**Background:** The cornea requires unique healing conditions to maintain transparency and avoid fibrosis (hazing) that can compromise vision. Calreticulin (CALR), an ER chaperone with extracellular roles, has previously shown tissue regenerative properties in skin wounds without scarring. This study investigates whether topical CALR can accelerate corneal wound closure and mitigate fibrosis in a rabbit partial keratectomy model, and explores its effects on corneal epithelial cells and keratocytes in vitro.
**Methods:** Two experiments were conducted using New Zealand White rabbits (n=5/group). In Experiment 1, CALR at 2.5, 0.25, and 0.025 mg/mL (87.5 μg, 8.75 μg, 0.875 μg per application) was applied twice daily for 14 days, compared to TBS vehicle and 0.1% dexamethasone (Dex). In Experiment 2, CALR at 0.0125 and 0.025 mg/mL (438 ng, 875 ng) was applied once daily for 4 days alone or with Dex (twice daily for 7 days), compared to PBS and Dex alone. Wound closure was assessed by fluorescein staining and ImageJ analysis. Ocular examination (OE) scores, optical coherence tomography (OCT) for corneal thickness and fibrosis, and histopathology (H&E, Masson's trichrome) were performed. Immunohistochemistry for α-SMA and vimentin was quantified. In vitro, telomerase-immortalized human corneal epithelial (CE) cells and primary human keratocytes were treated with CALR (0.01-100 ng/mL) to assess proliferation (CCK-8), migration (scratch assay), and protein expression (immunoblotting for TGF-β2, vimentin, α-SMA).
**Key Results:** In Experiment 1, all CALR doses achieved full wound closure by 96 hours, compared to 168 hours for TBS and 240 hours for Dex. Average closure times: CALR 2.5 mg/mL = 91.2 h, 0.25 mg/mL = 91.2 h, 0.025 mg/mL = 91.2 h, TBS = 141.6 h (p≤0.01), Dex = 174 h (p≤0.01). CALR accelerated healing 1.6-fold vs TBS and 2-fold vs Dex. In Experiment 2, 0.025 mg/mL CALR healed at 107 h (4.5 days) vs PBS at 123 h (5.1 days) and Dex alone at 190 h (7.9 days). CALR 0.025 mg/mL plus Dex healed at 152 h (6.3 days), 1.6 days faster than Dex alone. OE scores for lower CALR doses were similar to vehicle; only 2.5 mg/mL CALR showed elevated scores. OCT fibrosis scores: 2.5 mg/mL CALR = 3.6 (Day 17), 0.25 mg/mL = 2.7, 0.025 mg/mL = 2.5, TBS = 2.2 (p≤0.01 for 2.5 mg/mL vs TBS). Histology showed high fibrosis (score 3) in 4/5 rabbits with 2.5 mg/mL CALR, but mild fibrosis (score 0-1) in 0.025 mg/mL CALR plus Dex group, better than Dex alone. In vitro, CALR stimulated CE cell proliferation 1.5-fold at 0.05 ng/mL (p≤0.01) and keratocyte proliferation 1.4-fold at 10 ng/mL (p≤0.05). Migration: CE cells showed 72% wound closure at 10 ng/mL CALR vs 44% control (p≤0.01); keratocytes showed 83.6% closure at 1 ng/mL vs 59.4% control (p≤0.05). Immunoblotting revealed that CALR (10 ng/mL) decreased TGF-β2 by 50% (p≤0.001) and vimentin by 75% (p≤0.0001) in CE cells, and dose-dependently reduced α-SMA in keratocytes. Immunohistochemistry confirmed reduced α-SMA staining in 0.025 mg/mL CALR plus Dex corneas compared to Dex alone.
**Clinical Implications:** Topical CALR accelerates corneal wound closure and reduces fibrosis at low doses, potentially offering a therapeutic option for corneal injuries, including those from refractive surgery (PRK, LASIK), diabetic keratopathy, and infectious keratitis. CALR may counteract the delayed healing associated with corticosteroid use, as shown by its combination with dexamethasone. The anti-fibrotic effects, mediated by decreased TGF-β2 and α-SMA, suggest CALR could prevent corneal hazing. Further studies are needed to optimize dosing and explore delivery via nanofibers for sustained release. CALR's antimicrobial and anti-angiogenic properties may broaden its ophthalmic applications, including for macular degeneration.