The cornea is a transparent, avascular structure that forms the outermost layer of the eye and plays a key role in both protecting the intraocular contents and refracting light to enable vision(Wang et al., 2023; Zhang et al., 2022). Corneal ulcers, which frequently occur in small animals due to trauma, infection, or chemical injury, can rapidly progress and lead to vision loss if not treated effectively. Uncomplicated ulcers typically resolve within a week using topical antibiotics and cycloplegics. However, deeper or complicated ulcers—especially those involving stromal degradation due to endogenous or exogenous matrix metalloproteinase activity—often require more aggressive treatment. This includes targeted antibacterial therapy, matrix metalloproteinase inhibitors, and in severe cases, surgical intervention to prevent corneal perforation and preserve visual function (Feghhi et al., 2015) (Chow and Westermeyer, 2016) (Maggs et al., 2018).

In dogs and cats, corneal disease is commonly associated with trauma, chronic inflammation, or degenerative processes, often resulting in opacification, vascularization, and impaired epithelial integrity. Given the essential role of the limbal region in corneal epithelial regeneration, various therapeutic strategies have been developed to manage corneal defects. These include topical medical treatments, surgical procedures such as keratectomy, third eyelid or conjunctival flaps, corneal grafts, and the use of biologic materials like porcine intestinal submucosa or amniotic membrane (Gellat et al., 2022).

A conjunctival pedicle graft is one of the most commonly employed surgical techniques for managing large corneal defects in veterinary ophthalmology. These grafts deliver fibroblasts, blood vessels, epithelial cells, and antimicrobial agents directly to the site of injury, thereby promoting tissue regeneration. However, their opaque nature can impair vision, and they offer minimal structural support, which limits their utility for central or deep corneal wounds. To address these drawbacks, a variety of alternative biological and synthetic materials have been investigated, including cyanoacrylate adhesives, conjunctival island patches, tarsoconjunctival grafts, corneoscleral transpositions, dermal grafts, and biologic scaffolds such as equine pericardium, bovine pericardium, porcine intestinal submucosa, and amniotic membranes (Dulaurent et al., 2014; Gellat et al., 2022; Arcelli et al., 2009; Bussieres et al., 2004; Vanore et al., 2007). Despite their potential, many of these options are expensive, require specialized surgical tools, or are difficult to obtain, especially in veterinary settings(Chow and Westermeyer, 2016).

In recent years, the use of amniotic membrane (AM) has gained attention due to its unique biological properties. AM is the innermost layer of the placenta, rich in growth factors, anti-inflammatory proteins, and basement membrane components that facilitate epithelial migration and reduce stromal inflammation (Gheorghe et al., 2016). It has been applied successfully in managing corneal wounds in dogs, horses, and experimental models, functioning either as a protective barrier or a scaffold for epithelial regeneration. (Barros et al., 1998; Capistrano da Silva et al., 2021). Equine AM is commonly used due to its thicker structure and ease of handling; however, bovine AM offers a promising alternative with favorable biomechanical and biological characteristics, and has been reported to enhance corneal healing in both clinical and experimental settings (Dua et al., 2004) (Mcdaniel et al., 2021). (Gellat et al., 2022; Kang et al., 2006; Kim et al., 2009; Zhou et al., 2019).

The limbal region, situated at the junction of the cornea and sclera, harbors a population of stem cells essential for corneal epithelial regeneration, particularly in conditions such as limbal stem cell deficiency (LSCD) (Tseng et al., 1998). Amniotic membrane has been widely used as a scaffold for cultivating limbal epithelial cells (CLET method), and transplantation techniques such as simple limbal epithelial transplantation (SLET) have demonstrated clinical success by placing small limbal tissue onto AM, which is then applied to the damaged corneal surface (Sangwan et al., 2012; Ho et al., 2013; Stuart, 2016; Khorolskaya et al., 2023). However, such approaches often demand advanced surgical expertise, specialized instruments, and access to cell culture facilities, which may limit their use in routine veterinary practice. Furthermore, anatomical features in animals—such as the presence of a third eyelid—can present additional challenges for these procedures.

Consequently, there is a need for a simple, cost-effective, and practical method that can promote epithelial healing, improve tissue quality, and simultaneously serve as a mechanical barrier to protect the wounded cornea. In this study, we investigated the therapeutic potential of directly applying crushed limbal tissue fragments to corneal alkaline wounds in rabbits, in combination with bovine amniotic membrane or a conjunctival flap as physical bandages. We aimed to evaluate the wound healing response in terms of re-epithelialization, inflammation, and neovascularization, and to compare the biological effects of the two bandaging techniques.