Corneal bioengineering via electrospun nanofibers and nanoparticles

Hierarchical organization of corneal layers

The cornea is located at the front of the eye and surrounds the iris, pupil, and front chamber. The opaque white sclera marks the edges of this chamber (Figure 1). This clear, avascular tissue lets light through the pupil and into the lens. It also protects against mechanical damage and the entry of germs.^28,29 The cornea is the eye’s strongest lens, with a refractive power of about +43 diopters. It also helps light reach to the eye’s internal structures. The average horizontal diameter is between 11 and 12 mm, and the average vertical diameter is between 9 and 11 mm. The thickness is about 0.5 mm, and it becomes thicker as it grows closer to the edges. ³⁰

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Figure 1.

Schematic representation of the human corneal layered architecture. The illustration delineates the five distinct histological layers of the cornea: the outermost epithelium, Bowman’s layer, the substantial stroma, Descemet’s membrane, and the innermost endothelium.

The cornea is a thin but complex tissue made up of many layers that serves optical, protective, and mechanical purposes. It is typically divided into five parts: the corneal epithelium, Bowman’s layer, stroma, Descemet’s membrane, and endothelium. ²⁹ Each region has its cells and extracellular matrix, as well as its own physiological functions. Together, these things ensure that the cornea stays clear and works together.

The corneal epithelium is made up of non-keratinized, stratified squamous cells that act as the first line of defense against outside threats. The limbal region mainly feeds these cells. It has epithelial stem cells that help the corneal surface stay fresh. Basal cells proliferate and differentiate into wing cells, which then migrate to the ocular surface and further differentiate into superficial flat cells, enabling epithelial renewal approximately every 7 days. ³¹ The XYZ hypothesis describes this process: X represents the proliferation and stratification of basal limbal cells, Y indicates their centripetal migration, and Z pertains to the desquamation of superficial epithelial cells. ³² Tight junctions between epithelial cells and their connection to the basement membrane make a strong barrier against chemicals, microorganisms, and water. This keeps the epithelium clear and intact. Molecular signaling between the epithelium and stroma, facilitated by growth factors and paracrine molecules, is essential for maintaining structural and functional stability, in addition to its protective roles. The epithelial surface is covered by a tear film. It keeps the surface from drying out, helps move oxygen and nutrients, and protects the body from disease. The delicate interaction among stem cells, surface cells, basement membrane, and tear film facilitates swift tissue regeneration, protective mechanisms, and biochemical homeostasis, establishing a basis for corneal tissue engineering.^33,34

Bowman’s layer is an acellular structure that lies beneath the epithelium. It is made up of a dense network of collagen fibrils and extracellular matrix (ECM). Proteoglycans and collagen types I, III, V, and VII make up the matrix. These are randomly spread out in a framework that is not well-defined. Bowman’s layer connects the epithelium to the stroma, which is a mechanical function. By making it easier for stromal collagen fibrils and keratocytes to interact, this process improves the shape and strength of the corneal anterior. Unmyelinated nerve axons go through the structure, which helps separate the epithelium from the stroma. ³⁵ The intervention speeds up the healing of the stroma after photorefractive keratectomy and helps the epithelium grow back, which is important for corneal regeneration. Structural changes in Bowman’s layer, seen in keratoconus, are linked to less clear corneas. This phenomenon shows how important it is to accurately copy its physical and chemical properties in engineered corneal scaffolds. Bowman’s layer becomes thinner as you grow older, probably because of natural processes that break down or cross-link collagen. There is no regeneration after an injury, and any replacement happens slowly, which often leads to a structure that is different from the original. Bowman’s layer is a mechanical barrier and structural integrator that is crucial for keeping the cornea clear and working.^36,37

The stroma, which is the thickest layer of the cornea, makes up more than 90% of the cornea’s weight. It is made up of type I collagen fibers that are arranged in parallel layers. For optical transparency, the collagen fibrils need to be arranged in a certain way, and proteoglycans like lumican, keratocan, and decorin need to be present. Because the cornea has a hierarchical structure that goes from the nanoscale to the macroscale, it can stay clear. This structure also provides the cornea the mechanical strength it needs to stay healthy. The replication of this highly organized architecture in synthetic scaffolds remains a substantial challenge in corneal tissue engineering.^38,39

At the posterior region of the cornea lie Descemet’s membrane and the endothelium. Descemet’s membrane is a special basement membrane for endothelial cells. These cells control the hydration of the stroma by using ionic pumps, which keeps the cornea clear. ⁴⁰ Since endothelial cells in the adult human cornea have very limited proliferative capacity, any damage to this region can lead to corneal edema and visual impairment. Consequently, restoring endothelial pump function is a critical objective in advanced corneal tissue engineering research. ⁴¹

The cornea is a multilayered structure with a complex and organized architecture. This is because the cellular parts, structural proteins, and extracellular matrix all work together in a coordinated way. This integrated system makes sure that the cornea is clear, strong, and safe, which shows how complicated it is to make copies of it for regenerative purposes.

Key cellular components and their roles

The cornea can heal and work properly because different types of specialized cells are spread out across its layers and work together in a precise way. These cells not only keep the cornea clear and strong, but they also help the body heal after an injury and keep the immune system in balance. To make tissue-engineered scaffolds and identify new ways to treat corneal damage, you need to know where each type of cell comes from, what it does, and what its properties are.^42,43 Studying epithelial, stromal, endothelial, stem, and immune cells and how they work together presents us important information about how regeneration works and how to improve engineered corneal models.

Corneal epithelial cells, which come from limbal stem cells, make up the outermost layer of the cornea. These cells are the first line of defense against damage from the environment, microbes, and physical forces. They also control how cells grow and move, which lets the epithelium keep renewing itself. They release growth factors like EGF, TGF-β, and NGF, which work with stromal cells and the basement membrane to help repair tissue while also supporting the stromal keratocytes and the corneal nerve network.^44,45

Stromal keratocytes are the main type of cell in the stroma, and they come from mesenchymal tissue. Keratocytes, in normal circumstances, remain inactive and help the cornea stay clear and strong by making type I and V collagen and proteoglycans like decorin and lumican. When keratocytes are hurt or inflamed, they become active and change into fibroblastic or myofibroblastic phenotypes. These cells help change the extracellular matrix. However, prolonged activation may lead to scarring or stromal opacity. ⁴⁶

During embryonic development, endothelial cells come from the inner ocular layer and are found on Descemet’s membrane. These cells control the hydration of the stroma through Na⁺/K⁺-ATPase pumps and aquaporin channels, which are necessary to keep the cornea clear. Restoration of endothelial function often requires bioactive scaffolds or cell-based therapies because endothelial cells don’t divide very well. ⁴⁵

Limbal stem cells, located at the corneoscleral junction, are the primary source for epithelial regeneration. They come from mesenchymal and embryonic sources and can turn into mature epithelial cells. When these cells are harmed, the epithelium doesn’t work as well, and the cornea isn’t as clear. To keep their regenerative phenotype and function, they need artificial microenvironments and bioactive biomaterials like hydrogels and amniotic membrane. ⁴⁷

Resident immune cells, including dendritic cells, macrophages, and memory T lymphocytes, originate from the bone marrow and mesenchyme. Under physiological conditions, they maintain immune homeostasis and, upon injury or infection, activate inflammatory pathways. They play essential roles in pathogen clearance, tissue repair, and immune regulation. Tissue-engineered scaffolds must be designed to support controlled immune responses.^48,49

Corneal nerves and Schwann cells, which come from the trigeminal nerve, work closely with the epithelium and keratocytes. Neuropeptides such as Substance P and CGRP are released to encourage the regeneration of epithelial tissue, while the epithelium produces neurotrophic factors that support the survival of neurons. The reestablishment of neural networks within engineered scaffolds is a pivotal marker of functional corneal regeneration. ⁵⁰

The endothelial cells of limbal blood vessels, derived from embryonic endothelium and mesenchymal cells, perform nutritive and regulatory functions within the corneal microenvironment. In pathological conditions, they can promote neovascularization, potentially impairing corneal clarity. When making scaffolds to stop unwanted vascularization, two important things to do are to control VEGF expression and raise the levels of anti-angiogenic factors.

Corneal stromal stem cells (CSSCs), located in the anterior stroma and encircling the limbus, derive from mesenchymal tissue. These cells can become keratocytes or epithelial cells. Research on animals has shown that they can repair corneas without leaving scars. Corneal stromal stem cells constitute a viable cellular source for corneal tissue engineering. ⁵¹

In short, the coordinated work of all these cell types makes a dynamic network that keeps the cornea clear, helps it heal, and keeps the immune system in check. One of the main goals of corneal tissue engineering is still to recreate these cellular interactions in artificial settings.

The corneal extracellular matrix as a blueprint

The corneal extracellular matrix (ECM) is a complex and well-organized network of fibrillar and non-fibrillar molecules that are needed to keep the cornea’s shape, transparency, and mechanical stability. This system serves as a basis for designing scaffolds for tissue engineering. Most of the stromal extracellular matrix is made up of type I and V collagens. The type I and V collagens are arranged in parallel, highly organized lamellae. This setup provides strength and flexibility to the structure while letting in as much light as possible and cutting down on scattering. Type VI collagen surrounds keratocytes, which helps cells talk to each other and keeps the tissue’s structure.^52–55

Decorin, lumican, keratocan, and mimecan are all types of stromal proteoglycans that are important for controlling the stroma’s thickness and hydration. This is because they can absorb water and interact with collagen fibrils. These molecules serve not only a structural function but also participate in cell signaling through interactions with integrin receptors and growth factors. This process changes how keratocytes and stromal stem cells grow and work.^54,56

The epithelial basement membrane, which is made up of laminin, nidogen, perlecan, and fibronectin, holds the epithelium to the stroma and helps it heal after an injury. This membrane sends biochemical signals and growth factors to epithelial cells, which helps them stay alive and grow. ⁵⁷

Type VIII collagen and endothelial-specific proteoglycans in Descemet’s membrane are crucial for keeping the endothelial layer stable and the stroma hydrated. This unique ECM composition allows endothelial ionic pumps to work well in normal conditions, which keeps the cornea clear. ⁵⁸

The ECM is made up of structural elements, growth factors, cytokines, and signaling molecules that work with stem cells and keratocytes to start the processes of regeneration and repair. Pathological changes in the extracellular matrix, including heightened myofibroblast activity, proteoglycan degradation, and disorganization of collagen structure, can result in opacity, scarring, and compromised corneal function. ⁵⁹

The corneal extracellular matrix serves as a mechanical and optical framework while also establishing a crucial microenvironment that influences cellular behavior, facilitates tissue healing, and maintains corneal clarity. This is a key framework for making scaffolds for tissue engineering and finding ways to treat problems with the cornea.

Optical clarity and light transmittance

The cornea is the main part of the eye that reflects light, and its flat surface is important for clear vision. ⁶⁰ In cornea tissue engineering, particularly for corneal wound dressings and stromal regeneration, it is imperative to maintain the cleanliness of nanofibrous scaffolds. ⁶¹

Transparency is contingent upon various design elements, including the reduction of scaffold thickness and the utilization of polymers with refractive indices closely matching that of the human cornea.^61,62 Himmler et al. indicated that electrospun scaffolds measuring 10 µm in thickness demonstrated transmission rates between 15% and 75%, contingent upon the fiber diameter. The alignment of smaller fiber diameters with the refractive indices of both the medium and the fibers resulted in significantly enhanced light transmission. ⁶¹ Electrospinning presents numerous benefits, such as high output, affordable prices, and a simple, straightforward process. It contributes to developing ECM-like three-dimensional fibrous structures with aligned fibers that improve cell phenotype, migration, and proliferation. ⁶³ Nonetheless, restricted light transmission is a barrier owing to the short interfiber spacing in synthetic polymer fibers. ⁶³

Refraction is influenced by the refractive indices of both the cornea and the tear film. The human cornea generally possesses a refractive index of 1.376 across the visible spectrum of the eye. ⁶⁴ Engineered scaffolds must protect internal ocular tissues from UV radiation and pathogens, provide sufficient strength to endure intraocular pressure, and permit the diffusion of nutrients and oxygen to achieve a comparable refractive index. ⁶⁵

Alignment of fibers is necessary for cell growth, as fiber contact guidance is compatible with cell morphology and cytoskeletal stress in engineered scaffolds. ⁶⁶ Kang et al. created decellularized squid mantle scaffolds that maintained alignment and transparency through the CUBIC process. The scaffolds demonstrated biocompatibility, facilitated corneal cell proliferation, and exhibited effective integration in vivo, indicating potential for structured corneal regeneration. ⁶⁷ Tayebi et al. observed that the incorporation of chitosan nanoparticles (CSNPs) into chitosan/PCL scaffolds enhanced their optical transparency and wettability. The CSNP/PCL 50/25 formulation attained the optical transparency comparable to that of human acellular corneal stroma and minimum water contact angle. ⁶⁸ Zhang et al. proved that electrospun collagen nanofibers (eCFs) increased corneal healing by inhibiting inflammation, promoting cell proliferation, and reducing fibrosis; in vivo, they promoted epithelial wound healing without compromising transparency, thereby proving their viability for optical-functional regeneration. ⁶⁹ Zargar et al. developed multilayered nanofibrous scaffolds incorporating collagen, aloe vera, silk fibroin, and epithelial growth factor. The three-layered electrospun/electrosprayed configuration possessed appropriate thickness, structure, and mechanical strength, all of which were also tunable. In vitro assays depicted that the cells possessed greater adhesion, longer viability, and improved differentiation, which proves that the cells are capable of corneal epithelium regeneration. The confirmation was established through scanning electron microscopy. ⁷⁰

Optical transparency in corneal tissue engineering demands stringent control over fiber diameter, alignment, and refractive index matching. Hybrid nanofibrous scaffolds made of natural polymers and synthetic polymers, such as chitosan/PCL and collagen-based systems, are the optimal technique for achieving functional transparency and biocompatibility within the engineered corneal substitutes.

Mechanical integrity and suture retention

Biomaterials analogous to corneal stroma can be synthesized using vitrification, a regulated dehydration technique. These materials exhibit elevated collagen concentration and alignment, enhancing their mechanical strength and compatibility for in vivo implantation with simple interrupted sutures. ⁷¹ Peng et al. created graphene oxide/niobium carbide nanocomposite scaffolds with adequate mechanical strength and stability. These scaffolds have the potential to be strong and bioactive substrates for corneal tissue applications. They demonstrate its potential for increasing the number of stromal cells in vitro and lowering inflammation and fibrosis in vivo. ⁷² Through modifications to the annealing and manufacturing processes, Beena et al. improved silk fibroin sheets for corneal engineering. The films presented were compelling, mechanically sound options for corneal rehabilitation, demonstrating varying tensile capacities and degradation rates influenced by the solvent and processing conditions, while consistently preserving structural integrity, biocompatibility, and optical clarity. ⁷³ Jung and others created three-dimensional electrospun keratin/PVA nanofiber membranes to implant/transplant in the cornea. The three-dimensional structure was stronger, clearer, and more compatible with organisms than the two-dimensional versions. ⁷⁴ Kung et al. (2016) enhanced the assessment of suture retention strength for ocular scaffolds through the utilization of polycaprolactone (PCL) films and amniotic membranes. Their modified test precisely assessed resistance to suture pull-out, deformation under maximum load, and rupture. This method reliably assesses the mechanical strength and surgical handling of corneal biomaterials. ⁷⁵

An appropriate corneal scaffold must replicate the mechanical characteristics of a healthy cornea while ensuring biocompatibility, optical clarity, surgical stability, and non-immunogenicity. ⁷⁶ In 2022, Himmler et al. created electrospun nanofibrous structures with fiber diameters ranging from 35 to 549 nm. To augment the regenerating capabilities of the structures, heparin or hyaluronic acid coatings were subsequently applied in a methodical layer-by-layer manner. Scaffolds featuring 113 nm fibers exhibited enhanced drug-loading capacity and suture retention, indicating their potential as biomimetic alternatives to traditional amniotic grafts. ⁷⁷

In conclusion, achieving adequate mechanical integrity and suture retention in engineered corneal structures requires the modification of fiber diameter, hierarchical organization, and interfacial chemistry. Nanocomposite and electrospun 3D structures, particularly structures that utilize hybrid natural-synthetic polymers, can demonstrate a favorable use of mechanical strength while retaining transparency and biocompatibility within the operating room and while healing.

Biocompatibility and bioactivity

Corneal regeneration biomaterials must possess excellent mechanical stability, regulated biodegradability, transparency, and satisfactory biocompatibility. Nanotechnology has made corneal scaffolds more compatible and biologically effective, which has improved cell connections and decreased the risk of inflammation.^78,79 The protective barriers on the surface of the eye allow less than 5% of the active chemicals in eye drops to reach the interior tissues. Due to this issue, researchers are exploring both traditional and innovative methods for medication delivery. Nanoscale carriers have demonstrated superior efficacy in overcoming corneal permeability issues and enhancing ocular bioavailability. ⁸⁰ Selecting an appropriate scaffold material is crucial for the long-term success of corneal endothelial tissue engineering and transplantation. Biocompatible and bioactive substrates that enhance endothelial cell adhesion, proliferation, and phenotypic maintenance are essential in ensuring effective regeneration. ⁸¹ Nanofibers have been extensively utilized in healthcare environments due to the fact that they consist of biocompatible materials. They are also utilized for drug delivery and tissue engineering. Two of the greatest strengths for ophthalmology are their capacity to deliver sustained release of drugs and their application in research involving corneal tissue. ⁸²

Biocompatible nanoadhesives have been produced employing biomimetic materials from tissue engineering, leveraging cell adhesion, proliferation, and development throughout the healing process. These nanoadhesives facilitate the closure and healing of ocular tissues. ⁸³

Ultimately, the application of nanotechnology techniques, particularly through biomimetic polymers and nanofibers, synergistically enhances the bioactivity and biocompatibility of corneal scaffolds. Corneal substitutes derived from these pharmaceuticals safely and successfully replicate the characteristics of the original tissue by gradually degrading, releasing medications into the surrounding environment over an extended period, and improving the connections between cells and the matrix.

Permeability and nutrient diffusion

The eye surface acts as a complex biological barrier that prevents the majority of therapeutic agents from achieving clinically relevant penetration, which indicates that larger total doses should be given on a continuous basis. Conversely, inadequate efficacy and adverse effects are prevalent consequences of this method. The enhanced ocular permeability, retention time, and overall pharmacokinetic efficacy of nanoparticle-based approaches render them a viable alternative. ⁸⁴ Drug delivery through nanocarrier-mediated drug delivery systems offers targeted therapeutic effect, sustained release, and improved bioavailability; facilitated ocular barrier penetration and reduced systemic exposure are also offered. ⁸⁵ A nanosponge, a product of β-cyclodextrin crosslinking with diphenyl carbonate, is one such system. This technique normally yields low efficiency and unnecessary side effects. Nanoparticle-based strategies present a promising alternative with their increased ocular permeability, increased residence time, and improved pharmacokinetic behavior. ⁸⁴ The ideal scaffold for corneal regeneration would be able to exhibit flexibility, mechanical durability, moisture retention, and enhanced transparency to replicate the two-dimensional lamellar architecture of the natural cornea. The substrate must possess appropriate porosity to facilitate food and oxygen penetration, appropriate oxygen permeability to support cellular viability, and a controlled disintegration rate that promotes tissue remodeling without inflammation. ⁷⁰ Nanotechnology in ocular engineering applies physical and chemical advancements in designing surfaces for promoting cellular adhesion and growth. This technology constructs microenvironments that enable the accelerated delivery of nutrients to cells. ⁸⁶

In conclusion, making the scaffold more permeable and allowing nutrients to spread through it is important for keeping eye cells alive and helping tissue grow back. The combination of nanocarrier-mediated delivery and nanostructured scaffolds effectively circulates around diffusion barriers, making sure that oxygen and nutrients are delivered to the right places and stay there for a long time.

Biodegradability and integration with host tissue

Biodegradability is an essential criterion for corneal endothelium implants, guaranteeing that the scaffold degrades at a regulated rate in alignment with the regeneration of Descemet’s membrane to prevent detrimental ocular consequences.^87,88 Numerous natural biomaterials exhibit inherent biodegradability; nonetheless, their degradation rates may require modification. For instance, gelatin is rapidly degraded by enzymes, resulting in diminished long-term stability within the eye. ⁸⁸ Synthetic polymers allow for precise control over composition and degradation rate, providing customizable performance according to the needs of corneal repair. ⁸⁷ Kuragel, a hydrogel composed of gelatin and hyaluronic acid, was gradually degraded by enzymes, retaining around 69% of its mass after 1 month in an enzymatic environment. This ensured that the matrix was sufficiently stable to facilitate the regeneration of corneal tissue. ⁸⁸ Furthermore, poly (lactic-co-glycolic acid) (PLGA) membranes, a recognized biodegradable copolymer, were successfully utilized on rabbit corneas and exhibited total degradation after 29 days. ⁸⁹ However, excessive or uncontrolled degradation in similar polymeric materials may lead to medium acidification, compromising cell viability and inducing local inflammation.^90,91 Individuals commonly utilize polycaprolactone (PCL) due to its gradual degradation. When combined with other polymers such as chitosan in composite scaffolds, it can alter the degradation rate.^87,92,93

Biomaterials must exhibit biocompatibility and possess sufficient adhesive strength to be incorporated into host tissues without sutures. ⁸⁷ Kuragel was designed for sutureless integration and bioadhesion. Its adherence involves covalent bonds between methacrylate groups in the hydrogel and thiol groups on the wound surface. ⁸⁸ LC-COMatrix hydrogel reached a burst pressure of 542 mmHg, enabling seamless tissue merging in rabbit models.^94,95 Nevertheless, certain synthetic polymers, such as poly(hydroxyethyl methacrylate) (PHEMA), have been associated with the recruitment of inflammatory cells in vivo, which can impede regeneration.^96,97 Kuragel was engineered for bioadhesion and integration without the necessity of sutures. The adhesion results from covalent interactions between methacrylate groups in the hydrogel and thiol groups on the wound surface. ⁸⁸

The optimal equilibrium between biodegradability and tissue integration is crucial for effective corneal regeneration. Hydrogels and polymeric composites that degrade in alignment with natural tissue remodeling, while maintaining adhesion, transparency, and bioactivity, provide a viable approach for sutureless, regenerative corneal replacement. The integration of visual, mechanical, and biological components underpins successful corneal regeneration. Electrospinning offers a versatile technique for fabricating nanofibrous scaffolds that effectively replicate the native architecture of the cornea to meet these diverse requirements.

Fundamentals of the electrospinning process

Over the last several years, electrospinning has become the most widely used method for producing nanofibers. This technique utilizes electrostatic forces to extrude fibers from a polymer solution through a spinneret. The fundamental principle of electrospinning involves the administration of high voltage, which imparts an electric charge to the polymer solution and results in the establishment of a structure called the Taylor cone. A delicate stream of solution is discharged from the apex of the Taylor cone when the electrostatic forces overcome the solution’s surface tension. This jet travels along the electric field and deposits it onto a grounded collector, forming nanofibers. The process is affected by multiple parameters, including the conductivity and viscosity of the polymer solution, the imposed voltage, the space separating the collector and the needle tip, and the solution flow rate. Figure 3 depicts the electrospinning setup that produces nanofibers on a revolving drum.

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Figure 3.

Schematic diagram of a basic electrospinning setup. The illustration depicts the core components, including the syringe pump, spinneret, high-voltage power supply, and collector. The process begins with the formation of a Taylor cone at the needle tip, leading to the ejection of a polymer jet that thins and solidifies into nanofibers deposited on the collector.

One of the most intriguing benefits of the electrospinning procedure is the capacity to control the scaffold’s porosity and thickness. It can precisely and accurately create fibers with sizes spanning from 50 to 100 nm or larger, and it comes with an effortless experimental setup. The resultant nanofibers will be very porous and have a large surface area. Inside the scaffold, porous structures facilitate optimal cell interaction; wide pores limit cell attachment due to their reduced area, while small pores restrict how deeply cells can penetrate. Proper porosity facilitates vascularization, metabolite transfer, and cell division. Scanning electron microscopy (SEM) and mercury porosimetry are two methods utilized to study the scaffold’s porosity. Manufacturing-oriented nanofibers are an undeniable benefit of electrospinning over alternative manufacturing methods. Commercial applications for scaffolds and fibers created from electrospinning are numerous and include the creation of polymeric separation membranes, fibers as reinforcing agents in composite materials, nonwoven fabric substitution, and other biomedical applications.^98–101 Figure 4 shows the basic way that electrospun scaffolds interact with corneal cells. This diagram shows a three-dimensional nanofibrous network that has been improved with important adhesion proteins like fibronectin and collagen. These proteins replicate the structure of the natural extracellular matrix. The picture shows how these proteins act as ligands for integrin receptors on the surfaces of endothelial cells, keratocytes, and epithelial cells. This interaction makes it easier for important intracellular signaling to happen, which controls adhesion, proliferation, and phenotypic maintenance. All of these are necessary for successful corneal regeneration.

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Figure 4.

Schematic of corneal cell interaction with electrospun nanofibrous scaffolds. This figure shows how corneal cells (epithelial, stromal, and endothelial) bind to adhesion proteins attached to the nanofibers through integrin receptors. This binding leads to the activation of intracellular signaling pathways and regulates the processes of cell adhesion, proliferation, and differentiation. Furthermore, the controlled release of growth factors or drugs from nanoparticles in the scaffold provides a biological and therapeutic environment for corneal regeneration.

Because of its close similarities in structure to the native extracellular matrix (ECM), elevated porosity, and high ratio of surface area to volume, all of which support motion, proliferation, differentiation, and cell adhesion, in addition to its unique mechanical properties, simplicity of adjusting fiber properties, excellent handling, and suturability for implantation, electrospinning has attracted more interest recently in the fabrication of biomimetic efficient corneal tissue. ¹⁰²

Material selection: Natural & synthetic polymers

The shape of the electrospun fibers is significantly influenced by both the molecular weight of the polymers and the concentration of the polymer solution. The diameter of the electrospun fibers typically demonstrates a favorable association with the concentration of the solution, assuming all other parameters are held constant. Bead formation, or the incapacity to create fibers, may stem from an improper concentration. Scaffolds featuring different fiber configurations can be efficiently and swiftly produced to replicate the structure of the extracellular matrix (ECM) found in authentic corneal tissue (for example, fibers in the corneal stromal layer that are lined together) through the manipulation of the electrospun collector or the electrostatic field. The structure, fiber configuration, surface chemical modification (for example, plasma), and information molecules (for example, epidermal growth factor) can all influence how well electrospun scaffolds work. Through regulating these variables, scaffolds with superior qualities (for example, mechanical, biological, and high transparency) can be created that enhance corneal cell adhesion, movement, proliferation, and differentiation in addition to promoting the development of corneal tissue. ¹⁰³ For corneal tissue engineering, natural materials with high biodegradability, biocompatibility, and low immunogenicity, like collagen, gelatin, and silk, have been widely used.^104,105 But natural polymers are used together with a synthetic polymer for electrospinning due to their low strength. ¹⁰⁶ In this section, we discuss studies that have used natural polymers in corneal repair.

Applications of synthetic nanofibers in corneal tissue repair

Different synthetic biomaterials have been applied in regenerative medicine, and it is essential to note that only a limited number of them can generate nanofibers.^133,134 Although natural polymers have very good biological properties, they don’t have suitable mechanical properties as a result, so their clinical application properties are largely limited.^103,135 However, only a small number are suitable for corneal repair, like PCL, PLDLA, PLGA, PHBV, PEUU, and so on (Table 1). ¹⁰² The ratio of surface area to volume and porosity of electrospun nanofibrous scaffolds created from synthetic chemicals are similar to those made from natural components.^136,137

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Table 1.

Several synthetic polymers and their uses in corneal tissue engineering with electrospinning methods.

Polymers	Solvent	Attached cells	Advantage	Ref.
PCL	TFE/Chloroform/DMF	The corneal epithelial cells of either humans or rabbits, epithelial cells of the limbal layer	Biocompatible, supports corneal phenotype maintenance, and promotes corneal epithelium development	157–159
PLDLA	DMF and chloroform	Corneal epithelial cells	Enhances biocompatibility of corneal substrates; promotes fibroblast differentiation into keratocyte-like cells	1
PLGA	Dichloromethane and chloroform	Limbal fibroblasts and limbal epithelial cells	Biocompatible, supports multilayered cell formation	160,161
PHBV	Chloroform and DMF	keratocytes	Promotes cell attachment and proliferation	151,162
PEUU	HFIP	Corneal stromal stem cells	Facilitates collagen matrix formation and stem cell differentiation into keratocytes	102

The crucial aspect of corneal implants in tissue engineering is the scaffold’s transparency, so the method of electrospinning for corneal tissue engineering and the choice of material are so important to making a transparent layer.^106,138 Therefore, in this section, we will look at several FDA-approved polymers utilized for the repair of the cornea. In some cases, they are used solely, but in most of the studies, they were used in blending with other polymers.

Polycaprolactone (PCL) is a synthetic polymer for medical use because it is biocompatible and biodegradable. The intrinsic properties of PCL include its ability to produce nanofibers, which make it suitable for medical applications. The benefits of the nanofibers include a wide surface area, minimal pore diameter, and elevated porosity. ¹³⁹ The PCL nanofibers possess the capacity to promote the growth and attachment of limbal epithelial cells while maintaining their original characteristics. Moreover, these nanofibers can be utilized for the regeneration of impaired ocular surfaces. ¹³⁹ Additionally, PCL-based scaffolds can serve as a substitute substrate for scaffolds made from the human umbilical cord and can be a helpful option in ocular surface tissue engineering. ¹⁴⁰ Human corneal cells easily adhered to PCL-based nanofibers and demonstrated excellent adhesion and proliferation. The cells showed growth on aligned matrices, suggesting that the matrices effectively supported the expansion of corneal cells. These findings confirm the potential of the matrices as substrates for biomedical purposes. ¹⁴¹

Poly(L-co-D, L-lactic acid) (PLDLA) is a significant biomaterial due to its biocompatible features that encourage cell renewal and proliferation.^102,142,143 The electrospun-aligned PLDLA scaffolds, when cultivated with human corneal stromal cells, exhibited changes in matrix elasticity (elastic modulus) and dimensions, leading the cell phenotype to change from fibroblasts that are actively contracting to quiescent keratocytes. ¹ Serum-free medium and insulin supplementation have been demonstrated to alter the activity of mature human-derived corneal stromal cells in three-dimensional PLDLA multi-layered structured constructions, causing corneal fibroblasts to revert to keratocyte characteristics in a three-dimensional structure. ¹⁰²

Due to its advantageous properties, polylactic-co-glycolic acid (PLGA), a polymer that is biodegradable and developed through synthetic processes, has been extensively studied in the area of regenerative medicine. ¹⁴⁴ Furthermore, owing to its highly responsive behavior towards stimuli, it is also recognized as a variant of an intelligent polymer. ¹⁴⁵ This polymer is employed in PLGA-derived drug delivery strategies for the management and detection of certain illnesses.^146,147 Furthermore, this compound has been utilized in the area of regenerative medicine and has received investigation for its applications in corneal regeneration. ¹⁴⁴ The electrospun PLGA scaffold as a substitute for amniotic membrane in ocular surface regeneration may offer assistance for corneal epithelial stem cells and act as a protection against infectious diseases. ¹⁴⁸ A suitable carrier for Limbus stromal cells was applied with a combination based on PEGDA (poly(ethylene glycol) diacrylate) and then electrospinning of PLGA (poly(lactic-co-glycolic acid)) on it using a mix of stereolithography and electrospinning techniques. LSCs could easily adhere to and grow on PLGA sheets and induce epithelization in an ex vivo environment. ¹⁴⁹

PHBV (poly-3-hydroxybutyrate-co-3-hydroxyvalerate) belongs to the polyhydroxyalkanoate family. This material is a category of natural polyesters that can be obtained as by-products of microorganisms under specific circumstances with a supplementary carbon source. These polymers are promising materials to produce materials with biological applications. The large number of monomers in this group of materials allows changes to be made in their polymer and copolymer structure, which allows the production of copolymers with different compositions and ratios. ¹⁵⁰ Drug screening laboratory procedures offer expedited and efficient data analysis, resulting in a major decrease in animal sacrifices. Most of these methods use biomaterials that have been investigated in the cornea field, like PHBV (poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid)) and PHB (polyhydroxy butyrate). ¹⁵¹ Some studies have shown that human limbal stem cells (LSC) were able to maintain a typical corneal stem cell characteristic and remain tightly bound to the PHBV substrates. These cells have permeated the porous scaffold. Additionally, porous PHBV substrate acts as a good substitute carrier for tissue engineering of the ocular surface and possesses the capacity to replace other biological membranes as a substrate for LSC expansion. ¹⁵²

Polyurethane stands out as among the most widely utilized synthetic polymers in the area of medicine. The mechanical properties and thermoplastic elastomer characteristics of these polymers render them appealing. Studies done recently have looked into the application of a new category of polyurethanes that demonstrate natural and biological degradation in regenerative medicine and drug delivery systems.^153,154

Transforming corneal fibroblasts to a keratocyte characteristic in a three-dimensional structure is helpful for aligned fibrous formations. Alignment of cultured human corneal stromal stem cells (hCSSCs) induced by electrospinning resulted in aligned PEUU mats, which created a 7–9 μm thick collagen matrix on the substrate. Following the arrangement of collagen on the matrix, the PEU layer systematically detaches over time. The collagen fibers within this matrix demonstrated characteristic interfibrillar spacing and consistent width; they also showed a parallel alignment similar to that of the natural stroma.^155,156

Natural and synthetic nanofiber for corneal tissue engineering

Proper porosity, mechanical, and optical characteristics are crucial for scaffolds designed for corneal tissue reconstruction.^163,164 For corneal cell proliferation and differentiation, scaffolds made of natural polymers often work well because they are more like the natural environment. However, they break down more quickly and aren’t as strong as scaffolds made of synthetic polymers.^165,166 However, synthetic polymers’ light transmittances are not almost as high as those of the natural cornea. ¹⁶⁷ Nano scaffolds have been made using mixtures that include natural and synthetic polymers to achieve ideal light transmission and degradation qualities for the proliferation of corneal cells. These combining methods can generate a better imitation of the original cornea ¹⁶⁸ (Table 2).

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Table 2.

Several hybrid polymers and their application in corneal tissue engineering with electrospinning methods.

Component	Material type	Application in corneal regeneration	Ref.
PLGA/collagen type I	Polymer/Protein	Artificial cornea	169
PCL/silk fibroin	Polymer/Protein	Stroma	170
PCL/hyaluronic acid or heparin	Polymer/Polysaccharide	Corneal bandage	131
Gelatin methacryloyl (GelMA)/silk nanofibrils (SNF)	Polymer/Protein	Stroma	171
Nanofibrous polycaprolactone/gelatin (PCL/Gel)	Polymer/Protein	Corneal limbal	172
Hydroxyethyl cellulose/PVA/graphite/nano-hydroxyapatite	Polymer/Composite	Artificial cornea	173
PCL/anti-VEGF	Polymer/Protein	Endothelium	27
PCL/collagen type I	Polymer/Protein	Endothelium	174
PCL/Gelatine (type A)	Polymer/Protein	Endothelium	102
PCL/Chitosan	Polymer/Polysaccharide	Endothelium	102

To identify the most ideal and appropriate scaffold for ocular tissue engineering, Arabpour et al. developed and created triple-layer electrospun nanofibers of poly(D, L-lactide-co-glycolide) (PLGA, 50:50) and aligned type I collagen nanofibers. The scaffold’s center layer was electrospun with aligned type I collagen nanofibers, while its inner and outer layers were made of PLGA nanofibers. To improve the scaffold’s mechanical properties and biocompatibility, PLGA and collagen were incorporated. PLGA (50:50) nanofibers were utilized as a scaffold for corneal endothelial and epithelial cells, while aligned type I collagen nanofibers were electrospun in the intermediate layer to act as a platform for corneal stroma. Collagen fibers that were aligned were utilized to enhance the mechanical characteristics. In consequence of their alignment and small size, aligned collagen fibrils have greater mechanical characteristics than the random form with adequate stromal transparency, as established by previous studies. ¹⁷⁵ Under cell culture conditions, the shrinking of electrospun PLGA is an undesired characteristic.

Results demonstrated that the use of cross-linked scaffolds resulted in reduced scaffold shrinkage and a more gradual pace of degradation. The fibers at the nanoscale were in favorable form, according to the SEM assessment. Additionally, regarding both non-cross-linked and cross-linked materials, the in vitro examination showed enhanced proliferation and cell adhesion in the existence of endometrial stem cells of humans. According to this work, ¹⁶⁹ it may be possible to create biodegradable electrospun nanofibers for applications involving ocular tissue engineering.

Another investigation used an electrospinning approach to produce a polycaprolactone (PCL)/silk fibroin (SF) hybrid scaffold in various ratios (70:30, 60:40, and 50:50). For ocular tissue engineering, SF provides requisite cell adhesion, biocompatibility, and optical transparency, while PCL gives the mechanical strength needed (that has beneficial strength characteristics for surgical stitches). The findings show that adding SF resulted in scaffolds with an additional consistent structure that is aligned. ¹⁷⁶ The mechanical characteristics of hybrid electrospun scaffolds are directly affected by a number of things, such as the physical properties and interactions of the components, as well as the size of the fibers. ¹⁷⁷ The ultimate tensile strength of corneal stromal tissue in the natural state is around 3–5, while the modulus of the young of it ranges from 0.3 to 7 MPa.^178,179 For corneal applications, both nonaligned and aligned PCL-SF (50:50 and 60:40) fibers have a high tensile strength and Young’s modulus within an acceptable range. For corneal stromal engineering, pure PCL’s tensile strength is inadequate. The statistics show that the pure PCL had the least light transmittance out of every sample. It’s clear that the fiber orientation and the incorporation of SF into the PCL scaffold have an impact on the light transmittance of scaffolds. Therefore, it was shown that the optimum light transmission properties for corneal tissue engineering are found in aligned PCL-SF scaffolds (60:40 and 50:50). Notably, compared to the other scaffolds, the aligned PCL-SF scaffolds (60:40 and 50:50) showed greater levels of hydrophilic nature, transparency, in vitro deterioration pace, and water absorption.

HSKCs (human stromal keratocyte cells) are adhered to and proliferate on both non-aligned and aligned PCL-SF scaffolds, according to the cell compatibility data; however, the alignment of the structure has a direct and beneficial impact on the arrangement and gathering of cells. According to the MTT results, the scaffolds’ cell viability increases significantly and progressively when the SF ratio increases in comparison to the pure PCL scaffold. Improved cell viability is a consequence of SF’s hydrophilic characteristics, which greatly increase the adherence and proliferation of the cells. ¹⁸⁰ However, at every time point, the alignment also is a crucial part of the viability and growth of hSKCs. The results show that, in comparison to the non-aligned PCL-SF (50:50 and 60:40) scaffolds, the aligned scaffolds planted with the human keratocyte cells show significantly increased proliferation (p < 0.05).

Nanofibers that mirror the extracellular matrix (ECM) are getting increasingly favored as suitable scaffolds for drug delivery and tissue engineering.^173,181,182

In a study, polyvinyl alcohol (PVA) and hydroxyethyl cellulose (HEC) were used to generate nanofibers, which were then cross-linked with graphite and nano-hydroxyapatite conjugated with glutaraldehyde. In scaffolds for corneal tissue creation, two bioactive substances that have been utilized to enhance cell adhesion are graphite (GR) and nanohydroxyapatite (nHA). ¹⁷³ A clear PVA was surrounded by varied quantities of nHA on the scaffolds containing GR. The final products have undergone a comprehensive evaluation employing different methods to establish their suitability for artificial cornea applications. HEC is a polymer characterized by its hydrophilic and biocompatible properties, making it appropriate for usage in tissue engineering and as an ingredient in eye drops designed to alleviate severe dry eye conditions. ¹⁸³ Nonetheless, HEC is not an ideal polymer for the manufacture of microfiber scaffolds because of its inadequate tensile strength and the requirement for electrospinning. ¹⁸⁴ As a result, HEC and polymers such as PVA have been combined. ¹⁸⁵

PVA is a suitable polymer for ocular tissue engineering ¹⁸⁶ owing to its high-water content and great elastic modulus, ¹⁸⁷ as well as its excellent biocompatibility. ¹⁸⁸ Incorporating nHA into polymers at higher concentrations can lead to the dissolution of nHA, resulting in the formation of the non-bioactive compound Ca-Na6(PO4). ¹⁸⁹ Consequently, it has been advised to utilize low nHA concentrations for scaffolds. ¹⁹⁰

The water content of the translucent PVA core was approximately 79.5%, closely resembling that of the average human cornea, which is around 78%. ¹⁹¹ With an increase in nHA quantity, there was a corresponding decrease in the pore diameters of the scaffolds, which likely accounts for the observed decrease in water content. For every sample, the water contact angle was below ninety degrees, suggesting that the structures exhibit hydrophilic properties. ¹⁹² For the polymers to endure suturing during implantation, scaffolds intended for artificial corneas need to exhibit significant tensile strength. Recent findings suggest that scaffold nanofibers employed in artificial corneas exhibit a greatest tensile strength of 6 MPa, in contrast to the average tensile strength of human corneas, which is around 3.5 MPa.^102,193

Every scaffold nanofiber had a tensile strength of more than 7 MPa. Additionally, scaffold nanofibers with the least quantity of nHA usually didn’t break easily, as shown by the enlarged graph, indicating that the nanofibers’ flexibility was maintained. Furthermore, the composite nanofibers’ tensile strength was enhanced with an increase in nHA content. It was found that the scaffolds caused no harmful impacts on human corneal epithelial cells. Two distinct cell lines, the immortal HCE cell line (human corneal epithelial) and L929 mouse fibroblasts, were used to determine cell adherence on the scaffolds’ surface. Not many cells adhered to the PVA hydrogel. Proteins don’t stick to PVA hydrogels very well, which makes it hard for cells to bind to them. Even if they are biocompatible and hydrophilic, this is still the case. ¹⁹⁴ This is beneficial for an artificial cornea because it preserves transparency optimally when there is little to no cell attachment to the core. Because graphite can absorb proteins, applying graphite to scaffold structures may increase L929 fibroblast adhesion to polymers. ¹⁹⁵

Higher nHA concentrations resulted in fewer cells adhering to the scaffolds, suggesting that expanding the nHA content might cover the GR surface and reduce protein absorption. Smaller pore sizes in the polymers caused by an increase in nHA may also lead to a decrease in the cells’ number adhering to surfaces.^196,197 The sample containing 1.66% w/w of nHA demonstrated the greatest cell adhesion, or the overall quantity of cells that were attached to the scaffolds. The diameter of HCE was decreased via raising the scaffolds’ nHA content. L929 cells did not experience such a strong change. Furthermore, the scaffold twisted somewhat because of the high concentration of nHA; hence, it was deemed inappropriate to utilize for further in vivo studies.

The corneal stroma can be described as thick tissue that makes up nearly 90% of the cornea’s thickness among these layers. Several materials have been investigated for stromal cell regeneration and possible transplantation. ¹⁹⁸ Particularly, stromal cell attachment and proliferation are facilitated by collagen-based materials, even though their tensile strength is often lower than that of the human cornea. ¹⁹⁹ Additionally, functional sequences like arginine-glycine-aspartic acid (RGD) that promote cell adherence may be present in gelatin, one of the collagen derivatives. ²⁰⁰ Alteration of chemicals with different functional groups, such as acyl azide, ²⁰¹ aldehyde groups, ²⁰² and carbodiimides, ²⁰³ has been performed to enhance the gelatin’s physicochemical properties.

Gelatin methacryloyl (GelMA) has recently become known as an encouraging hydrogel for tissue engineering utilizations ²⁰⁰ due to its adjustable physical properties and degradation. ²⁰⁴ Bektas et al.’s investigation on GelMA hydrogel’s applicability for corneal stroma engineering^205,206 noted that the hydrogel produced high cell proliferation, stromal collagen, and proteoglycan production, as well as excellent transparency. Although GelMA has many useful qualities for a vast array of applications, it also has a number of disadvantages, including low mechanical strength and a quick rate of deterioration, which may require improvement based on the application. ²⁰⁵

In an additional study, they produced a hybrid scaffold with mechanical characteristics and increased transparency that might replace the corneal stroma. The scaffold was made from various ratios of silk nanofibers (SNF) to gelatin methacryloyl (GelMA). The toughness and tensile strength are notably enhanced through boosting the ratio of surface-to-volume, which is the outcome of SNF formation. ²⁰⁷ Furthermore, in comparison to raw silk fibroin, the existence of nanoscale-thinner silk strands may considerably improve the final film’s transparency and optical clarity. ²⁰⁸

According to the results, a desirable 30/70 SNF/GelMA ratio exhibits high mechanical characteristics that are similar to the native corneal stroma, hydrophilicity, and great transparency, having more than 85% transparency to light in the wet condition. This hybrid membrane has the following characteristics: toughness (0.46 ± 0.04 MPa), tensile strength of 3.8 ± 1 MPa, elastic modulus of 36.2 ± 7 kPa, and the capacity to absorb water as much as 138 ± 27% after 10 days (72 ± 1% in PBS, 62 ± 2% in synthetic tear solution) without exhibiting mineralization. ²⁰⁹

Corneal neovascularization (NV) is a medical disorder that leads to blood vessels proliferating too much from the stroma to the limbal zone. This disorder makes eyesight less clear over time. This syndrome is linked to inflammation, which can happen in conditions like atopic conjunctivitis and corneal graft rejection; trauma from things like chemical burns; infections from viruses, bacteria, or fungi; degenerative diseases; developmental disorders that are present at birth, like limbal stem cell deficit; and other diseases.^210,211

Controlling corneal NV requires the utilization of anti-vascular endothelial growth factor (anti-VEGF), a medicinal medication that blocks the function of VEGF. ²¹² Known as intravitreal drug administration, the anti-VEGF is typically injected straight into the vitreous (posterior) chamber of the eye. Regrettably, the administration of drugs in this manner may be associated with several severe adverse effects, such as systemic and infectious endophthalmitis, rhegmatogenous retinal detachment, ocular hemorrhage, intraocular inflammation, and an increase in intraocular pressure. ²¹³ According to reports, the anti-VEGF drug delivery method must meet the requirements listed here: biocompatibility, transparency, sufficient concentration, bioactivity, and prolonged and targeted delivery over a month. ²¹⁴ In order to avoid abnormally high ocular vascularization, VEGF-blocking drugs are integrated into engineered biomaterials to enhance their performance and sustained target release. Studies that have been recently reported on the creation of fibrous scaffolds electrospun primarily use VEGF or, in a small number of cases, anti-VEGF chemicals, usually for tissue engineering goals.

In this work, an anti-VEGF substance was added to electrospun PCL by simple physical adsorption. The physicochemical characteristics and final function of anti-VEGF/PCL scaffolds in supporting limbal stem cells (LSCs) for corneal tissue engineering were evaluated. The anti-VEGF/PCL electrospun scaffold has a notably elevated modulus of elasticity. The findings indicate that the scaffolds’ flexibility is directly impacted by the biological component’s inclusion.

At both stresses of 5 and 10%, the anti-VEGF completely reduced the hydrophobicity of PCL and almost tripled its Young’s modulus, which improved the material’s tensile and wetting characteristics. The incorporation of the anti-VEGF leads to a notable rise in the storage modulus, indicating that the biological component’s strengthening impact has enhanced the scaffold’s mechanical behavior. The findings show that the anti-VEGF particles are positioned in the PCL’s amorphous phase and that their mobility has decreased. The results additionally confirmed the greater biocompatibility of anti-VEGF/PCL compared to a single PCL, which was discovered to be more beneficial to LSC attachment, differentiation, and growth as seen by the SEM, as well as the CK₃ (a corneal epithelial cell differentiation) and p₆₃ (presence of stem cells) markers following cell staining. The findings will aid in the development of electrospun scaffolds aimed at preventing angiogenesis during the LSC ocular tissue building process, facilitating long-term anti-VEGF administration. The upcoming investigations will focus on assessing the ongoing bioactivity of the biological material, gathering data regarding its release rate, and conducting a quantitative analysis of cell proliferation via the identification of CK3- and p63-positive stem cells or cells that are differentiated within the corneal epithelium. In the same way, certain limitations should be acknowledged and addressed. ²⁷

The ailment known as limbal stem cell deficiency (LSCD) affects the cornea and is characterized by damaged or dysfunctional limbal epithelial stem cells (LESCs). Limbal stem cells are essential for corneal epithelial renewal, so LSCD can lead to corneal neovascularization, chronic epithelial defects, reduced vision, and pain. ²¹⁵ The state described as LSCD (limbal stem cell deficiency) affects the cornea and is characterized by damaged or dysfunctional limbal epithelial stem cells (LESCs). Because for the corneal epithelium to regenerate, limbal stem cells are essential, LSCD can cause chronic epithelial defects, corneal neovascularization, reduced vision, and pain. ²¹⁶

When it comes to ocular surface problems, amniotic membrane (AM) is the most frequently utilized and renowned membrane for cellular regeneration. ²¹⁷ AM treatment carries a substantial risk of viral transmission, despite its unique ability to induce cell proliferation and epithelialization.^216,218

Results about how the PCL/gel mix affects various cell types are debatable. According to some research, combining PCL with gel might improve cell adhesion.^219,220 Other research also suggests that gel might not significantly change the PCL’s biological characteristics. ²²¹

This study looked at how nanofibrous polycaprolactone (PCL) and PCL/gelatin (PCL/gel) affected limbal epithelial stem cells (LESC) and how well they transplanted in an animal model of alkaline damage. PCL and PCL/gel were evaluated with regard to LESC proliferation and adhesion, biocompatibility with the ocular, and ease of transfer after surgery. The electrospinning method produced PCL and PCL/gel with mass ratios of 50:50 and 70:30.

According to this study, PCL was better for LESC adhesion, stimulation of proliferation, and epithelial morphology than PCL/gel. Superior to PCL/gel, PCL may also be able to preserve the viability and morphology of LESCs. The finding may be the result of the differences in PCL and PCL/gel scaffold morphology. The broad range of sizes and irregularities of the fibers in PCL/gel composites might affect the pore diameter of PCL/gel relative to PCL. According to previous studies, various scaffold architectural characteristics can promote various cell adhesion and proliferation.^222,223

The larger fibers will provide broader pore interformations, which will aid in cell adhesion and further penetration in the inner structure, according to Mijovic et al. ²²⁴ Therefore, the PCL fiber’s bigger diameter than that of the PCL/gel mixture may be advantageous for LESC adhesion and proliferation. The inability to blend PCL with gel to enhance PCL surface characteristics in LESC culture highlights PCL’s aptitude for limbal cell proliferation and adhesion in contrast to PCL/gel. Histopathologic examination of corneal sections from animals that had undergone transplantation revealed that whereas epithelial regeneration was nearly identical in the PCL and PCL/gel groups, there was a notable decrease in inflammation and vascularization in the PCL group. The histopathologic analysis of corneas post-surgery, alongside the ease of transfer during the procedure, offers compelling evidence supporting the effectiveness of PCL when contrasted with PCL/gel blends and amniotic membrane. ²²⁵

Female placenta tissue and human amniotic membranes (HAM) are currently the wound dressings’ highest standard for following ocular surgery. Their well-known beneficial characteristics include improving re-epithelization and having antibacterial and anti-inflammatory properties. ²²⁶ Usually, corneal ulceration or persistent epithelial abnormalities need suturing of the tissue in the patient’s eye. ²²⁷ Similar to the overwhelming number of allogeneic tissues utilized in tissue engineering, there are challenges with quality differences, a lack of donors, and possible infections. ²²⁸ Artificial wound dressings are the focus of study to solve these drawbacks. Electrospun nanofibrous scaffolds seem like a good approach, as they combine the qualities of permeability and high-specific-surface scaffolds, the latter of which is dependent on the fiber diameter. Numerous methods for modifying nanofibrous scaffolds have been written about in the literature, such as, ²²⁹ where, for better ocular tissue regeneration after surgery, scaffolds made by electrospinning with fiber diameters of 549 nm, 167 nm, 113 nm, and 35 nm were generated and coated using the method known as layer-by-layer (LbL) using heparin or hyaluronic acid. In contrast to nanoparticles coated using layer-by-layer (LbL) techniques for drug administration (e.g., Ref. 230), our approach included the direct immobilization of medicines onto the nanofiber surface. Using the application-oriented suture retention test (SRT), this work examines how fiber diameter affects medication loading and release kinetics and mechanical characteristics to further create customized nanofibrous scaffolds for the healing of corneal wounds.

Although a scaffold’s specific surface increases as fiber diameter decreases, there may be a threshold beyond which the scaffold’s modification by the LbL-coating technique is no longer effective. The best-performing scaffolds are PCL scaffolds, which have a 113 nm mean diameter, an increased capacity for drug loading, and a strong resistance to suture pull-out. ²³¹

To see well, the cornea must be clear. The five main layers that make up the cornea all work together to keep it functioning well. It is around 550 μm thick. A highly organized monolayer of human corneal endothelial cells (HCEC) is formed on top of the Descemet’s membrane. Individual cells are approximately 5 μm thick and 18–20 μm wide. The endothelium establishes a diffusion barrier to the surrounding aqueous humor to keep the intraocular pressure of the anterior chamber under control. ²³²

Here, the pump function of the corneal endothelium (aquaporin 1 water channels and Na+/K+-ATPase) simultaneously balances the leakage of aqueous humor into the stroma, removing excessive stromal fluid and maintaining transparency. The density of endothelial cells (ECD) varies with age. However, there is an acceptable level of 500–600 cells per m m 2 , wherein the endothelium layer can carry on with its drainage activity.^233–235

A human corneal endothelium-containing artificial biomimetic transplant could reduce reliance on corneas from human donors, enabling more patients to access this vision-restoring procedure. Four types of scaffolds made of electrospun nanofibers constructed from polycaprolactone were compared in a study to cultivate corneal endothelial cells. Endothelial cells in the human cornea (HCEC-B4G₁₂) were used to tissue-engineer PCL/collagen, PCL, PCL/chitosan, and PCL/gelatin scaffolds.^174,236 Regarding their possible use as artificial posterior lamellar grafts, each scaffold was examined. Similar to the Descemet’s membrane, their very permeable fibrous network creates a solid but permeable scaffold. ²³⁷

Na⁺/K⁺-ATPase and ZO-₁ antibody staining showed that cell functions were intact. A varied blend morphology might be revealed by showing that blending led to a decreased contact angle. By using live/dead staining, scaffold cytocompatibility could be checked for every group. The collagen and gelatin blended matrices showed notably higher cell viability, with 97 ± 3% and 98 ± 2% viable cells, independently. The scaffolds’ surface attaching the HCECs is visible on TEM images. The advantages of combined PCL nanofibrous scaffolds for endothelial keratoplasty of the cornea are emphasized in this work. A few hundred nanometer-diameter fibers make up nanofibrous scaffolds when PCL is blended with collagen, gelatin, or chitosan. For application in lamellar keratoplasty, PCL-GEL and PCL-COL solutions improve characteristics of nanofibrous scaffolds that are electrospun. Comparing these mixtures to PCL and PCL-chitosan scaffolds, more cells survive. While endothelial cells were visible by antibody labeling, no cytotoxic risk could be seen in any of the examined samples. Therefore, PCL-blend and PCL electrospun scaffolds appear to be viable alternatives to posterior lamellar keratoplasty.

Achieving structural mimicry through fiber alignment and porosity

A fundamental approach to attaining structural mimicry in engineered scaffolds involves the precise alignment of fibrous components, which significantly affects cell orientation and mechanical anisotropy. Aligned fibers can mimic the directional signals present in natural tissues such as muscle, tendon, and nerve. This alignment facilitates cellular elongation, extracellular matrix deposition, and the comprehensive organization of tissue. For instance, recent advancements in 3D printing and additive manufacturing have made it possible to control the orientation and geometry of strands with precision. This positioning allows for scaffolds to have hierarchical pores that encourage contact guidance and direct cell migration. In one study, researchers created a grid-in-grid polycaprolactone (PCL) scaffold using precision extruding deposition, which provided human mesenchymal stem cells (hMSCs) with alignment cues. ²³⁸ This experiment demonstrated that incorporating alignment at different scales can significantly influence how cells organize themselves. ²³⁹

Furthermore, adding a gradient in fiber orientation that goes from aligned to random areas has shown that it is better at replicating the gradual changes seen in native tissue structures. This technique enhances functional integration at the interfaces. Along with orientation, changing the pore structure and how they connect to each other is also important for achieving structural mimicry. The porosity of a scaffold affects how mass moves, how nutrients diffuse, and how well it can bend, all of which are important for how cells behave and how tissues mature. Reviews of the literature emphasize that scaffolds with interconnected porosity and optimized pore sizes—typically ranging from 50 to 300 µm for bone-like tissues—are essential for promoting vascularization and deep cellular infiltration. ²⁴⁰ Additionally, the interplay between fiber alignment and pore geometry can be effectively utilized: aligned fiber packing can create anisotropic channels, while larger macropores facilitate bulk transport. Take biomimetic hydrogels, for example. They’ve been crafted to align nanofibrils in a way that creates a flow-induced structure, allowing for directional ordering and the right amount of porosity to facilitate transport. ²⁴¹ Scaffold designs can copy the complexity and function of natural tissues by using techniques like managing fiber alignment and making hierarchical porosity. This method works well to link synthetic materials to their biological counterparts.

Morphological analysis (SEM, TEM)

Scanning electron microscopy (SEM) ought to be employed as the principal method for recording the surface and mesoscale structure of corneal scaffolds. It is important to report fiber-diameter distributions, fiber alignment, surface imperfections (like beading), and pore interconnectivity in full, rather than just using single-value summaries. The quantification obtained via SEM facilitates direct comparisons with previous studies on corneal scaffolds, associating aligned or lamellar microarchitectures with enhanced optical transmission and anisotropic mechanical properties. Recent investigations into electrospun and 3D-fabricated corneal constructs have utilized SEM to illustrate lamellar alignment and the absence of bead formation in nanofibers, correlating with improved transparency and cellular coverage. ¹³¹

In addition, transmission electron microscopy (TEM) works well with SEM because it shows intrafiber and ultrastructural features that have a big effect on optical properties and bioactivity. Transmission electron microscopy can distinguish between core-shell morphology in coaxial fibers, fibrillar packing, collagen-like banding, and the state of dispersion or aggregation of incorporated nanoparticles or drugs at a scale of 1–10 nm. Recent comparative studies demonstrate that TEM validation of uniform nanoparticle incorporation or fibrillar organization forecasts enhanced mechanical homogeneity and more reliable epithelial/endothelial cell monolayers on scaffolds. ²⁴³ This necessitates the existence of paired SEM data sets, including quantitative distributions, alignment indices, and porosity estimates, along with TEM data focusing on the ultrastructural organization and localization of nanoparticles. Morphometric data should then be analyzed in conjunction with optical and cellular results.

Mechanical testing (tensile strength, Young’s modulus)

Uniaxial tensile testing is usually used to make stress-strain curves, which are used to test the mechanical performance of engineered corneal scaffolds. This process yields important quantitative metrics, like ultimate tensile strength and strain at break, as well as elastic properties, like Young’s modulus, which comes from the first linear segment. These factors have a big impact on how well the surgery goes, how well the sutures hold, and how well the graft can handle intraocular forces without becoming too misshapen. It is important to report both tensile strength and the method for calculating Young’s modulus because the method and the sample’s properties have a big effect on the values that come out. The hydration level, sample shape, testing speed, and type of test (uniaxial, biaxial, or indentation) can all affect modulus estimates by a wide range of values. Comprehensive reviews suggest integrating standard uniaxial tensile tests with an exhaustive account of sample preparation (including thickness, hydration, and gauge length) and additional tests (such as burst or suture pull-out tests) to enable significant comparisons of scaffold data with native corneal benchmarks.^244,245

Comparative experimental studies demonstrate the influence of the fabrication method on mechanical properties and the relationship between reported values and those of native tissue. Kong et al. created a compressed-collagen/laser-perforated electrospun hybrid that, when set up correctly, showed an ultimate tensile stress of about 3.42 ± 0.22 MPa, which is similar to what has been found for native corneal stroma. The research demonstrated that macro-perforation, alignment of electrospun fibers, and collagen compression can be adjusted to either enhance or diminish stiffness and strength as needed. Recent evaluations of polymer scaffolds for corneal applications demonstrate that softer hydrogel-based Descemet/Endothelium substrates must achieve a balance between reduced Young’s moduli, essential for compatibility with the fragile posterior lamella, and adequate handling strength. Thin supportive mats or controlled cross-linking techniques are often used to address this compromise. These comparative results suggest that authors should provide both tensile strength and the Young’s modulus obtained from the slope, along with comprehensive test conditions. Furthermore, the reasons for choosing materials, alignment, crosslinking, and perforation for the scaffold should be backed up by showing how the construct fits in with the native corneal ranges that have been reported in the literature.^246,247

Optical property assessment

Optical transparency and light-scattering properties are critical functional parameters for engineered corneal scaffolds, as even minimal opacity can markedly affect visual acuity post-implantation. It is common to use spectrophotometric measurements of transmittance and absorbance in the visible spectrum (380–750 nm) to test optical properties. These measurements include the total and diffuse transmittance, as well as the haze and scattering indices. In an experimental study conducted by researchers, collagen scaffolds at a concentration of 10% were tested as a measure of optical property transparency. The transmittance of these scaffolds at different wavelengths of visible light (450, 500, 550, 600, 650, and 700 nm) was evaluated using UV/vis spectroscopy and directly compared to normal mouse corneas. The results indicated that, despite satisfactory apparent transparency in all samples, the scaffold with a 10% collagen concentration exhibited the greatest similarity to the natural cornea in terms of leveling. This study underscores a significant engineering dilemma: lower collagen concentrations enhance transparency, whereas higher concentrations (e.g., 20%) provide superior mechanical strength, which is crucial for suturing and surgical procedures. This finding confirms that simultaneously achieving high optical clarity and sufficient mechanical strength remains a major obstacle in the development of corneal scaffolds. ²⁴⁸

Photographic-based optical assessment methods, along with spectrophotometry, provide a direct, image-based depiction of transparency and blurring, making them suitable for swift evaluations. Gonzalez-Andrades et al. devised a photographic-based method (PBM) that involves capturing a standardized stripe pattern from scaffold samples. They figured out a “transparency ratio” (TR) and a “blurring index” (BI). They discovered a robust correlation between TR (approximately 80.3 ± 2.8%) and the gold-standard inverse adding-doubling (IAD) measurements of diffuse transmittance across wavelengths (ρ > 0.6). Researchers in scaffold development can determine the optical purity of high spectral transmittance by contrasting these two methodologies. ²⁴⁹ Then they can decide if the best way to optimize is to reduce scattering (by aligning fibers, matching the refractive index, or separating the micro-phase) while also increasing raw throughput.

In vitro degradation and swelling studies

In vitro degradation and swelling assays are critical functional assessments for corneal scaffold candidates, forecasting alterations in thickness, porosity, and optical performance in the initial post-implantation phase. Hydrogels and protein-based membranes generally undergo degradation through enzymatic cleavage, involving collagenases and proteases, alongside hydrolytic processes in buffered saline. When looking at how well a scaffold can support cell engraftment without causing long-term inflammation, it’s important to identify the right balance between network stability and controlled mass loss. After 14 days in PBS, a gelatin electrospun nanofiber membrane made to replace Descemet’s membrane showed a thickness reduction of about 88.6% and changes in permeability. This example shows that quick degradation can make it easier to move donor cells quickly, but it could also hurt the structure if it doesn’t happen at the same time as the biological timeline. The kinetic results are significantly influenced by factors such as crosslinking chemistry and density (physical vs covalent), polymer composition (natural vs synthetic), and scaffold microstructure (including fiber diameter and porosity). Comparative in vitro protocols must document mass loss over time in conjunction with additional metrics, such as thickness, permeability (e.g., FITC-dextran flux), and variations in mechanical stiffness throughout the incubation period.^18,247

Swelling is a crucial process because small changes in how much water is absorbed can change the alignment of the refractive indices and the curvature of corneal implants, which can then change how clear and powerful they are. Recent comparative analyses and experimental studies highlight two principal considerations: (1) Dual-crosslinked hybrid hydrogels, which have both physical and photo- or thermally activated covalent crosslinks, break down more slowly and in a more controlled way than single-mode networks. This improves dimensional stability and optical clarity. (2) Electrospun proteinaceous membranes usually lose mass faster, but they also have better initial permeability and cell compatibility. You can change the amount and type of cross linker to get the desired in vitro residence time.^250,251

A thorough characterization of engineered corneal scaffolds, encompassing morphological, mechanical, optical, and degradation assessments, creates a fundamental basis for forecasting in vitro performance and facilitating in vivo application. Morphological profiling through SEM and TEM confirms microstructural integrity and cell-interactive topographies. Mechanical testing checks the cornea’s elasticity and strength, which should be similar to the cornea. An optical assessment makes sure that things are clear and that light doesn’t scatter too much. In vitro degradation and swelling studies corroborate dimensional and compositional stability during the healing process. The interconnected evaluations show how processing variables affect structural integrity, transparency, and biological performance. This approach makes it possible to optimize scaffold design in a logical way. Corneal tissue-engineering strategies can only reach clinically significant levels of transparency, biomechanics, and biodegradability that meet the physiological needs of the human cornea through multi-parametric and comparative characterization.

Naturally biomaterials-derived nanoparticles

Natural nanoparticles’ use in drug delivery methods for corneal disease is being studied. Since biodegradable nanocarriers have demonstrated sufficient capabilities for this purpose, they are among the most appealing and challenging techniques for recurrent corneal drug delivery. However, drugs may be able to stay in different areas of the cornea for a longer period by using these types of nanoparticles (Figure 6).

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Figure 6.

Schematic illustration showing the preparation of nanoparticles from natural polymers such as chitosan, gelatin, collagen, and hyaluronic acid for corneal tissue engineering applications.

Synthetic nanoparticle

Composite nanoparticle

Synthetic and natural polymers can be combined to achieve improved functional properties. Synthetic polymers are utilized for their stable mechanical properties, while natural polymers are applied to improve cellular behavior. Biodegradable polymers, such as PCL, PLA, PEG, and PLGA, are widely studied for ophthalmic applications owing to their capacity to enhance the residence time of formulations on the precorneal surface.

Polymeric nanoparticles are promising carriers for ocular drug delivery. They have many benefits, such as being biodegradable, biocompatible, and non-toxic. They also help poorly permeable drugs get into cells better, keep drugs in cells and tissues longer, and release drugs more slowly. They possess the capacity to transform ocular drug delivery, overcoming traditional limitations and improving therapeutic outcomes. This section summarizes the most common composition of natural-synthetic polymeric nanoparticles for corneal treatment.

Ex vivo corneal culture models

Corneal tissue transplantation is the standard treatment for corneal injury, but it faces limitations, including limited donor tissue resources.^353,354 Therefore, it is essential to develop alternative approaches to simulate various corneal injuries and wounds, investigate the penetration of ocular drugs, evaluate the efficacy of various drugs, and investigate the efficacy of cell therapy that ultimately leads to the restoration of normal corneal function. Accordingly, the use of ex vivo corneal culture models can be considered as a suitable option. These models offer a 3D structure to explore topical or systemic drugs to test their toxicity, penetration and effect under near-to-physiological conditions, free from ethical and biological limitations of animal research. Moreover, these models can be used to assess cell adhesion, proliferation, differentiation, and study wound healing and scarring processes in the human cornea.^355–357 Ex vivo corneal culture models serve as the bridge between in vitro cell cultures and in vivo animal experiments, since they allow for a more reproducible and safe environment, presenting a microenvironment that physically resembles that of native cornea with lesser ethical or practical problems compared to live humans or animals These models can be basically divided into two groups: whole-organ corneal culture models and partial- organ corneal culture models. Whole-organ corneal culture models are cultured for weeks in cell-culture medium the entire cornea and keep it transparent with multilayered tissue under sterile conditions. This culture system provides the means to study functional epithelial regeneration, endothelial viability and stromal response in a more physiological environment. In partial organ culture models the cornea is also isolated in smaller strips or pieces of tissue which will undergo culturing conditions promoting cell growth and migration. These models are particularly useful for studying specific corneal cell types and investigating wound healing dynamics at the cellular level. The choice between whole-organ and partial-organ culture models depends on the purpose of the experiment. Examples of studies conducted in recent years based on these methods are listed in the Table 3.

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Table 3.

Overview of recent ex vivo and organ culture corneal models highlighting their design principles, applications in wound healing, infection, and endothelial studies, and corresponding cell or tissue sources.

Model type	Description		Main applications	Corneas/cells source	Reference/year
Whole Organ Corneal Culture Model	Development of an ex vivo human corneal epithelial wound-healing model using 1-heptanol and assessment of bone marrow-derived mesenchymal stromal cell small extracellular vesicles (MSC-sEV) treatment effects on repair		To investigate epithelial healing and the therapeutic potential of MSC-sEV in corneal repair	Human cornea; MSC-sEV drived from human bone marrow mesenchymal stromal cells	355
					2025
	Investigated expression and localization of Yes-associated protein (YAP) in human corneal endothelial cells before and after storage in organ culture, to understand how mechanical cues and culture conditions affect cell behavior and homeostasis		To study biomechanical regulation and homeostatic mechanisms of human corneal endothelial cells during organ culture storage, aiming to improve corneal preservation and transplantation outcomes	Human corneal endothelium	358
					2024
	An ex vivo bovine cornea model was used to study infection caused by several bacterial pathogens and to investigate antibacterial efficacy		Reliable ex vivo model for studying bacterial corneal infection and testing antimicrobial treatment, cost-effective	Bovine corneas	359
					2023
	Establishment of a simple ex vivo organ culture model using porcine corneas to study epithelial wound healing. The model used free-floating corneal disks with a punch defect and allowed observation of wound closure under different media conditions, wound sizes, and limbal presence. Modulation of healing was tested with Mitomycin C		Evaluation of epithelial wound healing, drug/toxicity testing, and regenerative or pharmacological studies (e.g., testing mitosis inhibitors like Mitomycin C)	Porcine (pig) corneas	360
					2021
	Bacterial keratitis is induced by infecting the corneas with Pseudomonas aeruginosa, and the system can be used to study drug treatment		Evaluation of drug efficacy and corneal tissue response to bacterial infection (bacterial keratitis)	Porcine (pig) corneas	361
					2020
	Anterior keratectomy wound created on corneas while still in the globe, and then corneas are excised and cultured on a collagen/agar base for 2 weeks in supplemented-serum free medium with stabilized vitamin C		Assessment of wound closure, myofibroblasts development, fibrotic markers, and testing of drugs or gene knockdown	Porcine (pig) corneas	357
					2019
	Tissue engineered corneal endothelium was developed by seeding porcine corneal endothelial cells onto a compressed collagen I hydrogel (RAFT). The graft was transplanted onto ex vivo human cornea to evaluate attachment and restoration of endothelial function		Restore corneal endothelial function and normal thickness	Porcine corneal endothelial cells (PCECs)/ex vivo human cornea	362
					2025
	Immersed culture model	Human donor corneas were wounded using an excimer laser, with or without prior removal of epithelium and limbus. Histological and immunofluorescence analyses were performed to mimic corneal scarring	Developing reproducible ex vivo models to study human corneal scarring and test pharmacological agents that prevent or treat haze formation	Human donor corneas	356
	Air-liquid interface culture model				2012
Partial organ culture Model	Perfused corneal rim organ culture model	Developed a glue-based ex vivo perfusion organ culture system using human corneal rims containing trabecular meshwork (TM) to study aqueous humor outflow and TM biomechanics. The model used 3D-printed perfusion plates and measured outflow facility, TM stiffness, and morphology	To establish a low-cost and accessible ex vivo perfusion model for studying trabecular meshwork function, intraocular pressure regulation, and drug effects in glaucoma research	Human corneal rim with intact trabecular meshwork	363
					2022
	Establishment of an ex vivo porcine corneal endothelial organ culture model using split corneal buttons. In this method, after partial corneal trephination, the outer corneal layers (epithelium, Bowman’s layer, part of stroma) were removed to reduce stromal swelling and preserve the endothelium. The model allows stable cultivation up to 15 days without using deswelling agents like dextran		Investigation of corneal endothelial cell physiology, preservation, and response to external factors or drugs; provides a stable alternative to human donor corneas for endothelial studies	Porcine (pig) corneas (split corneal buttons)	364
					2019

In vivo assessment in animal models of corneal injury

Analysis of corneal regeneration, transparency, and immune response in animal models is an important step in pre-clinical tests for ocular therapies. In vivo research offers the possibility of studying corneal healing biological events in an intact physiological setting, which involves interactions between epithelial, stromal and endothelial cells as well as from nerves, tear film and immune elements. Such studies are suitable for experiments across several animal models but predominantly on rabbits, rats and mice primarily because of the size of the cornea and due to its ease in handling and organized ocular anatomy, respectively. Corneal damage is usually achieved through mechanical debridement, chemical burns thereof or laser ablations. After injury it is possible to apply the therapeutic strategies including cell therapy, biomaterial scaffolds and pharmacological agents. The animals are followed over time to analyze corneal wound healing, re-epithelialization and stromal remodeling. These evaluations are confirmed by histology, molecular and imaging approaches for a more complete understanding of the healing response.

Evaluating regeneration, transparency, and immune response

Regeneration: Corneal re-epithelialization, stromal repair and corneal thickness restoration are used to measure the degree of regeneration. Imaging techniques that include slit-lamp biomicroscope, optical coherence tomography (OCT) and fluorescein staining permit visualization of epithelial integrity and stromal remodeling. Histological analysis and immunohistochemical evaluation are also used in the evaluation of corneal regeneration.^365,366

Transparency: Corneal transparency is an important criterion to evaluate functional recovery. Clinically, it can be assessed by slit-lamp examination, measuring light transmission and haze scoring. Quantitative techniques such as in vivo confocal microscopy and densitometry to analyse corneal images taken allow for objective measurement of opacity.^367,368

Immunological Response: Infiltration of inflammatory cells and cytokine production are measured in order to investigate immune reactions. Methods include immunohistochemistry using immune cell markers (CD45, CD11b), flow cytometry of isolated corneal cells, pro-inflammatory cytokines ELISA and in vivo imaging fluorescently labeled immune cells. Such analyses can aid in the assessment of the biocompatible effects for therapeutic interventions and associated side-effects specifically of immune origin.^369–371 Table 4 summarizes the in vivo evaluation for corneal regeneration, transparency, and immune response.

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Table 4.

In vivo assessment of corneal regeneration, transparency, and immune response.

Evaluation aspect	Parameter/Marker	Method/Technique
Regeneration	Epithelial healing rate (re- Epithelialization)	• Fluorescein staining
		• Slit-lamp photography
		• Digital image analysis
	Stromal regeneration	• Histology (H&E, Masson’s trichrome)
	Epithelial/stromal/corneal thickness	• OCT imaging
	Corneal cell density	• SHG microscopy
	Cell proliferation	• Ki67 immunostaining
	Cellular structure and regeneration	• qPCR of proliferative genes
		• F-actin staining
	α-SMA, fibronectin, collagen I, collagen III proteins	• Immunofluorescence Staining
Transparency	Corneal opacity scoring	• Slit-lamp biomicroscopy
		• Photographic grading (0–4 scale)
	Light transmission	• UV-Vis spectrophotometry
		• Densitometry
	Stromal organization	• OCT
		• Confocal microscopy
		• SHG imaging
	Haze quantification	• Image analysis (ImageJ)
		• Backscatter measurement
Immune Response	Cytokine expression (such as TNF-α, IL-1β, IL-6, IL-10)	• qPCR
		• ELISA
	Macrophage polarization	• Flow cytometry
		• CD68/CD163 immunostaining