The influence of amniotic membrane proteins on corneal regeneration when delivered directly or using hydrogel platforms

Ayla Basasoro ^1,2 Javier Mendicute ^1,2 Marta Rezola ² Jorge Burgos ³ Mercedes Fernández ⁴ David Esporrín-Ubieto ⁵ Ana Sonzogni ⁵ Marcelo Calderon ^5,6 Itxaso Calafel ⁴ Aitor Díaz ⁷ Juliana De Souza ⁷ Ana Aiastui ¹ Mikel Azkargorta ⁸ Felix Elortza ⁸ Elena Vecino ⁹ Arantxa Acera ^1,6,9*

• ¹ Biogipuzkoa Health Research Institute, DONOSTIA-SAN SEBASTIÁN, Spain
• ² Donostia University Hospital, San Sebastian, Spain
• ³ Cruces University Hospital, Barakaldo, Spain
• ⁴ Department of Polymers and advanced materials: Physics, chemistry and technology, Faculty of Chemistry, University of the Basque Country, Donostia-San Sebastián, Spain
• ⁵ Department of Applied Chemistry, Faculty of Chemistry, University of the Basque Country, Donostia, Spain
• ⁶ IKERBASQUE Basque Foundation for Science, Bilbao, Basque Country, Spain
• ⁷ CIDETEC, Basque Research and Technology Alliance (BRTA), DONOSTIA-SAN SEBASTIÁN, Spain
• ⁸ Proteomics Platform, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), CIBERehd, Derio, Spain
• ⁹ Department of Cell Biology and Histology, University of the Basque Country, Bilbao, Basque Country, Spain

Background/aims: Chemical burns to the eye cause extensive ocular surface damage, often resulting in permanent visual impairment. The amniotic membrane (AM), with its regenerative properties, has shown great potential in treating such injuries. Recent innovations, including hydrogel-based delivery systems, have been developed to improve drug retention and promote corneal healing. This study aimed to evaluate whether hydrogels loaded with proteins from human AM could enhance corneal wound healing effectively Methods: Alkaline burns (8 mm diameter) were induced in the cornea of anesthetized male New Zealand White rabbits (n=44) by placing filter paper soaked in 1 M NaOH on them for 60 s. After rinsed the corneas immediately with a balanced salt solution, the wounds then received: 1) no treatment; 2) AM transplantation; or 3) a dynamic hyaluronic acid hydrogel based on gold thiolate loaded with an AM protein extract (AME); or 4) a physically cross-linked ocular hydrogel insert loaded with the same AME. The contralateral uninjured eye served as a control. The wound area and proportion of healed corneas was assessed in microphotographs. In addition, corneal histology was evaluated by haematoxylin-eosin and Masson's trichrome staining, examining epithelial and stromal thickness, the endothelial layer, and inflammatory infiltration in the early (day 2) and late (day 28) phases of healing.Results: There was more frequent corneal wound closure in animals treated with the hydrogels (treatments 3 and 4) on day 14 (44.4% and 55.5%, respectively) than in the untreated controls (33.3%). Histologically, abnormal re-epithelialization and altered epithelial junctions were observed, with no significant differences in epithelial thickness. Endothelial damage correlated with significant thinning (p=0.001), with treatments 2 and 3 producing significant differences in the inflammatory infiltrate (p=0.01).Applying new biocompatible hydrogels to the ocular surface that release AM proteins may help close corneal wounds caused by caustic burns. The aggressive nature of burns hinders the detection of differences in the wound area between treatments 28 days after injury. However, improving the adhesion of a solid hydrogel to the mucosa of the ocular surface could enhance these outcomes by keeping the implant in contact with the wound for a longer period.