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Chi-Chin Sun, Su Jong-Hwei Pang, Kuo-Ying Lee; Effects of chitosan membrane-cultivated keratocyte spheres on corneal stromal regeneration. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1947.
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To investigate the regenerating effects of chitosan membrane-cultivated keratocyte spheres on corneal stromal wound healing in rebbits.
Corneas of New Zealand white rabbits stripped of both endothelium and epithelium were placed in a solution of collagenase overnight at 37°C. Confluent cell layers were treated with trypsin-EDTA and seeded onto the chitosan coatings or plastic dishes. A central superficial keratectomy of the rabbit cornea was performed with a 7-mm trephine and dissecting approximately 50% of the anterior stroma. Intracorneal injection of PBS, rabbit corneal keratocytes (RCK) or chitosan membrane-cultivated keratocyte spheres were performed with 30-gauge needle immediately after wounding. Animals with corneal stromal wounding but no treatment were served as controls. External eye photos, IOP and corneal thickness measurements were performed at pre- and post-operative day 1, 3, 7, 14, 28 and 56, respectively. At time points of 2 weeks, 4 weeks and 8 weeks after wounding, rabbits were killed. The corneas were removed and subjected to different analyses including H&E stain, SEM, TEM, immunohistochemical staining, and light transmission assay.
Corneal wound reepithelialization was completed by 8 weeks. There is no significant difference in corneal re-epithelialization rate among the groups. The IOP fell immediately after wounding in all groups and gradually returned to baseline levels. The corneal thicknesses gradually returned to normal from day 14 after wounding. Less corneal scarring was noted in sphere group at day 56 post-op. The same finding was noted H&E stain, which showed a smooth collagen arrangement pattern. From SEM, discorganized collagen and scarring are profound in all groups except for the sphere group. TEM of the corneal specimens also demonstrate typical rectangular collagen lamellae arrangement. α- SMA was expressed in control group rather than in sphere group, demonstrating the potential anti-fibrosis effects of keratocyte spheres on chitosan membrane. Sphere group has the highest light transmittance percentage at all wavelength, a direct evidence supporting the anti-scarring effects of chitosan membrane-expanded keratocyte spheres on stromal wounds.
Our studies have shown that chitosan membrane-expanded corneal keratocyte spheres may be applied as a potential tissue engineering strategy in treating corneal stromal wounds.
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