**Background:** Neurotrophic keratopathy (NK) is a degenerative corneal disease caused by damage to trigeminal nerve innervation, leading to loss of corneal sensation, epithelial defects, and potential perforation. Current treatments are limited; topical nerve growth factor (NGF) is effective but requires high-frequency dosing (up to 6 times daily) and is expensive. This study aimed to develop a chitosan-based thermosensitive hydrogel that provides sustained release of murine NGF (CTH-mNGF) to reduce dosing frequency and improve patient convenience.
**Methods:** CTH-mNGF was prepared by mixing 3% chitosan solution with 8 mM genipin at a 1:1 ratio, then adding mNGF to a final concentration of 100 ng/mL. The hydrogel is liquid at 4°C and solidifies at 32°C. Release kinetics were assessed in vitro (supernatant of solidified gel at 37°C) and in vivo (murine cornea after topical administration) using ELISA. In the animal study, a corneal denervation (CD) model was created in C57BL/6 mice by clamping the ciliary nerve. Thirty-six mice were divided into three groups (n=12 each): CD (no treatment), CD+CTH (hydrogel without mNGF), and CD+CTH-mNGF. Treatments were given twice daily for 10 days. Outcomes included corneal perception (Cochet-Bonnet esthesiometer), Schirmer's test, epithelial wound healing (2-mm debridement), and whole-mount corneal nerve staining (anti-TUBB3 immunofluorescence). In the clinical study, 7 eyes from 6 patients with stage 2 or 3 NK (causes: LASIK, diabetes, alkali burn, corneal dystrophy, trigeminal nerve injury) were enrolled. Patients received one drop (~50 μL) of CTH-mNGF twice daily for 8 weeks. Assessments at baseline and every 2 weeks included slit-lamp examination, fluorescein staining score, corneal perception, Schirmer's test, BCVA, and IOP. In vivo confocal microscopy (IVCM) was performed at baseline and every 4 weeks.
**Key Results:** In vitro, mNGF concentration in supernatant was maintained at ~1500 pg/mL for >36 hours, with cumulative release of 41.2% at 24 hours and 73.9% at 72 hours. In vivo, murine corneal mNGF was >1300 pg/mL for >20 hours, with cumulative release of 39.1% at 24 hours and 77.8% at 72 hours. In the CD mouse model, CTH-mNGF treatment significantly improved temporal corneal perception (P<0.05 vs. CD and CD+CTH groups) and increased corneal nerve area and total nerve length (P<0.05). Schirmer's test and epithelial wound healing showed trends of improvement but were not statistically significant. In the clinical study, all 7 eyes showed decreased corneal epithelial defect area after 8 weeks; 6 of 7 eyes had a fluorescein staining score <5. Cases 2 and 3 achieved full recovery. Schirmer's test improved at 4 weeks but not at 8 weeks. Corneal perception and BCVA showed trends of improvement without statistical significance. IOP remained stable. IVCM revealed increased corneal nerve density in 3 of 7 eyes (cases 1, 2, and 6); in case 1, nerve density significantly increased at week 8 (P<0.05) and branch points increased at week 4 (P<0.05). Adverse events were minimal: orbit pain (1 day), unusual blink sensation (1 week), and corneal epithelial exfoliation (1 week, managed with bandage contact lens).
**Clinical Implications:** CTH-mNGF is a transparent, thermosensitive hydrogel that provides sustained release of mNGF, allowing twice-daily dosing instead of the 6-times daily regimen required for cenegermin. The study demonstrates that CTH-mNGF is effective in healing corneal epithelial defects in NK eyes, with a favorable safety profile. The reduced dosing frequency and use of murine NGF (which is more affordable and accessible than recombinant human NGF) may improve patient compliance and expand treatment access. However, corneal nerve regeneration was variable and slower than epithelial healing, suggesting that longer treatment or follow-up may be needed for nerve recovery. Limitations include the small sample size, heterogeneous causes of NK, and lack of a concurrent control group. Further studies with larger cohorts and longer follow-up are warranted to optimize dosing and confirm long-term benefits.