Fibroblasts isolated and expanded from ReLEx SMILE lenticules can be a source of human keratocytes. Since corneal keratocytes are quiescent cells, it is difficult to expand them in vitro in suitable numbers for clinical and experimental use. In the present study, this problem was solved by isolating and growing corneal fibroblasts (CFs) with a high proliferative potential and their reversion to keratocytes in a selective serum-free medium. Fibroblasts reversed into keratocytes (rCFs) had a dendritic morphology and ultrastructural signs of activation of protein synthesis and metabolism. The cultivation of CFs in a medium with 10% FCS and their reversion into keratocytes was not accompanied by the induction of myofibroblasts. After reversion, the cells spontaneously formed spheroids and expressed keratocan and lumican markers, but not mesenchymal ones. The rCFs had low proliferative and migratory activity, and their conditioned medium contained a low level of VEGF. CF reversion was not accompanied by a change with the levels of IGF-1, TNF-alpha, SDF-1a, and sICAM-1. In the present study, it has been demonstrated that fibroblasts from ReLEx SMILE lenticules reverse into keratocytes in serum-free KGM, maintaining the morphology and functional properties of primary keratocytes. These keratocytes have a potential for tissue engineering and cell therapy of various corneal pathologies.

Cell-based therapies using corneal stromal stem cells (CSSC), corneal keratocytes, or a combination of both suppress corneal scarring. The number of quiescent keratocytes in the cornea is small; it is difficult to expand them in vitro in quantities suitable for transplantation. This study examined the therapeutic effect of corneal fibroblasts reversed into keratocytes (rCF) in a mouse model of mechanical corneal injury. The therapeutic effect of rCF was studied in vivo (slit lamp, optical coherence tomography) and ex vivo (transmission electron microscopy and immunofluorescence staining). Injection of rCF into the injured cornea was accompanied by recovery of corneal thickness, improvement of corneal transparency, reduction of type III collagen in the stroma, absence of myofibroblasts, and the improvement in the structural organization of collagen fibers. TEM results showed that 2 months after intrastromal injection of cells, there was a decrease in the fibril density and an increase in the fibril diameter and the average distance between collagen fibrils. The fibrils were well ordered and maintained the short-range order and the number of nearest-neighbor fibrils, although the averaged distance between them increased. Our results demonstrated that the cell therapy of rCF from ReLEx SMILe lenticules promotes the recovery of transparent corneal stroma after injury.

The article presents a literature review of the modern concept of anatomical and physiological structure and functioning of the cornea. The strict morphological structure and corneal tissue homeostasis ensure its transparency. Studying the mechanisms that regulate the constancy of the corneal tissue internal environment allows us to get closer to understanding the prospects forregenerative therapy for the corneal stroma pathology. The article discusses in detail the role and functional potential of corneal stromal cells, which are capable of reverse cytologic differentiation, which primarily ensures the maintenance of tissue homeostasis and corneal transparency. The functional activity of corneal cells can change for a number of reasons, which may be exogenous, iatrogenic (trauma, infection, etc.) or endogenous. Endogenous causes include: cell autoregulation pathologies (for example, enzyme defects); defects in transport systems leading to tissue hypoxia; disorders of the neuro-humoral regulation of trophism. The physical reason forthe violation of the corneal transparency is an increase in the light scattering. The article presents five main causes of increased light scattering in the opaque cornea, and also provides an overview of the main substances – components and products of cellular synthesis of corneal stromal cells: cytokines and growth factors (complex of the signal molecule and the SDF1/CXCR4 receptor, insulin-like growth factor 1, tumor necrosis factor alpha, intercellular adhesion molecule 1, erythropoietin, neurotrophic factors, etc.). Thus, corneal opacity can be caused by a single pathogenic mechanism or be the result of a complex effect of several factors. The main processes of tissue homeostasis regulation are aimed at maintaining the unique morphological structure of the cornea.

Relevance. Around the world, corneal blindness caused by impaired transparency is one of the leading causes of vision loss. Damage to the cornea can also lead to the development of opacities. The pathogenesis of corneal opacities is based on pathomorphological changes in the corneal stroma. To explore the therapeutic potential of biomedical products and develop new treatments, a standardized and reproducible in vivomodel of corneal stromal injury is needed. In experimental ophthalmology, mouse models of various corneal injuries are widely used, but the dynamics of posttraumatic restoration of the ultrastructural organization of the stroma have been little studied. Purpose. To evaluate structural and functional changes in the corneal stroma of mice in the dynamics of post-traumatic recovery. Material and methods. Injury to the corneal stroma was performed using a sharp 33G needle in the form of a tunnel defect in the thickness of the cornea of C57BL/6 mice. The presence of post-traumatic damage and the dynamics of corneal recovery were assessed using instrumental methods (biomicroscopy of the anterior segment of the eye with a fluorescein test, optical coherence tomography (OCT) before, immediately after the injury, and after 2 and 4 weeks after injury. Transmission electron microscopy was performed using a JEM 1400 electron microscope. Morphometry of digital images was performed using Image J software, calculating the minimum nearest interfibrillar distance, diameter and fibril density. Results. The formation of a corneal stromal wound was confirmed by the development of reactive edema according to OCT data and the accumulation of fluorescein in all damaged corneas. 2 weeks after the injury, complete epithelization of the wound surface was observed. According to OCT data, 2 weeks after the injury, an increase in corneal thickness by 34% and an increase in the opacity index by 1.5 times were recorded compared to a healthy cornea. By the 4th week of observation, the corneal thickness and opacity index remained at a high level in the injured cornea. After the formation of injury to the corneal stroma, a decrease in the diameter of collagen fibrils, an increase in fibril density and the shortest nearest interfibrillar distance were revealed throughout the observation period. onclusion. A mechanical model of corneal trauma performed using a sharp 33-gauge needle induces the formation of stable stromal opacities. Instrumental and morphological research methods did not reveal stromal restoration during the entire observation period (4 weeks). Key words: corneal trauma, optical coherence tomography, corneal thickness, corneal transparency, corneal morphology, corneal fibrils