PURPOSE: Donor tissue shortfalls and postsurgical complications are driving novel corneal tissue regeneration approaches. Corneal stromal keratocytes (CSKs) have shown promise in promoting corneal repair and restoring transparency. We investigated the impact of intrastromal CSK injection on corneal ultrastructure and proteoglycan (PG) distribution in a rat injury model. METHODS: Rats were divided into four groups: normal (n = 12), injured (irregular phototherapeutic keratectomy centrally
n = 6), CSK (injured + human CSK intrastromal injection
n = 6), and PBS (injured + PBS injection
n = 6). Three weeks after treatment, corneas were examined by slit-lamp and optical coherence tomography. Corneal ultrastructure was analysed via small-angle x-ray scattering (collagen fibril diameter, interfibrillar spacing and matrix order), transmission electron microscopy with cuprolinic blue before and after chondroitinase digestion (CS/DS and KS PGs), and immunofluorescence staining (lumican and decorin). RESULTS: Irregular phototherapeutic keratectomy caused corneal opacity and significantly disrupted stromal ultrastructure, characterized by increased haze density (P <
0.0001), change in central corneal thickness (P = 0.0005), and interfibrillar spacing (P <
0.0001), along with decreased fibril diameter (P <
0.0001), matrix order (P <
0.0001), CS/DS (P <
0.0001) and KS (P <
0.0001) PGs, lumican, and decorin. CSK injection recovered corneal clarity and native stromal ultrastructure, with haze density (P = 0.8086), change in central corneal thickness (P = 0.9503), fibril diameter (P = 0.1139), interfibrillar spacing (P = 0.5879), matrix order (P = 0.9999), CS/DS (P = 0.9969) and KS (P = 0.2877) PGs, lumican, and decorin returning to normal. In contrast, the PBS group exhibited similar corneal injury responses to the injured group. CONCLUSIONS: CSK injection resolved early stage corneal scarring by restoring stromal collagen arrangement and PG distribution, further endorsing its potential for treating corneal opacities.