribution). The PdI at this concentration was low, indicating good stability of the complexes. At 100 ppm, the Z-average particle size increased to 1.584–1700 nm, with secondary aggregates (~ 5499 nm) contributing up to 3.2% of the particle intensity. The PdI also increased slightly, reflecting reduced stability at higher concentrations.Fig. 1Physicochemical analysis of Q, HaP, and QHaP (100 ppm). Visualization of materials used by transmission electron microscopy (TEM): A Q, B HaP, C QHaP. Zeta potential graph: D Q, E HaP, F QHaP. Size distribution: G Q, H HaP, I QHaP.Full size imageIn vitro experimentEnzymatic antioxidant activityThe CON group displayed the highest catalase (CAT) activity at 0.427 µmoles/min/mL, but all experimental groups demonstrated a significant decrease (P