**Background:** Reactive oxygen species (ROS) are produced in excess after corneal injury and can fragment hyaluronan (HA), a key extracellular matrix component that regulates wound healing. High-molecular-weight HA (HMWHA) is associated with tissue homeostasis, while low-molecular-weight HA (LMWHA) is linked to inflammation. Although HA is known to be susceptible to oxidative fragmentation, it was unknown whether the resulting oxidized HA fragments (oxHA) have altered physiological properties compared to size-matched native HA. This study aimed to characterize oxHA generated by hydrogen peroxide (H2O2) or hypochlorous acid (HOCl) and compare their effects on corneal epithelial wound healing in vitro and in vivo.
**Methods:** HMWHA (2.67 × 10^6 Da) was incubated with H2O2 or HOCl at molar ratios of 1:50, 1:250, 1:1250, and 1:6250 for 4 h at 37°C. Oxidized products were purified by gel filtration. Molecular weight was assessed by agarose gel electrophoresis and gel filtration HPLC. Chemical structure was analyzed by 1H-NMR. Human corneal epithelial cells (hTCEpi) were treated with 0.02% (w/v) oxHA, native HMWHA, LMWHA, ULMWHA, or PBS. Viability was measured by CCK-8 assay, proliferation by BrdU incorporation, and migration by scratch wound assay over 30 h. In vivo, C57BL/6J mice received 1.5 mm corneal debridement wounds and topical eye drops (2 mg/mL HA or oxHA) every 15 min for 2 h, then at 12 and 16 h. Wound closure was assessed by fluorescein staining at 0, 12, 16, and 24 h.
**Key Results:** H2O2 at 1:250 produced fragments of 250–100 kDa; HOCl at 1:1250 produced 300–50 kDa fragments, and at 1:6250 fragments under 100 kDa. NMR confirmed that HOCl, but not H2O2, introduced chloramide groups (shift from 2.0 to 2.3 ppm). HMWHA significantly increased cell viability by ~20% vs. PBS (p<0.05), but all oxHA (except HOCl 1:6250) lost this effect. HMWHA and LMWHA increased proliferation by ~20% vs. PBS (p<0.05); oxHA from H2O2 or HOCl at 1:1250 and 1:6250 significantly inhibited proliferation (p<0.05 vs. PBS). In scratch assays, LMWHA and H2O2-generated oxHA (1:250) significantly promoted migration at 12 h (p<0.05 vs. PBS), while HOCl-generated oxHA did not. In vivo, HMWHA, MMWHA, and LMWHA significantly accelerated wound closure at 24 h vs. PBS (p<0.05). H2O2-oxHA at 1:50 showed a trend toward faster healing but not significant; H2O2-oxHA at 1:250 lost the pro-healing effect. HOCl-oxHA promoted healing but did not reach significance.
**Clinical Implications:** This study demonstrates that ROS-mediated fragmentation of HA not only reduces its molecular weight but can also introduce chemical modifications (e.g., chloramides) that alter its biological activity. In the context of corneal injury, where ROS levels are elevated, native HMWHA's beneficial effects on cell viability, proliferation, and wound healing may be compromised or reversed. Oxidized HA fragments, particularly those with chloramide groups, can inhibit proliferation, potentially delaying healing. These findings suggest that HA-based therapies for ocular surface wounds may be less effective in the presence of oxidative stress, and that strategies to limit ROS production or scavenge oxidants could preserve HA's pro-healing properties. Future research should explore how oxHA interacts with HA receptors (e.g., CD44) and whether these altered interactions underlie the observed functional changes.