Advanced 3D scaffolds for corneal stroma regeneration: a preclinical progress

Abstract

Corneal stromal defects represent a significant global cause of blindness, necessitating innovative therapeutic strategies to address the limitations of conventional treatments, such as corneal transplantation. Tissue engineering, a cornerstone of regenerative medicine, offers a transformative approach by leveraging biomaterial-based solutions to restore damaged tissues. Among these, three-dimensional (3D) scaffolds fabricated using advanced techniques like 3D printing have emerged as a promising platform for corneal regeneration. These scaffolds replicate the native extracellular matrix (ECM) architecture, providing a biomimetic microenvironment that supports cell proliferation, differentiation, and tissue integration. This review highlights recent advances in the design and fabrication of 3D scaffolds for corneal stroma engineering (CSE), emphasizing the critical interplay between scaffold architecture, mechanical properties, and bioactive signaling in directing cellular behavior and tissue regeneration. Likewise, we emphasize the diverse range of biomaterials utilized in scaffold fabrication, highlighting their influence on cellular interactions and tissue reconstruction. By elucidating the complex relationship between scaffold design and biologics, this review aims to illuminate the evolution of next-generation strategies for engineering functional corneal tissue. Eventually, this review will provide a comprehensive synthesis of the current state-of-the-art in 3D scaffold-based corneal tissue engineering (CTE), offering insights that could advance progress toward effective vision restoration therapies.

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Amin Orash Mahmoudsalehi

Amin is a PhD candidate in Nanotechnology at Tecnológico de Monterrey (Tec), Mexico, specializing in corneal tissue simulation and advanced biomaterials for corneal stromal regeneration. Initially trained in Dr Mamidi's lab at Tec, Amin has developed multidisciplinary expertise in biomaterials, tissue engineering, electrospinning, scaffold fabrication, and drug delivery systems. Currently, he is working in Dr Wendy's lab. His research extends beyond ophthalmology, exploring engineered living drug delivery systems for advanced disease therapeutics. Amin has secured competitive research grants from institutions such as the Shahid Beheshti University of Medical Sciences and Isfahan University of Medical Sciences, enabling innovative work on biomaterial-based regenerative strategies. His contributions are documented in high-impact peer-reviewed publications, advancing knowledge in tissue engineering, regenerative medicine, and drug delivery. Actively engaged in international collaborations, Amin also serves as a reviewer for scientific journals, further solidifying his standing in the research community. Recognized for his academic excellence, Amin holds a fully funded scholarship from Tecnológico de Monterrey, underscoring his passion for nanotechnology and regenerative medicine. His work exemplifies the transformative impact of interdisciplinary research, bridging biomaterials science with clinical applications to address complex biomedical challenges. Amin's dedication to innovation and collaboration positions him as a promising contributor to the future of regenerative therapies and advanced drug delivery systems.

Maryam Soleimani

Maryam is a PhD student at the Silesian University of Technology, Faculty of Mechanical Engineering, Department of Didactic Laboratory of Nanotechnology and Material Technologies. Her research focuses on the development of dental implants utilizing 3D printing and selective laser melting (SLM) techniques. Maryam has expertise in biomaterials, tissue engineering, scaffold fabrication techniques, advanced 3D printing methods, bioceramics, and biometals. Her work aims to enhance implant performance through innovative material design and fabrication strategies. Her research contributions have been featured in high-impact dental journals, showcasing advancements in dental implants, bone regeneration, and tissue engineering. Maryam has secured multiple research grants, including support from the Excellence Initiative – Research University program at the Silesian University of Technology, providing funding for researchers initiating new scientific projects. She is actively engaged in cutting-edge research and collaborates on interdisciplinary projects that contribute to advancements in regenerative medicine and biomedical engineering. Her academic excellence and potential are further acknowledged through a fully funded scholarship from the Silesian University of Technology.

Kevin Stalin Catzim Rios

Kevin Stalin Catzim Rios is a PhD candidate in Nanotechnology at Tecnológico de Monterrey, specializing in the fabrication of polymeric scaffolds using electrospinning for cardiac tissue engineering applications. He holds a bachelor’s degree in Nanotechnology and Chemical Sciences, along with a master’s degree in Nanotechnology. Throughout his academic career, he has published an article in a high-impact scientific journal that focuses on tissue engineering, biomaterials, and nanotechnology. His research aims to contribute to the development of advanced biomaterials for regenerative medicine applications. Due to his outstanding academic performance, he has received a full tuition scholarship from Tecnológico de Monterrey.

Wendy Ortega-Lara

Dr Ortega serves as a distinguished Professor at the School of Science and Engineering at Tecnológico de Monterrey in Mexico. Her expertise lies in the design, synthesis, and characterization of bioceramics. She focuses on developing advanced bioinks for 3D printing, innovative biomaterials for bone regeneration, and specialized meshes for medical applications. Dr Ortega has a research stay at the University of Cambridge and Addenbrookes Hospital. Her projects aim to advance the development of bioceramics and their application in various manufacturing techniques, including forcepinning®, electrospinning, microinjection, and additive 3D manufacturing, utilizing the unique properties of nanoparticles. Additionally, Dr Ortega is recognized as a Level 1 member of the Mexican System of Researchers. She holds the 2024 patent for “Hydroxyapatite obtained from wastewater, its production method, and its uses” and the patent for “Method to produce a customizable collagen membrane for biomedical purposes.” Dr Ortega is also the co-founder of Synthetic Ocular Restoration.

Narsimha Mamidi

Dr Mamidi is a distinguished scientist at the University of Wisconsin-Madison, USA, where his research focuses on the development and validation of cutting-edge nanobiomaterials and nanotherapeutics to enhance their therapeutic efficacy in cancer immunotherapy and gene delivery, pushing the boundaries of translational science. Prior to his current role, Dr Mamidi served as an Assistant Professor at Tecnológico de Monterrey (Tec), leading a dynamic team that engineered advanced biomaterials with tailored properties for diverse applications, including drug delivery, orthopedic implants, tissue engineering, and biomedical devices. His research drives the understanding of tissue-material interactions under clinically relevant conditions, accelerating the transition of innovative technologies from bench to bedside. With over 59 peer-reviewed publications in prestigious journals, Dr. Mamidi's work has made a significant impact, amassed more than 1800 citations, and achieved an H-index of 27 and an i10-index of 45. His contributions have been recognized with numerous accolades, including the 2015 Eli Lilly Award, the 2018 and 2021 National Research System (SNI) awards, and the distinction of being named an outstanding research professor at Tec. Beyond his research, Dr Mamidi actively engages in editorial roles for leading nanomedicine and biomaterials journals, demonstrating his commitment to advancing scientific knowledge and fostering innovation. Dr. Mamidi continues to shape the future of nanobiomaterials and their transformative therapeutic applications through his interdisciplinary approach and unwavering dedication to translational science.

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1. Introduction

The cornea, a transparent tissue composed of three cellular layers and two membranes, serves as the eye's primary refractive element.¹ Beyond its optical function, the cornea acts as a critical barrier, protecting the eye from chemical and mechanical injury, pathogenic infections, and ultraviolet radiation.^1–4 Disruptions to the corneal structure, whether due to immune disorders, genetic conditions, or trauma, can lead to severe visual impairment or blindness.^5–7 Corneal transplantation remains the gold standard for treating corneal diseases, with over 10 million individuals globally and 40 000 in the United States requiring the procedure, as reported by the World Health Organization (WHO).^8–10 However, the widespread application of corneal transplantation is hindered by the limited availability of donor tissues, the high cost of surgical intervention, and the risk of immune rejection.^8–13 Furthermore, aging-associated corneal endothelial cell senescence exacerbates corneal dysfunction, compromising the outcomes of transplantation.^14–16 Refractive surgeries, such as laser-assisted in situ keratomileusis (LASIK), while effective in correcting vision, render the cornea unsuitable for donation, further limiting the donor pool.^17–19

To address these challenges, regenerative medicine approaches, including stem cell therapy and tissue engineering, have emerged as promising alternatives for generating bioengineered corneas or individual corneal layers.^20–22 Tissue engineering, which integrates cells, bioactive molecules, and scaffolds to create functional tissues, has shown particular potential in this regard.^23–25 Notable progress in corneal surgery, human corneal cell keratoplasty (HCCK), employs transparent carriers to enhance the behavior of human corneal cells (HCCs), demonstrating success in both lamellar and full-thickness corneal transplants.^26–28 However, these approaches still depend on donor corneas, which remain limited in supply and are susceptible to immune rejection.^29–31 Recent research has highlighted the potential of maintaining the proliferative capacity of corneal epithelial cells (CEpCs), offering a promising strategy for CTE and the repair of corneal damage (Fig. 1).^32–37

Fig. 1 Illustration of the corneal bioengineering process. After isolating and identifying a suitable cell source, cells undergo in vitro culture to support their differentiation, proliferation, and/or expansion. A carefully selected medium and scaffold are essential to provide the necessary microenvironment for tissue formation. The engineered cells are then subjected to preclinical animal testing and clinical trials to validate their efficacy and safety before being considered for therapeutic applications in treating corneal disorders.

Significant progress has been made in developing tissue-engineered scaffolds that replicate the structural and functional properties of the cornea.³⁸ This review focuses on the anatomy and physiology of the corneal stroma, common ocular disorders affecting this layer, and the latest strategies for engineering stromal layer scaffolds. We emphasize the structural and biological requirements for designing effective stromal scaffolds and provide a comprehensive analysis of recent advances in scaffold design, characterization, and evaluation for CSE. Our discussion offers a novel perspective, highlighting the engineering principles underlying scaffold fabrication and the incorporation of functional additives. While fabrication methods alone may not fully dictate scaffold properties, the interplay between fabrication processes and additive selection critically influences the mechanical and biological performance of CSE scaffolds. We begin by emphasizing the structural properties essential for effective 3D scaffolds, drawing on recent studies that have explored various fabrication techniques and their impact on scaffold characteristics. Subsequently, we explore the biological engineering aspects of 3D scaffolds, reviewing the components that can enhance stromal tissue regeneration. By integrating structural and biological considerations, this review aims to advance the field of corneal stroma engineering and contribute to the development of innovative solutions for restoring vision in patients with corneal disorders.

2. Stromal anatomy and physiology

The cornea, serving as the eye's window, is remarkably transparent, characterized by specialized avascular tissue with a unique structure. Positioned at the anterior part of the eye, the cornea assumes a dome-shaped structure, providing adequate mechanical stability to withstand pressure and eye movements (Fig. 2(A)).³⁸ Comprising three cell layers and two membranes, namely epithelium (outermost), stroma, endothelium (innermost), Bowman's membrane, and Descemet's membrane, the cornea exhibits a complex organization (Fig. 2(B)).³⁹ Functioning as the eye's last superficial barrier, it shields against external hazards and infections. Sensory innervation and corneal nerves are essential for maintaining and safeguarding the ocular surface.³² Post-surgery corneal recovery relies significantly on factors such as corneal sensation and nerve density.⁴⁰ Notably, the eye's anterior surface houses the aqueous humor, contributing to the malleability essential for corneal function.⁴¹ Additionally, the tear film in the outermost part of the eye serves as a reservoir for growth and antibacterial factors.² Enveloping the corneal smooth surface, the tear film plays a critical role in maintaining homeostasis, supporting proliferation, and aiding in repair.¹⁸

Fig. 2 (A) Illustration of the eye showing its anatomical structures, including the cornea. Reprinted with permission.¹⁴ Copyright © [2024], Elsevier. (B) Diagram of the human corneal layers: the outermost stratified squamous epithelium, followed by Bowman's layer, the stroma with keratocytes, Descemet's membrane, and the single-layer endothelium. Reprinted with permission.¹⁴ Copyright © [2024], Elsevier. (C) Overview of corneal tissue transplantation techniques: PK involves full-thickness corneal replacement of the central part; ALK replaces only the stroma; DSAEK and DMEK are partial-thickness transplants targeting the endothelium, with DMEK providing a pure endothelial replacement without stromal cells. Reprinted with permission.¹⁴ Copyright © [2024], Elsevier. (D) Progression of in vitro HCCs culture techniques. The shift from conventional 2D CEC culture to an advanced 3D corneal structure requires accurately mimicking the natural in vivo environment. Critical factors include scaffold-cell interactions, cell–cell communication—such as endothelial-stromal signaling—and endothelial-Descemet's membrane connections, as well as cell-surface interactions, all of which contribute to developing a 3D corneal graft. The concept of 4D CEC culture introduces a dynamic aspect, where the corneal structure evolves. This self-adapting transformation depends on carefully engineered bioactuators, and dynamic cell-scaffold interactions. Reprinted with permission.⁴² Copyright © 2020, Springer Nature. (E) Summary of bioengineering strategies for corneal tissue replacement. Reprinted with permission.¹⁴ Copyright © 2024, Elsevier. (F) Depiction of an injured human cornea undergoing either uncontrolled or controlled CSE. Uncontrolled regeneration (top) may result from untreated severe stromal injuries or unsuitable implants, leading to irreversible keratocyte-to-(myo)fibroblast transitions and disorganized ECM production. Controlled regeneration (bottom) is supported by a well-integrated bioengineered implant that promotes keratocyte infiltration, reversible keratocyte-to-fibroblast transition, and organized ECM deposition. Reproduced with permission.⁴³ Copyright © 2024, Wiley. (G) Common materials, modifications, and fabrication techniques used for designing stromal constructs with optimal mechanical, optical, and biocompatible properties. Reproduced with permission.⁴³ Copyright © 2024, Wiley.

The stromal layer, at approximately 500 μm, is the thickest, while the epithelium and endothelium are less than 50 μm thick. Occupying over 90% of the cornea, the stromal layer is crucial for corneal main functions due to its transparency and mechanical strength, influenced by COL alignment and fibril cross-linking. Its mechanical properties vary across the peripheral and central sections, affecting COL orientation and corneal cell behavior.⁹ The stroma contains almost 8% CKCs and is an acellular but dense connective layer derived from neural crest cells.⁴⁴ Consisting of over 200 noncellular collagenous lamellae with uniformly aligned small COL fibers, the stroma's flattened fibroblasts are activated during injuries to produce COL, stabilize collagenous lamellae, and secrete stromal components.⁴⁵ A healthy stroma exhibits optical transparency and suitable mechanical strength, which is crucial for maintaining light transmittance. Challenges for tissue engineers include achieving equal mechanical stability and high optical transparency.⁴⁶

The corneal stroma comprises both extracellular and cellular elements. Cellular components within the mature corneal stroma are known as CKCs.⁴⁰ These cells, with a dendritic morphology, play a crucial role in maintaining the ECM of the stroma.⁴⁷ CKCs actively produce keratocan and lumican, essential factors in preserving the shape and transparency of the stroma. Keratocan and lumican, belonging to the small leucine-rich protein family, serve as vital keratan sulfate proteoglycans in the corneal stroma.⁴⁸ While keratocan is uniquely found as a proteoglycan in the cornea, lumican may also exist as a glycosylated protein in various tissues. Both keratocan and lumican interact with COL fibrils, regulating the tissue's structure within defined limits for specific properties. Notably, keratocan is crucial in preserving the corneal structure, as previous studies have shown.^48–50

During wound healing processes, the dendritic morphology of CKCs transforms into a fibroblastic appearance. This transformation is associated with a decrease in two critical functions of keratocytes—expression of keratocan and synthesis of keratan sulfate-highlighting changes during fibroblast/myofibroblast transformation.⁴⁶ Both isolated keratocytes from the corneal stroma and cultured keratocytes exhibit fibroblastic/myofibroblast phenotypes, demonstrating decreased keratocan expression and keratan sulfate synthesis, mirroring in vivo wound healing processes. This underscores that keratocan can serve as an indicator of the native keratocyte phenotype.^43,51,52

3. Corneal scarring: an in-depth examination

A thorough exploration of the cornea's structural foundations is imperative to comprehend deficiencies or diseases within the visual system. This underscores the need for a significant focus on individualized medical and surgical regenerative therapies. Vision impairment hindrance of light to the eye has unveiled valuable biological insights into ophthalmic diseases, ranging from superficial layer damage and limbal cell issues to corneal injuries.⁹

3.1. Keratoconus

Keratoconus is a progressive, non-inflammatory corneal disorder characterized by a generalized weakening of the corneal connective tissue, which leads to corneal thinning and protrusion, resulting in a change from its normal dome shape to a cone-like structure with gradual bulging.⁵³ Patients may experience blurred vision in its early stages, displaying symptoms like irregular astigmatism and refractive errors.⁵⁴ As keratoconus advances, the visual impairment becomes more pronounced, depending on the severity of the condition. With further progression, keratoconus becomes easier to diagnose, as affected individuals report issues such as diminished night vision, sensitivity to light (photophobia), severe headaches due to eye strain, and frequent eye itching. The condition generally affects both eyes and typically begins during adolescence. In advanced stages, corneal scarring develops, contributing to additional vision deterioration, often reaching a stage where corneal transplantation becomes necessary to restore vision.^55,56 Keratoconus leads to stromal scarring, axial thinning, disintegration of the epithelial basement membrane, and ruptures in Bowman's membrane. Clinical reports suggest that the progression of keratoconus transforms regular astigmatism into irregular astigmatism.⁵⁷

3.2. Dry eye disease

Dry eye encompasses a diverse array of ocular surface disorders. As outlined in the 2007 International Dry Eye Workshop, dry eye is a multifactorial condition characterized by symptoms such as tear film instability, visual disturbances, ocular discomfort, and potential damage to the eye surface.^58–60 Based on earlier research, it is estimated that around 4.91 million individuals in the United States are affected by dry eye disease. Moreover, millions more experience milder symptoms, which, if left untreated, may progress to more severe dry eye, often exacerbated by factors such as prolonged use of digital screens or wearing contact lenses.⁶¹ The underlying pathophysiology involves either increased tear evaporation, reduced tear production, or a combination of both, which leads to tear film hyperosmolarity and inflammation of the ocular surface. In moderate to severe cases, the integrity of the corneal epithelium is compromised, evidenced by punctate epithelial erosions, which can be identified through fluorescein staining. Standard treatment for moderate to severe dry eye includes tear supplementation, anti-inflammatory eye drops, eyelid hygiene, punctal plugs, and oral tetracyclines.⁵⁹

3.3. Bacterial keratitis

Bacterial keratitis is recognized as a severe corneal infection that arises when the ocular defense mechanisms are compromised. Consequently, its progression leads to inflammation and gradual vision loss. Notably, epithelial defects and reduced corneal sensitivity are key factors contributing to developing severe ulcers, stromal necrosis, and bacterial proliferation. Disruption of the protective barrier and the loss of ocular surface integrity allow bacterial pathogens to penetrate the cornea.⁶² The primary causative agents of bacterial keratitis are Staphylococcus aureus, Streptococcus pneumoniae, and Staphylococcus epidermidis.⁶³

3.4. Light and chemical injuries

Exposure to UV light from various sources can damage the corneal epithelium, which is often detected through fluorescein staining. Some of the primary causes of corneal surface lesions include snow blindness, the use of tanning beds, direct exposure to lightning, and direct observation of the sun. Other factors, such as chemical burns, can weaken the corneal surface cells, hinder epithelial regeneration, and may even result in blindness.⁶⁴ Current treatments for minor conditions typically focus on enhancing the recovery of the corneal epithelium. However, for severe cases, advanced cell-based therapies are required. These may involve stem cell transplants, allografts, and limbal autografts to support re-epithelialization.⁶⁵

3.5. Corneal abrasion and foreign body

Corneal abrasions are frequently encountered in patients with epithelial layer damage, as this layer is highly susceptible to injury. These abrasions are often accompanied by symptoms such as a foreign body sensation, pain, tearing, light sensitivity, and blurred vision, typically linked to a history of eye trauma.⁶⁶ If a foreign body is present in the cornea, immediate treatment is required to prevent permanent scarring and extensive epithelial damage. Deep injuries involving contaminated foreign material can lead to serious complications, including traumatic iritis, recurrent erosion syndrome, bacterial keratitis, and corneal ulcers.⁶⁶

4. Structural and biological characteristics in corneal stroma equivalent

Although therapies such as HCCK offer treatment options (Fig. 2(C)), regenerative medicine has emerged as a modern approach for reconstructing various layers of the cornea, including the epithelium, stroma, and endothelium.⁶⁷ While the epithelium layer encompasses differentiated states of epithelial cells, the endothelium layer adopts a monolayer cell structure. In contrast, the corneal stroma is a thick layer with a highly organized structure.⁶⁸ Tailoring cell delivery techniques based on the native structure of each layer, both flat, 2D, and/or 3D approaches have been employed to facilitate tissue healing.^69–75 Among these approaches, cell sheet-based delivery has gained attention for CSE. For instance, Chen et al. reported a small molecule-based method to differentiate hiPSCs into CKCs using an EB induction approach. The differentiated cells exhibited significantly upregulated expression of keratocyte markers ALDH1A1, lumican, and keratocan, confirmed by quantitative RT-PCR (p < 0.05), immunostaining, and western blot analysis (p < 0.01). Importantly, hiPSC-CKCs retained their keratocyte phenotype without transitioning into fibroblasts or myofibroblasts, highlighting the potential of this approach for CSE.⁷² While 2D scaffolds may offer suitable mechanical properties and fibril structure for cultured cells in the epithelium and endothelium layers, they fall short of mimicking the intricate 3D structure of stromal tissue.⁷⁵ Although methods like those proposed by Chen et al. demonstrate promising in vitro keratocyte differentiation, replicating the complex stromal microenvironment requires further innovation. In addition to considering mechanical properties and cell adhesion, ensuring proper permeability of the microenvironment for nutrient and glucose transfer is paramount.⁷⁶ Transplantation methods, especially for addressing ocular surface disorders, are crucial considerations.⁷⁷ Noninvasive delivery methods, such as LSC/growth factor injection, are preferred over invasive surgical approaches, known for their potential inflammatory responses and manipulation of the soft corneal surface.⁷⁸ The trajectory of regenerative medicine is evolving towards noninvasive procedures for epithelium and endothelium layer regeneration, shifting from 2D to 3D cell delivery methods for CSE. This progressive shift reflects a holistic approach aimed at enhancing the efficacy of CSE while minimizing invasive interventions (Fig. 2(D)).⁴²

Viewing it from a material perspective (Fig. 2(E)), studies have demonstrated that natural biomaterials and dC tissues enhance critical aspects like cell adhesion, viability, and differentiation.^42,79–81 However, limitations such as poor mechanical properties and a high degradation rate are associated with these natural biomaterials.^82,83 Conversely, synthetic biomaterials exhibit favorable attributes like acceptable mechanical strength, a low degradation rate, and tunable geometry, making them appealing to researchers.^80,83 Nonetheless, challenges such as the absence of cell binding sites and potential inflammatory responses leading to graft rejection are considerations with synthetic biomaterials.⁸³ Consequently, many studies explore the synergistic use of various biomaterials to regenerate damaged corneas, with some even opting for approaches that eliminate the necessity for any biomaterial in the CSE process.⁸⁴ Characterizing scaffolds, encompassing factors such as stiffness, surface topology, degradation rate, and cytocompatibility, plays a pivotal role in influencing cell differentiation and growth in scaffold-based cell delivery methods.⁷⁹ Optimal mechanical properties are essential to emulate the microenvironment of the host tissue and promote the differentiation of cells into the desired cell type.⁸⁰ Considering the tensile strength of the cornea, adjusting the scaffold's stiffness through variations in polymer/crosslinker concentration or crosslinking time significantly impacts cell differentiation and the reconstruction of damaged tissue.⁷⁵ The corneal stroma's mechanical properties, such as strength, elasticity, and viscoelasticity, are primarily governed by its unique architecture and composition at microscopic and macroscopic levels. COL fibers provide strength and elasticity, while proteoglycans and cellular components contribute to viscoelastic properties. However, most bioengineered stromal tissues lack cellular elements, making viscoelasticity less relevant and highlighting the importance of strength and elasticity. The Young's modulus and ultimate tensile strength are commonly used metrics to assess these properties, where the Young's modulus represents resistance to elastic deformation and varies widely (0.1 to 57 MPa) depending on the stromal region, donor age, and testing methods.⁸¹ The tensile strength, indicating the force required to break the tissue, is typically around 3.8 MPa. These mechanical parameters are crucial for maintaining corneal structure and supporting keratocyte functions such as adhesion, proliferation, and differentiation. Changes in stiffness can affect keratocyte behavior. Consequently, designing bioengineered stromal implants with suitable mechanical characteristics is essential to ensure long-term functional integration, provide biomechanical cues for keratocyte activity, and withstand clinical handling and surgical procedures without compromising structural integrity.⁸¹

4.1. Scaffold-driven cellular reprogramming

Biomaterials play a pivotal role in guiding corneal stromal cell behavior by modulating transcriptional and epigenetic landscapes. Scaffold properties such as mechanical stiffness, topographical features, and biochemical composition influence keratocyte phenotype, ECM synthesis, and fibrosis-related pathways.⁸⁵ These biophysical and biochemical cues activate key signaling cascades that regulate transcription factors, chromatin remodeling, and non-coding RNA activity, ultimately dictating cellular reprogramming and functional integration within the corneal microenvironment. Scaffold stiffness is a primary determinant of keratocyte fate, largely through the TGF-β/Smad signaling axis. Increased stiffness enhances Smad 2/3 phosphorylation, promoting myofibroblast differentiation and excessive ECM deposition, which contributes to stromal fibrosis and corneal opacification.⁸⁶ Conversely, scaffolds optimized for physiological stiffness can suppress Smad activation, maintaining keratocyte homeostasis and preventing fibrotic remodeling. This highlights the importance of tailoring scaffold stiffness to mimic native corneal tissue properties, thereby promoting regenerative outcomes. Integrin-mediated mechanotransduction, via FAK and MAPK pathways, plays a pivotal role in translating scaffold topography and ECM stiffness into intracellular signals that regulate cell adhesion, migration, and ECM synthesis.⁸⁷ For example, scaffolds with aligned fibrillar structures can enhance integrin clustering and downstream signaling, promoting keratocyte alignment and ECM organization. This mechanistic insight underscores the potential of scaffold design to guide cellular behavior and tissue regeneration. During corneal injury, IL-1 triggers keratocyte apoptosis and fibroblast activation, while TGF-β and PDGF-BB drive myofibroblast differentiation, leading to fibrosis and opacity.⁸⁸ Recent studies have demonstrated that restoring the EBM function can regulate cytokine homeostasis and inhibit fibrosis. For instance, Huang et al. reported that corneal fibrosis inhibition depends on EBM restoration, which regulates cytokine homeostasis. Their bioactive hydrogel (Hep@Gel) mimicked EBM function, blocking 83.8% of IL-1, reducing keratocyte apoptosis, and inhibiting myofibroblast transition (αSMA downregulated from 18.1 to 3.6). In animal models, Hep@Gel reduced fibrosis by 73%, preserving transparency without surgery. This highlights biomaterial-driven cytokine modulation as a promising strategy for scar-free corneal regeneration.⁸⁹ These pathways converge on transcription factors such as YAP/TAZ, which are mechanosensitive regulators of fibroblast-to-keratocyte reprogramming.⁹⁰ Beyond transcriptional regulation, biomaterials also induce epigenetic modifications, including DNA methylation, histone modifications, and non-coding RNA regulation, which collectively influence stromal cell fate. Studies have shown that histone acetylation changes at fibrosis-associated gene loci can suppress myofibroblast activation, preventing excessive ECM deposition and maintaining corneal transparency.⁹¹ Additionally, miRNAs, such as miR-29 and miR-200, modulate ECM remodeling by targeting pro-fibrotic genes, thereby fine-tuning cellular responses to scaffold-mediated cues.⁹¹ Certain biomaterial formulations, particularly decellularized ECM hydrogels and nanostructured electrospun fibers, have demonstrated the ability to reprogram corneal stromal fibroblasts toward a keratocyte phenotype, restoring physiological ECM secretion patterns and minimizing fibrotic scarring.⁹² The integration of biomaterial-based strategies with advanced tissue engineering techniques holds promise for precise control over cellular reprogramming in corneal regeneration. Future approaches leveraging engineered scaffolds functionalized with epigenetic modifiers or miRNA-loaded nanoparticles may offer novel therapeutic avenues for reversing corneal fibrosis and promoting stromal tissue repair with minimal scarring.⁹³

The structural features of scaffolds significantly influence cellular behavior, as CSCs in vivo are situated between layers of well-organized COL fibrils (Fig. 2(F)). To simulate these topographical cues in vitro, researchers have employed channels at both micro- and nanoscale dimensions. Such aligned substrates have been shown to enhance stromal cell alignment and direct migration, as well as support the maintenance of a keratocyte phenotype. Similarly, nanoscale channels designed to mimic the architecture of the corneal basement membrane have demonstrated the ability to regulate corneal cell elongation, adhesion, proliferation, and gene expression. To create a more physiologically relevant environment, 3D scaffolds incorporating topographical features have been developed. For example, Wilson et al. incorporated aligned nanofibers into hydrogels, resulting in an upregulation of keratocyte-specific genes and a reduction in myofibroblastic gene expression.⁹⁴ Beyond surface topography, recent research has shown that surface curvature can also influence the orientation and phenotypic behavior of both corneal stromal and epithelial cells, further underscoring the importance of scaffold design in modulating cellular responses.

When designing scaffolds for corneal regeneration, it is essential to consider their light transmittance properties, ensuring they mimic the natural cornea's ability to transmit visible light (400–780 nm) while limiting UV light (<400 nm). UV transmission varies between the central and peripheral regions of the cornea, and excessive UV exposure can damage the retina.⁷⁶ Additionally, the cornea's shape is critical for focusing light onto the retina, with irregularities like astigmatism causing blurred vision. The transparency of the cornea largely depends on the highly organized COL fibril arrangement in the stroma. Small, evenly spaced fibrils allow light to pass through; any disruption from injury or disease reduces transparency.⁶² Moreover, keratocytes in the stroma contain crystalline proteins, such as ALDH1A1 and ALDH3A1, which minimize light scattering. When these cells are activated, the reduction in these proteins leads to increased light scattering, affecting corneal clarity.^65–67

Biodegradability stands out as a critical aspect of scaffold-based CSE.⁹⁵ An ideal cell carrier should undergo degradation as transported cells secrete ECM, allowing the produced matrix to replace the biomaterial.⁹⁶ The interplay between mechanical properties and degradation is noteworthy, and this relationship can be carefully controlled to ensure proper healing of damaged tissue.⁵⁰ Additionally, the swelling ratio is influenced by the stiffness and hydrophilicity of the scaffold, directly impacting the material's water content.⁷⁹ This water content, crucial for biocompatibility and cell growth, is directly related to the scaffold's hydrophilicity and inversely related to its mechanical strength.⁹⁷ Notably, the equilibrium water content of the cornea is reported to be 80%.⁸² Scaffold design not only influences cellular behavior but is also subject to remodeling by the cells through the application of mechanical forces, enzymatic activity, and the secretion of ECM components. Therefore, scaffolds must be engineered to support cellular remodeling while maintaining their structural integrity, ultimately resulting in tissue formation that closely resembles the native cornea.²⁴ As the scaffold degrades, it should ideally be replaced by newly synthesized ECM, ensuring that the tissue retains its functional properties and transparency. Proper alignment of COL fibrils is crucial for maintaining corneal clarity. It can be enhanced by initially structuring the scaffold in a manner that mimics the organization of natural COL fibers.³⁶ It should be noted that COL hydrogels with low polymer concentrations tend to contract into opaque masses unless stabilized by external supports such as rings. Parameters like cell seeding density, culture medium composition, scaffold degradation rates, material concentration, and porosity must be meticulously tuned to guide the remodeling process. While certain scaffolds are designed to be stable and resistant to degradation, they may lack the ability to integrate with host tissue and facilitate long-term regeneration.³⁴ Although such designs may offer advantages, such as minimizing the risk of premature degradation and reducing variability in clinical outcomes, they are likely to fail due to the absence of dynamic tissue regeneration. Therefore, scaffolds engineered to promote controlled remodeling and support tissue regeneration present a more viable approach for CSE.

The cytocompatibility of biomaterials emerges as another critical parameter influencing not only cell viability but also the material's ability to support cell growth, migration, and ECM deposition during tissue regeneration factor, significantly influencing cell–cell and cell–tissue interactions. The careful consideration and optimization of these scaffold characteristics are imperative to enhance the overall effectiveness of CSE. Therefore, ensuring appropriate surface topology and properties becomes crucial for promoting cell growth and differentiation in the context of CSE.^50,80,81 Thus, the careful selection of materials with suitable properties, combined with advanced scaffold fabrication techniques, is essential to developing constructs that closely mimic the unique characteristics of corneal stromal tissue. Achieving the appropriate balance of transparency, mechanical strength, and biocompatibility is critical to creating structures that meet the functional and structural requirements of the corneal stromal tissue. By addressing these considerations, researchers can advance the field of CSE and contribute to the development of innovative solutions for treating corneal disorders (Fig. 2(G)).

5. Biomaterials in corneal stroma equivalent

Biomaterials utilized in tissue engineering are categorized as natural or synthetic, each presenting distinct advantages and drawbacks (Tables 1 and 2). The tissue engineer faces the crucial task of discerning the most suitable biomaterial, ensuring that its benefits outweigh any associated drawbacks. Natural biomaterials offer high biocompatibility, controllability, and molecular-level functionality.³⁰ However, they may trigger an immune response, elevating the risk of host rejection, and can degrade prematurely, leading to inadequate mechanical support. On the other hand, synthetic biomaterials prove valuable in situations requiring meticulous control over physical and chemical properties, enabling manufacturers to closely mimic native tissue characteristics, including porosity and other structural components.⁹⁸ Careful consideration and evaluation are imperative before in vivo testing, as certain synthetic biomaterials may elicit adverse reactions upon degradation or exposure to specific environments.⁹⁹ A strategic combination of natural and artificial materials allows for enhanced control over properties and incorporation of functional aspects, such as improved strength and reduced degradation rates.¹⁰⁰ The ideal biomaterial for a tissue-engineered cornea scaffold should closely emulate the native cornea ECM functionally, minimizing potential side effects.⁴⁶

Table 1 Biomaterials employed in corneal stromal equivalent—a comparison of natural and synthetic variants—highlighting their pros and cons

Biomaterials	Pros	Cons	Ref.
COL	• Biodegradable	• Unstable	101–106
	• Biocompatible	• Degrades rapidly
	• Encourages cell adhesion	• Expensive
	• Easy to produce	• Shrinks at cell introduction
	• Component of stroma, therefore compatible with corneal cells	• Poor mechanical toughness and elasticity
	• Can be crosslinked to improve properties
	• Can be organized to match native tissue
GEL	• Cost-effective compared to COL	• Degrades at a faster rate than COL due to the removal of cross-linkages.	102 and 107
	• Derived from COL
	• Possesses the same amino acid structure as COL
	• Exhibits superior transparency compared to COL
	• Demonstrates a good elastic modulus
CHI	• Found naturally in insects, algae, fungi, and crustacean shells	• Can swell in aqueous conditions if not crosslinked or combined with a stabilizer	108–110
	• Non-toxic	• Degrades in less than 8 weeks
	• Biodegradable	• Compact internal structure leading to reduced cell proliferation
	• Encourages wound healing
	• Transparent
	• Exhibits good mechanical strength
HA	• Naturally occurring in the vitreous humor of the eye	• Occasionally, it may trigger allergic reactions. Additionally, when injected into the eye, it has the potential to elevate intraocular pressure.	111
	• Demonstrates effective electrospinning capability
SF	• Highly versatile material that can be molded into various scaffold types, and cost-effectivy	• Vulnerable to UV radiation, lacking durability	112–114
	• Biodegradable with good cell viability	• Exhibits opacity as a material characteristic
	• Robust in water	• Limited biocompatibility
	• Exhibits good mechanical properties
	• Provides porous scaffolds
	• Can be combined with COL to enhance cell attachment and proliferation
CS	• Resembles the structure of HA	• During storage, there has been an observed increase in corneal thickness. No reported side effects or disadvantages have been noted.	115
	• Naturally occurring in the ECM
	• Employed for therapeutic treatment of disorders
	• Transparency progressively enhances over time
dC	• Exhibits properties comparable to the native cornea	• Dependent on donations from human corneas	116–118
	• Demonstrates low rates of immune rejection	• Mandates screening for diseases
	• Offers improved transparency
PLA	• Biodegradable	• Takes 10 months to fully degrade	119 and 120
	• FDA approved	• Exhibits poor cell adhesion without surface modification
	• Naturally passes from the body during degradation
	• Flexible
	• Can be produced from plant extracts
	• Can be either semi-crystalline or amorphous
PLGA	• Lower stiffness in comparison to PGA	• Shrinkage may occur upon cell introduction	121
	• Degradation rate can be adjusted by altering the PLA to PGA ratio	• Exhibits poor cell adhesion without surface modification
	• Non-harmful to the body	• Displays inadequate mechanical strength
PCL	• Biocompatible	• Extremely slow degradation speed	122 and 123
	• Biodegradable	• Low-melting temperature
	• Exhibits good cell viability	• Highly hydrophobic
	• Approved as a cell carrier for the retina and conjunctiva due to non-toxicity	• Poor transparency
	• Flexible	• Exhibits poor cell adhesion without surface modification
	• Cost-effective
	• Capable of producing fine fibers when electrospun
PVA	• Biocompatible	• Water-soluble	124
	• Exhibits good transparency	• Rapid degradation
	• Capable of producing nanofibers when electrospun	• Limited mechanical strength
		• Poor cell adhesion without surface modification
PGS	• Elastic	• Challenging to obtain	125
	• Transparent polymer	• Requires crosslinking
	• Biocompatible	• Rapid degradation
	• Biodegradable	• Poor cell adhesion without surface modification
	• Adaptable for controlling degradation rates and mechanical strength
	• Utilizable as a shape memory polymer
PLLA	• Isomer form of PLA	• Less transparent than PLA	126
	• Capable of producing fibrous scaffolds for wound healing	• Degrades at a slower rate than PLA
	• Transparency increases over time	• Poor cell adhesion without surface modification
	• Exhibits good biocompatibility
pNIPAM	• Promotes cell adhesion and growth	• Monomer is toxic to neural tissue	127–129
	• Non-cytotoxic and biocompatible	• Considered expensive
	• Easy to manufacture
	• Relatively simple to adjust mechanical properties
	• Attachment properties can be altered by changing tissue temperature
	• Exhibits thermosensitive hydrophobic/hydrophilic changes
	• Can be combined with other materials to enhance properties
PGA	• Biodegradable	• Excessively rigid	109 and 110
	• FDA approved	• Mechanical properties deteriorate after 6 weeks
	• Naturally passes from the body during degradation	• Exhibits poor cell adhesion without surface modification
	• Exhibits greater strength compared to PLA
	• Requires 4 months for complete degradation
	• Achieves transparency approximately 8 weeks into the degradation process

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Table 2 Presents an overview of natural-based scaffolds in Corneal Stromal Equivalent

Type of material	Material source	In vivo test	Ref.
COL	Porcine Type I	Rabbit	130
	Rat Type I	Rabbit	131
	Bovine Type I	Rabbit	132
	Bovine Type I	Dog	133
	Rat Type I	Rabbit	134
	Bovine Type I	Pig	135
	Porcine Type I	Pig	136
	Recombinant human Type I and III	Mini-Pig	137
	Recombinant human Type III	Human	138
SF	Antheraea mylitta	Rabbit	139
	Bombyx mori and CHI	Rabbit	140
	Bombyx mori and COL	Rabbit	141
	Bombyx mori	Rabbit	142
	Bombyx mori and CHI	Rabbit	143
	Bombyx mori and RGD peptides	Rabbit	144
GEL	GelMA	Rabbit	145
	GEL	Rabbit	146
	GEL + GAG	Rabbit	147
dC	PdCT	Rabbit	148
	PdCT	Dog	149
	HdCT	Rabbit	150
	HdCT	Human	151
	HdCT	Rabbit	152
	PdCT	Rabbit	153
	PdCT	Human	154
	PdCT	Nonhuman primate	155
	PdCT	Rabbit	156
	HdCT	Rabbit	157
	PdCT	Rabbit	158

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5.1. Natural biomaterials

5.1.1. Collagen. COL is widely recognized as the primary component of the ECM in most tissues, playing a pivotal role in cell adhesion, migration, and proliferation through its interaction with cell surface integrin receptors.⁸⁸ Type I COL is extensively used in tissue engineering due to its abundance, biocompatibility, and cost-effectiveness, making it an essential material for fabricating biomimetic corneal stroma equivalents. COL's natural biochemical properties, such as the presence of RGD (Arg-Gly-Asp) motifs, facilitate cell adhesion by binding to integrin receptors on cell surfaces, thereby promoting cellular attachment and spreading. This interaction is critical for the initial stages of tissue regeneration as it provides a foundation for subsequent cell proliferation and ECM deposition. Studies have demonstrated that COL scaffolds enhance the proliferation of corneal stromal cells, epithelial cells, and endothelial cells, which are essential for corneal tissue repair.^88,89 Moreover, COL scaffolds create a biomimetic microenvironment that supports ECM secretion, including key components such as fibronectin and laminin. These ECM proteins are crucial for tissue remodeling and provide structural and biochemical cues that guide cell behavior. For instance, crosslinked COL membranes have been shown to promote ECM secretion by corneal stromal cells, leading to improved tissue organization and functionality.^90,100

Though COL's mechanical strength and elasticity may be insufficient for certain applications, crosslinking techniques have been employed to enhance its properties without compromising its biological benefits.^89,92 Chemical crosslinkers such as glutaraldehyde and genipin are widely used; however, their application in corneal bioengineering is limited due to the potential for toxic residues.^94,98 In contrast, physical crosslinking methods, such as UV treatment and enzymatic crosslinking using transglutaminase, offer alternatives with fewer toxic by-products (98). For example, Duan et al. demonstrated that different crosslinking methods yield varying optical and mechanical properties of COL membranes. Among these, EDC crosslinking has been noted for producing superior results, closely resembling the mechanical properties of the native human cornea.⁹⁰ UV-induced glucose crosslinking has also been shown to produce COL membranes with favorable mechanical and optical characteristics while supporting corneal stromal cell adhesion, proliferation, and ECM secretion.⁸⁸ Recent advances in COL-based scaffolds have focused on improving their mechanical resilience and transparency. For instance, COL composites incorporating substances like chondroitin sulfate and tobramycin have been developed to enhance mechanical properties and transparency. Additionally, COL-vitrigel composites offer improved strength and clarity, making them suitable for corneal tissue engineering applications. These modifications not only enhance the mechanical properties of COL scaffolds but also improve their ability to support cellular interactions, ECM deposition, and tissue remodeling.^89,100,159 Despite these improvements, challenges remain in accurately replicating the complex structure of the cornea. Researchers are actively exploring methods to mimic the intricate organization of COL fibrils in the corneal stroma, which is critical for achieving transparency and functionality. For example, techniques such as electrospinning and 3D bioprinting are being investigated to create COL-based scaffolds with aligned fibrillar structures that closely resemble the native corneal ECM.^160–170

5.1.2. Gelatin. GEL, derived from COL, is widely used in corneal engineering to create membranes for corneal cells through cross-linking. Two common crosslinking methods are dehydrothermal and chemical approaches. Dehydrothermally crosslinked GEL provides transparent sheets with enhanced properties compared to COL, while chemically crosslinked GEL promotes cell adherence and ECM deposition. By adjusting crosslinking parameters, GEL membranes with varied characteristics can be produced.¹⁷¹ Hybrid GEL and chondroitin sulfate scaffolds have been found to promote cell growth, although they may exhibit reduced mechanical strength. In contrast, innovative GEL scaffolds incorporating heparin have been shown to enhance growth factor absorption and release, supporting transplantation and cell growth.¹⁷² Additionally, HA-functionalized GEL microspheres have demonstrated the capacity to support large-scale corneal cell growth in a 3D culture system, showing compatibility with various cell types and good tolerance in rabbit eyes.¹⁷³ Semisynthetic GelMA is commonly used in tissue engineering due to its biocompatibility and adjustable physical properties, achieved through radical polymerization with a photoinitiator.¹⁷⁴ GEL, like COL, contains an NH₂ functional group in its molecular structure. As noted earlier, GelMA is a prevalent method for crosslinking GEL. Fig. 3(D and E) illustrates how NH₂ functional groups in GEL interact with methacrylic anhydride, forming GelMA with methacrylic acid as a byproduct. Additionally, in the presence of a photoinitiator, the methacrylated portions of GelMA molecules undergo reactions that bond multiple methacrylated GEL chains.¹⁷⁵ GEL and COL can also be physically crosslinked. It highlights the intermolecular interactions between GEL chains, emphasizing the functional groups (N–H and O–H) that can participate in hydrogen bonding.¹⁷⁶

Fig. 3 Typical crosslinking reaction mechanisms: COL–COL reaction in the presence of transglutaminase enzyme (A). Reaction mechanism using genipin as crosslinker (B). Reaction mechanism using glutaraldehyde as crosslinker (C). GelMA crosslinking mechanism, physical crosslinking by hydrogen bonding (D) and (E). Chemical molecular structures of CHI (F). Chemical molecular structures of SF (G).

5.1.3. Chitosan. CHI, a biopolymer derived from chitin, has emerged as a promising alternative to the AM in corneal bioengineering due to its ability to support cell growth and regeneration. Studies have demonstrated that primary BCECs exhibit enhanced proliferation and attachment on pure CHI membranes compared to AM while retaining their typical phenotypic characteristics.¹⁷⁷ Moreover, surface modifications of CHI have been shown further to promote the formation of a uniform monolayer of HCECs, highlighting its potential for CSE. Blending CHI with PCL has been explored to create composite membranes with adjustable biodegradation rates, supporting BCEC growth and forming continuous monolayers for endothelial regeneration.¹⁷⁸ Additionally, incorporating CHI into COL membranes has been found to improve both optical transparency and mechanical strength, making these hybrid scaffolds suitable for corneal applications. However, the inherent limitations of CHI, such as its low solubility in physiological solvents, have prompted the development of derivatives like HECTS, which is water-soluble and more versatile for biomedical use.¹⁷⁹ Membranes fabricated from derivatives such as HPCHI, GEL, and chondroitin sulfate exhibit properties closely resembling the human cornea, including optical transparency, water content, and enhanced glucose permeability. These membranes have been shown to promote the growth of HCECs and rCEN, supporting epithelial restoration in vitro and demonstrating excellent biocompatibility in rabbit models. Despite the promising attributes of CHI for corneal bioengineering, it may perform more effectively when combined with other biomaterials rather than being used as a standalone scaffold.¹⁸⁰ This synergistic approach can leverage CHI's functional groups, such as NH₂ and OH, which enable diverse chemical and physical interactions similar to those seen in COL and GEL.¹⁸¹Fig. 3(F) illustrates CHI's molecular structure, highlighting its potential for chemical modifications that enhance its properties for CSE.

5.1.4. Silk. SF, a protein commonly extracted from silkworm cocoons, is widely used in tissue engineering and regenerative medicine due to its excellent biocompatibility and mechanical properties. In corneal engineering, SF membranes provide optical clarity and support the growth of CECs, comparable to the current standard, AM. However, SF lacks the natural ECM proteins present in AM, which may result in slightly inferior cell attachment capabilities. To overcome this limitation, the surface of SF membranes can be modified with the RGD peptide sequence found in COL or PDL to enhance the attachment and proliferation of corneal cells, such as HCECs, hCFs, and HECs. These modifications make SF a promising alternative to AM for certain applications.^172,173 RGD-functionalized SF films have shown the ability to maintain their integrity and transparency for extended periods without adverse reactions in rabbit stroma. Another silk-derived protein, SS, has also garnered attention as a substrate for corneal epithelium regeneration, demonstrating improved HCLE attachment compared to SF. Furthermore, silk photocrosslinking using FMN represents an innovative method for corneal bioengineering, leveraging biologically and environmentally friendly materials.^182–184 Both silk and COL offer distinct advantages in terms of production and modification. Silk is easier to produce and manipulate, which can influence corneal cell behavior. For instance, the alignment of hCFs can be controlled by adjusting the depth and width of grooves in SF scaffolds.¹⁸² Additionally, stacked porous silk films provide a useful 3D model for constructing multilayered corneal structures, although further optimization is required to replicate the complexity of native corneal tissue. While chemical surface modifications aim to enhance biocompatibility, the multifunctional nature of corneal proteins presents challenges, indicating a need for more in-depth research. Further studies on in vivo performance, particularly focusing on the impact of porosity and surface patterning, would be beneficial.^182,185Fig. 3(G) illustrates the chemical structure of SF, characterized by the presence of N–H functional groups, which can undergo various chemical reactions, as discussed in earlier sections.¹⁸⁶

5.2. Synthetic biomaterials

Various synthetic materials, including PVA, PHEMA, PEGDA, PLGA, and PEG/PAA-based hydrogels, are used in corneal bioengineering as substrates or artificial cornea substitutes. These materials offer excellent permeability and controllable mechanical and optical properties, although they typically require surface modification to improve cell adhesion. Enhancements can be achieved by adding COL type I, phosphate groups, RGD sequences, or amine groups, which have been shown to promote cell attachment and growth. Notable advancements include ZnS/PHEMA/PAA hydrogels, which offer a high refractive index, and PEG/PAA hydrogels, which provide high glucose permeability and maintain optical clarity for up to two weeks. Additionally, PGS films have demonstrated the ability to support the viability of HCECs. However, more data is needed to fully understand their mechanical and optical properties and their in vivo performance.^187–191

6. Tissue engineering techniques and different scaffolds

6.1. 3D bioprinting technique

The scaffold creation process generally remains consistent across various 3D printed models.¹⁹² Commencing with developing a high-quality 3D model from the desired object, the subsequent step involves printing the 3D structure in 2D layers, with the thickness defined by the 3D image. The data serves as the blueprint for layer-by-layer printing, with commands transferred to the printer's desktop. This flexible manufacturing process allows tailored structures for specific target tissues. Additionally, graphical methods such as CAD and MRI can design structures mirroring patient-derived data (Fig. 4(A)).¹⁹²

Fig. 4 (A) Pathway for creating patient-specific devices using 3D printing technology. Reprinted with permission.¹⁹² Copyright © [2024], Elsevier. (B) Schematic of the basic techniques of 3D printing: Droplet printing (thermal (a), piezoelectric (b)), Extrusion printing (piston (c), pneumatic (d)), and Laser printing (stereolithographic (e)).¹⁹³

On the other hand, bioprinting, which utilizes cellular encapsulated biological materials as bioinks is considered a suitable platform for the simulation of various tissues.¹⁹⁴ Scaffolds printed with cells are produced in situ, necessitating a sterile environment compatible with the cells. Maintaining structural integrity and desired mechanical properties in the printed structure limits the selection of cell-compatible materials.¹⁹⁵ Appropriate rheological parameters must be chosen to minimize shear pressure during the printing process. However, the resolution of the printed substrate is compromised in cell-loaded bioprinting. Additionally, achieving a critical cell density relative to the surface area is paramount.^11,196 The standard healthy threshold for cell density in solid organs is approximately 10⁹ to 10¹⁰ cells per cell culture well. Bioprinted hydrogel scaffolds, however, typically exhibit a cell density ranging between 10⁵ and 10⁷ cells per cell culture well, falling short of this threshold.¹⁹⁷

Bioprinting techniques have been successfully applied by researchers, demonstrating reliable properties for generating ex vivo constructs and membranes.¹⁹⁸ Various approaches, including microextrusion, laser-based methods, and droplet-based techniques, have shown promising results in creating constructs for neural studies, 3D models of interacting HECs, cancer studies, and replication of the native ECM of cartilage.^199–201 Bioprinted scaffolds, considered a top-down approach, is recognized as a biofabrication technology for artificially fabricating various ex vivo membranes and tissues through consecutive deposition of cell-loaded layers.^202–205 Different bioprinting methods, such as laser-based, droplet-based, and extrusion-based techniques, are compatible with various bioinks, each with unique challenges in optimization according to specific bioprinting techniques (Fig. 4(B)).¹⁹³ Extrusion-based methods are particularly popular due to their compatibility with most injectable hydrogel platforms for biomedical engineering and regenerative medicine applications.^206–209 In brief, this method involves extruding pre-polymerized bioink through a single nozzle under pressure. Pressurized air can be applied to the printer head, producing a 3D construction layer by layer.

Corneal bioprinting presents a promising avenue for addressing contemporary challenges and requirements in CSE, encompassing a controllable structure, properties akin to natural mechanical strength, and the ability to fabricate fully organized corneal constructs.¹⁹³ As delineated in prior sections, the cornea. Comprises three transparent layers, with the stromal layer being the thickest (∼500 μm), and the delicate epithelium and endothelium layers measuring less than 50 μm.⁴ The stromal layer, constituting over 90% of the corneal structure, is of paramount importance in CTE due to its transparency, resulting from aligned COL lamellae and proteoglycan expressions, and mechanical performance, facilitated by cross-linked COL fibrils.⁴⁶ It should be noted that the mechanical properties vary between the peripheral and central sections of the stromal layer, influencing COL content orientation, corneal cell differentiation, and alignment.^8,210 Hence, accurate replication of the micro and macrostructure is crucial for enhancing the functionality of the stromal part, as the physical, mechanical, and chemical properties significantly impact biological factors.²¹¹ Recent studies (Table 3) have explored various bioprinting techniques for CSE, considering factors such as structure, transparency, and mechanical properties.^{1,44,87,212–222} Sorkio et al. investigated the potential of laser-assisted bioprinting for CSE using COL type-I and LSCs. Their study demonstrated the feasibility of constructing 3D cornea-like structures by combining hESC-LESCs and hASCs. By employing optimized bioinks based on recombinant laminin and human COL type-I, the researchers successfully bioprinted layered structures, including stratified epithelium, lamellar stroma, and combined epithelial-stromal tissues. The constructs showed promising cellular viability, proliferation, and key protein expressions, with CKCs organizing horizontally, mimicking native stromal structures. However, the sensitivity of CKCs impacted the mechanical properties and structural integrity of the bioprinted constructs, highlighting challenges in achieving fully functional tissue substitutes. Although the stromal layers adhered to porcine corneal organ cultures and showed initial signs of integration, the mechanical performance and stability of the bioprinted tissues require further refinement. This study underscores the promise of laser-assisted bioprinting in CSE while emphasizing the need for continued optimization to achieve structures with enhanced mechanical and biological properties (Fig. 5).²¹²

Table 3 Summary of different experimental studies based on 3D printing techniques in corneal stromal equivalent

Bioprinting method	Material	Cell source	Results	Ref.
Extrusion	SOALG, COL bioink,	CKCs	• Exhibits a structural resemblance to the native corneal architecture, with stromal cells encapsulated utilizing a COL-based bio-ink	192
	FRESH support		• Demonstrates notable viability of stromal cells within the construct
Laser	Matrigel, COL bioink	LECs	• The printed membranes exhibited commendable cell viability and positive COL labeling	212
			• Encountered challenges associated with insufficient transparency
Extrusion	COL, dC	CKCs	• The differentiation potential of hTMSCs was exclusively evident when using the dC-COL membrane.	220
			• The COL-dC scaffold demonstrated both adequate mechanical flexibility and enhanced transparency properties, surpassing those of the conventional COL scaffold
Droplet	COL, AG	CKCs	• Maintaining the native keratocyte phenotype along with appropriate cellular elongation was achieved	213
			• Comparable transparency was observed in relation to the stromal layer
Extrusion	GelMA, reinforced with PEG, PCL	LSSCs	• Creating an optimal environment conducive to the preservation of the keratocyte phenotype	219
	Fibers
Extrusion	GelMA	CKCs	• Keratocytes exhibited sustained preservation of their phenotype	44
			• Demonstrated transparency akin to the native cornea
			• Possessed satisfactory mechanical stability
Extrusion	dC	CKCs	• Determining the ideal nozzle diameter for the bioprinting of cornea-like aligned COL fibrils	223
			• Identifying the optimal nozzle diameter essential for maintaining the morphology and phenotype of keratocytes
			• Achieving outstanding transparency
			• Preserving the keratocyte phenotype throughout the process
Extrusion	HA	hASCs and hASC-dCSKCs	• The bioink exhibited remarkable characteristics, including superior shear-thinning properties, viscosity, printability, shape fidelity, and self-healing capabilities, all while maintaining high cytocompatibility.	224
			• The cells within the printed structures demonstrated robust tissue formation, and the 3D bioprinted cornea structures showcased exceptional ex vivo integration with host tissue, along with notable in vitro innervation.
Extrusion	GelMA and MC	CKCs	• GelMA7/MC8 hydrogel exhibited superior mechanical strength, with a maximum compressive strength of 69 kPa, compared to 54 kPa for GelMA5/MC8, attributed to higher cross-linking density.	225
			• Light transmission improved to ∼80% by day 14, while biocompatibility assessments showed excellent cell adhesion and proliferation, with GelMA5/MC8 supporting higher viability due to its larger pore size (∼674 nm) compared to GelMA7/MC8 (∼412 nm).
Laser	dC and GelMA	hCFs	• In vitro experiments underscored the hydrogel's ability to sustain elevated cell viability and express core proteins.	226
			• In vivo assessments suggested that the hydrogel holds potential for fostering epithelial regeneration, maintaining matrix alignment, and restoring clarity.
			• dC plays pivotal role in creating a conducive environment that promotes the transformation of cellular function.
Laser	GelMA	CSCs	• The cytocompatibility and elongation of CSCs within 12.5% GelMA scaffolds denote robust cell attachment, growth, and integration within the scaffold.	227
			• Over time, there was a noteworthy increase in the gene expression levels of COL type I, lumican, and keratan sulfate in cells cultured within 12.5% GelMA scaffolds compared to those cultured on conventional plastic tissue culture plates.
Laser	HA-aldehyde HA-ALD	CKCs	• The storage modulus of cell-laden constructs increased from 0.069 ± 0.005 kPa to 0.141 ± 0.184 kPa after 14 days, demonstrating that cell proliferation and tissue formation significantly enhanced the mechanical properties of the bioprinted structures.	228
			• Despite high cell proliferation, bioprinted constructs retained transparency within a 67–84% transmittance range, aligning with corneal transparency classification and supporting their potential application in corneal tissue engineering.
Extrusion	dC	CKCs	• Developed a corneal stroma-specific bioink using hASC–CKCs-derived ECM, eliminating donor corneas and enabling scalable, cost-effective production.	229
			• Ensured low immunogenicity with chemical and enzymatic methods, preserving ECM composition for clinical use.

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Fig. 5 (A) Illustration of the laser-assisted bioprinting system to create 3D structures that mimic the corneal stroma. These structures consist of ten alternating layers of hASCs and a cell-free bioink. Each cell-containing layer has two layers of hASCs, while the acellular layer comprises four printed layers. (B) 3D bioprinted stroma within a porcine corneal organ culture model after seven days (a)–(c), compared with blank Matriderm® sheets (d)–(f) and acellular bioink used as controls (g)–(i). Immunohistochemical staining for the human cell marker TRA-1-85 (shown in brown) indicates the successful integration of hASCs into the porcine corneal stroma in the bioprinted structures. In contrast, Matriderm® and acellular bioink showed limited stromal interaction. HE staining of the acellular bioink in the organ culture revealed porcine epithelial overgrowth—scale bars: 200 μm. (C) 3D cornea bioprinted from hESC-LESCs and hASCs using laser-assisted bioprinting. The 3D cornea printed on a PET substrate (a) demonstrates moderate transparency, whereas printing on the non-transparent Matriderm® substrate (b) was necessary to prevent structural shrinkage during culture. Panel (D) compares the bioprinted corneal tissue with a native human cornea. Immunofluorescence staining on cryosections shows multilayered corneal progenitor marker p40 (red)-positive hESC-LESCs on the 3D scaffold two days post-printing. HE staining highlights the tissue structure in the bioprinted sample, while cryosections of the human cornea serve as a control. Scale bars 200 μm. Reprinted with permission.²¹² Copyright © [2024], Elsevier.

Isaacson et al. explored the application of 3D bioprinting in CSE, employing a bioink composed of ALG-COL type-I with encapsulated CKCs. Using the FRESH method, the bioink was injected into a 3D mold to fabricate corneal structures mimicking the native human corneal stroma. The constructs achieved promising transparency and maintained high CKC viability, with over 90% viability observed on day 1 and 83% on day 7 post-printing. These results highlight the feasibility of 3D bioprinting as a novel method for creating synthetic corneal prostheses. However, despite the initial success in transparency and cell viability, the constructs were unable to adequately support the CKCs in adopting their native dendritic morphology, a critical characteristic for functional corneal stroma. This limitation underscores the challenges in replicating the intricate cellular architecture and biomechanical properties of the native tissue. While the study demonstrated significant progress in using 3D bioprinting to engineer artificial corneal structures, further optimization of the bioink composition and printing parameters is required to achieve fully functional and clinically translatable corneal tissue substitutes (Fig. 6A and B).¹⁹²

Fig. 6 (A) Process of support structure creation: (a) Human cornea, showing natural size and curvature; (b) Initial corneal model converted to a solid for Boolean operations; (c) Cornea sealed with a planar circle for volume subtraction; (d) Wireframe view of cornea centered within a cuboid before subtraction; (e) Digital support structure after volume subtraction; (f) 3D-printed plastic support structure. Reprinted with permission.¹⁹² Copyright © [2024], Elsevier. (B) Utilizing the support structure for 3D printing a corneal model with 3% alginate (200 μm nozzle diameter) and refining bio-inks for corneal printing. (a) Digital cornea loaded into 3D printer software slicer, showing concentric print direction preview; (b) support structure coated with FRESH hydrogel to aid in 3D bioprinting of corneal structures; (c) image of 3D printing in progress, using 3% AG bio-ink with trypan blue for visibility; (d) 3D-printed corneal structure before incubation; (e) FRESH hydrogel aspirated after 8 minutes of incubation, with corneal structure carefully removed, though structure begins to unravel one day post-printing with keratocytes in alginate bio-ink; (f) images of corneal structures printed with composite bio-inks; (g) Correlation between nozzle diameter and printed thickness of corneal structures (left) and transparency of structures printed with COL Type I bio-ink; (h) brightfield image of 3D-printed corneal structure with cells on day 1 and cell viability over 7 days (right); (i) live/dead staining fluorescence images at days 1 and 7 in COL Type I bio-ink. Reprinted with permission.¹⁹² Copyright © [2024], Elsevier. (C) CAD design and slicing for corneal bioprinting: (a) dome-shaped artificial cornea designed in CAD; (b) model sliced using custom software compatible with DoD bioprinting, where alternating blue and red drops are printed with spaces calculated for green and beige filling drops; (c) and (d) 3D structures bioprinted with human corneal stromal keratocytes showing transparency and optical qualities. Transparent dome-shaped artificial corneas consist of 0.5% AG and 0.2% COL Type I. (e) Live/dead staining of human CKCs one day after printing. Immunohistochemical and immunocytochemical staining on human corneal tissue and CKCs-loaded bioprinted samples: (f) keratocan (Kera), (g) lumican (Lum), and (h) SMA staining on human corneal tissue section (5 μm); (i) Kera, (k) Lum, and (m) SMA staining on CSK-loaded AG-COL blends 7 days post-printing. Reproduced with permission.²¹³ Copyright © 2024, Wiley.

Campos et al. tackled the challenges of maintaining CKCs vitality and proliferation by employing a droplet-based bioprinting technique to fabricate a COL type-I-AG bioink construct. Using a layer-by-layer printing strategy, they successfully created a dome-shaped structure miming the native corneal stromal architecture. The printed constructs displayed cell vitality and proliferation rates comparable to control samples, maintaining high levels of CKCs viability throughout the culture period. Notably, the bioprinted keratocytes expressed lumican and keratocan, essential markers for keratocyte functionality and stromal tissue integrity, underscoring the suitability of the approach for producing biomimetic corneal tissue (Fig. 6(C)). This innovative drop-on-demand technique enabled the fabrication of translucent corneal stromal equivalents with optical properties comparable to native tissue, as demonstrated by optical coherence tomography. The bioprinting method preserved CKCs phenotypes, maintaining their native characteristics for up to 7 days in vitro. These results highlight the potential of droplet-based bioprinting to generate 3D corneal models as viable alternatives to donor tissue for patients with corneal stromal diseases. While the study demonstrated substantial progress in replicating the structural and functional aspects of the human corneal stroma, further work is required to optimize the constructs for long-term functionality and integration in clinical settings.²¹³

Regenerating the stromal layer poses a considerable challenge due to its intricate microstructure comprising randomly oriented COL lamellae.⁸ Kong et al. developed a novel approach for CSE by fabricating an aligned PCL-PEG microfibrous scaffold infused with 15% GelMA hydrogel. This 3D fiber-hydrogel construct was engineered to mimic the intricate architecture of the native corneal stroma, with fiber spacing optimized for structural and functional resemblance. The scaffold demonstrated enhanced mechanical strength, suture-ability, and high transparency, making it a promising candidate for CSE. LSCs seeded on the construct exhibited improved expression of keratocyte-specific markers, suggesting that the scaffold provided an optimal environment for maintaining the keratocyte phenotype while preventing fibroblast transformation (Fig. 7(A)). The study also explored the synergistic effects of scaffold topology and serum-free media on keratocyte phenotype maintenance and stromal regeneration. The fiber-hydrogel scaffold, combined with serum-free culture conditions and chemical factors like ascorbic acid, insulin, and β-FGF, created a microenvironment conducive to LSC differentiation into keratocytes. Both in vitro and in vivo evaluations revealed that the construct facilitated the regeneration of damaged corneal stroma with properties closely resembling native tissue. These findings highlight the potential of aligned PCL-PEG fibers infused with GelMA as a biomimetic platform for corneal repair, addressing key challenges in CSE through mechanical robustness and biological compatibility.²¹⁹

Fig. 7 (A) The custom-designed direct writing device is shown along with a schematic of the direct writing process, illustrating a polymer jet ejected from a Taylor cone. (b) Images of the produced curly lines (scale bar: 500 μm), grids (scale bar: 500 μm; enlarged image: 150 μm), straight lines, and a specially designed pattern (scale bar: 10 mm). (c) Images demonstrate stable jet deposition at different substrate moving speeds under specific conditions (scale bar: 100 μm). (d) SEM images of grid scaffolds with fiber spacings ranging from 50–500 μm at varying magnifications (scale bar: 50 μm). (e) LSSCs inoculated on the grid scaffolds align along the fiber direction, stained with phalloidin (red) and DAPI (blue) (scale bar: 100 μm). (f) Macroscopic and microscopic morphology of GelMA hydrogels at 5%, 10%, and 15% concentrations (scale bar for macroscopic images: 10 mm; SEM images: 80 μm). (g) Viability of LSSCs within the 5% GelMA hydrogel, with live cells in green and dead cells in red (scale bar: 500 μm). (h) Cytoskeleton (red) and DAPI (blue) staining of LSSCs within the 5% GelMA hydrogel (scale bar: 100 μm). (i)–(l) Fabricated 100 μm-spaced grid fiber-reinforced GelMA hydrogel, with scale bars in (i)–(k) at 10 mm, the enlarged image in (k) at 1 mm, and (l) at 100 μm. Staining in (e), (g), and (h) was repeated three times with consistent results. Reprinted with permission.²¹⁹ Copyright © 2024, Springer Nature. (B) Schematic illustration of the preparation process for human keratocyte (HK)-loaded hydrogels, showing (a) GelMA slabs and (b) 3D bioprinted GelMA hydrogels Reproduced ref. 44 with permission from the Royal Society of Chemistry. (C) (a) Schematic of dC ECM gel preparation and validation, (b) H&E-stained images using a rabbit model, optical micrographs, OCT images with H&E staining on day 28, and (c) quantification of immune cells on days 14 and 28 (scale bar: 50 μm). Reproduced with permission.²²¹ Copyright © 2024, SAGE Publications.

Recent advancements in bioprinting have enabled the creation of corneal stroma equivalents with enhanced biological and mechanical properties. A notable study developed 3D bioprinted constructs using GelMA hydrogels optimized for CKCs printing by fine-tuning parameters such as nozzle speed and spindle speed. These constructs exhibited exceptional stability in physiological conditions, with only an 8% weight loss over three weeks and a 98% cell viability on day 21. Mechanical properties of the cell-loaded hydrogels increased twofold during incubation, reaching a tensile strength close to that of the native cornea. The constructs also maintained transparency above 80% at 700 nm, comparable to the native cornea. They supported the expression of key corneal markers such as COL types I and V and decorin, demonstrating their potential to replicate the biological and optical properties of the corneal stroma (Fig. 7(B)).⁴⁴

Another innovative approach employed a cornea-derived dC ECM bioink for CSE, leveraging its composition rich in COL and glycosaminoglycans to mimic the native cornea closely. The dC ECM bioink supported the differentiation of hTMSCs into CKCs, showcasing its cornea-specific capabilities. In vivo evaluations in mice and rabbits demonstrated excellent biocompatibility and safety comparable to clinical-grade COL, with no cytotoxic effects observed. Additionally, the dC ECM constructs preserved CKCs-specific characteristics, maintained proper transparency for vision, and offered design flexibility through 3D cell printing. These findings position dC ECM as a promising bioink for treating various corneal diseases, addressing the challenges of donor shortages and material deterioration in artificial corneas (Fig. 7(C)).²²¹

Recently, Vijayaraghavan et al. developed a 3D-printed GelMA-MC hydrogel for CSE, demonstrating stable optical activity, biocompatibility, and printability at room temperature. Rheological analysis confirmed a steady decrease in complex viscosity with increasing shear rate, ensuring shape fidelity, while optimal printing conditions for GelMA 5 wt%/MC 8 wt% and GelMA 7 wt%/MC 8 wt% were determined as 120 kPa and 145 kPa at 2 mm s⁻¹. FE-SEM analysis revealed that GelMA7/MC8 had smaller pores (∼412 nm) than GelMA5/MC8 (∼674 nm), leading to improved mechanical properties, with GelMA7/MC8 achieving a maximum compressive strength of 69 kPa compared to 54 kPa for GelMA5/MC8. The increased GelMA concentration likely contributed to higher cross-linking densities, enhancing stress–strain behavior, which is crucial for implantation and surgical applications. Additionally, light transmission improved to ∼80% by day 14. Biocompatibility assessments with goat stromal keratocytes confirmed excellent adhesion and proliferation, with MTT assays indicating superior viability in GelMA5/MC8, attributed to its larger pore size. Keratan sulfate and vimentin expression confirmed sustained keratocyte phenotype up to day 14. While the study presents promising advancements, limitations include the need for in vivo validation, the exclusive use of goat stromal keratocytes, and the necessity for further exploration of hydrogel-cell interactions, such as gene expression analysis, to enhance translational potential for corneal stromal disorders.²²⁵

Building upon previous studies, Puistola et al. reported the development of a novel multi-material 3D bioprinting strategy to fabricate biomimetic corneal stromal structures. Utilizing hASCs and hyaluronic acid-based bioinks of varying stiffness, they bioprinted alternating soft and stiff filaments in perpendicular layers to replicate the native stromal microarchitecture. The soft bioink supported cell proliferation and tissue formation, while the stiff bioink provided mechanical integrity and guided cellular organization. Notably, the storage modulus of cell-laden constructs increased from 0.069 ± 0.005 kPa to 0.141 ± 0.184 kPa after 14 days, demonstrating cell-driven mechanical enhancement. Additionally, transparency remained within an acceptable range (67–84%) despite high cell proliferation. Ex vivo cornea organ culture confirmed good integration of the constructs, with host porcine stromal cells migrating towards the bioprinted composite. These findings highlight the potential of multi-material 3D bioprinting in corneal tissue engineering, though further in vivo studies are required to validate long-term performance.²²⁸

6.2. Electrospinning technique

Electrospinning emerges as a versatile technique with substantial promise in crafting scaffolds that closely emulate the intricate nanostructured architecture of the cornea. This method integrates polymeric material properties, fiber diameter, and orientation to fabricate scaffolds possessing the requisite mechanical strength and transparency crucial for corneal applications. The resulting nanofiber scaffolds can exhibit high surface-to-area ratios and porosity, mirroring corneal stromal structure.²³⁰ The electrospinning process involves a pumping system and spinneret options, transmitting solute current through the needle conveyor and forming nanofibers on the collector plate (Fig. 8). The fiber diameter is influenced by environmental parameters, solvent choice, and electrospinning device settings.^231–233 The versatility of electrospinning lies in its ability to utilize a broad spectrum of materials- both synthetic and natural materials (such as COL, chitosan, GEL, SF, PCL, and PGS) to meet optical requirements, careful consideration is given to fiber orientation and diameter. Also, incorporating additional materials, modifying surfaces through plasma treatment, and reinforcing hydrogels with electrospun fibers to enhance biocompatibility, transparency, and mechanical properties are some other important aspects of electrospinning.⁴⁶ This strategic manipulation of parameters enables the engineering of scaffolds with superior biological and mechanical attributes, promoting optimal interactions with corneal cells and fostering tissue formation.²³³

Fig. 8 Schematic illustration of the electrospinning setup highlighting different collector configurations and their corresponding fiber orientations: (a) rotating mandrel collector. (b) parallel plate collector. (c) four-post collector. (d) pin-and-cup collector.²¹¹

Over polymers have been successfully electrospun into nano or micro-scale fibers, marking significant progress in tissue engineering. However, the widespread application of electrospun scaffolds hinges on exceptional biocompatibility and specific properties tailored for CSE.^231–234Table 4 serves as a comprehensive compilation of polymers investigated for this purpose. Among the commonly employed materials are both single polymers and blended combinations. The properties of the polymer solution, including concentration and molecular weight, play pivotal roles in shaping the electrospun fiber morphology. Precise control over electrospinning parameters allows the creation of scaffolds with varied fiber arrangements, mirroring the corneal stromal layer's structure. Material components, fiber morphology, surface chemical modifications, and incorporated information molecules collectively impact scaffold functionality. The strategic manipulation of these factors facilitates the engineering of scaffolds with superior biological and mechanical properties and high transparency, thus propelling advancements in CSE.⁵

Table 4 Summary of different experimental studies based on Electrospinning technique

Scaffold type	Cell Source	Application	Ref.
COL/PCL membranes	HCECs	• Biodegradable COL/PCL membranes could facilitate cell attachment and proliferation	235
		• Substrate showed promise as corneal grafts for CTE
GEL and PLLA fibers	CSCs	• Uniaxially aligned fibers can induce mechanical and transparency properties and enhance polarized ingrowth of keratocytes	126
PCL and PCL/dC ECM electrospun scaffold	CSCs	• Promising cell-free implant with aligned electrospun PCL fibers with incorporated ECM that encourage endogenous repopulation by neighboring cells	49
PCL/GEL electrospun scaffold	HCSCs	• PCL-GEL composition can be considered as an option in the selection of CTE application	236
Aligned electrospun PCL/PGS	HCSCs	• Promising as corneal's replacement and promote cell organization and immunocompatibility; consequently, this PGS-PCL composition could be applied to support corneal tissue repair	106 and 237
Aligned and non-aligned electrospun PCL/SF/AV	HCKCs	• Aligned scaffold act as an alternative scaffold for corneal stromal layer regeneration	234, 238 and 239
		• Membrane has a sufficient property to plays like native stromal structure
PLDLA	HCSCs	• Biocompatible and facilitates the transformation of corneal fibroblasts back to a keratocyte phenotype	240
		• Utilizes orthogonal multilayers with aligned fibers for each layer.
COL	CKCs and Fibroblast Cells	• Facilitates cell attachment, growth, and increased ECM deposition	241–244
		• Demonstrates biocompatibility, leading to a decrease in myofibroblast phenotype expression on the aligned scaffold
		• Exhibits high transparency, attributed to the presence of aligned fibers
PEUU	HCSCs	• Facilitates the differentiation of stem cells into keratocytes and stimulates the production of a COL matrix	28
PHBV	CKCs	• Enhances both cell attachment and proliferation	245

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Natural materials like COL, SF, and GEL, known for biocompatibility, biodegradability, and low immunogenicity, have found extensive use in CSE. Notable examples include COL-based scaffolds that replicate the corneal microenvironment. However, natural materials often exhibit limitations in mechanical strength. Synthetic polymers like PLGA, PCL, and PLA offer excellent mechanical properties, allowing for various cell types of inoculation on electrospun surfaces.²⁴⁵

Blended electrospun scaffolds, crafted from polymer blends, aim to balance mechanical, chemical, and biological properties for optimal CSE. These scaffolds offer a versatile solution by combining the biocompatibility of natural polymers like GEL with the mechanical prowess of synthetic polymers like PHBV or PCL. Adjusting polymer ratios within blends allows fine-tuning mechanical properties and degradation rates to align with the native ECM. COL/HA/PEO and SF/P (LLA-CL) exemplify successful blended scaffolds, exhibiting excellent biocompatibility, mechanical properties, and the ability to stimulate corneal regeneration. These advancements bridge the gap between the biological and mechanical demands of CTE, bringing us closer to clinically viable solutions.^246,247

Fabricating electrospun scaffolds tailored for CSE involves meticulous control over structural properties. Key considerations include fiber morphology, diameter, and orientation, each impacting the scaffold's performance.⁸ Notably, the use of special collectors (Fig. 8) allows for creating either isotropic or anisotropic structures, influencing cell behavior in response to micro- and nano-topography cues. Traditional collectors like cylinders or metal mesh yield random fibers suitable for non-woven scaffolds.⁴⁹ Scaffolds with aligned fibers offer unique properties that are beneficial for guiding cell growth in desired anisotropic directions. Various collection methods align fibers on the collector surface, including high rotational speed, wire drum collectors, auxiliary electrodes, and parallel double-thin plate collectors. Aligned fibers, mimicking the natural orientation of tissues, promote favorable cell adhesion, migration, and proliferation. In CSE, where alignment is crucial for mechanical and transparency properties, studies show that aligned COL nanofibrous scaffolds lead to down-regulated expression of smooth muscle actin in corneal fibroblasts.^241–244 Other investigations with aligned PEU and poly (L, D lactic acid) scaffolds demonstrate their potential for reverting corneal fibroblasts to a keratocyte phenotype. These aligned electrospun scaffolds serve as ideal matrices for guiding corneal cells into organized tissues that closely resemble the native corneal environment.²⁸

Constructing corneal substitutes in vitro requires meticulous attention to both the microstructure and crucial characteristics like transparency and mechanical properties in electrospun scaffolds.²³⁸ Optical transparency is a pivotal consideration when developing bioengineered corneal constructs. The cornea's role in clear vision and its contribution of two-thirds of the eye's refractive power underscore the significance of achieving transparency in corneal equivalents. A successful tissue-engineered cornea should efficiently transmit visible light, mirroring the natural behavior of the native cornea.²³⁰ Despite the versatility of polymers for electrospinning into nanofibrous scaffolds, not all materials are suitable for CSE due to opacity or low transparency.²²⁹ Three primary methods address this challenge. Firstly, post-modification via plasma discharge treatment proves effective. Plasma treatment modifies surface chemistry in an eco-friendly manner and enhances cell adhesion properties.⁸ For instance, He/O₂ plasma treatment of electrospun PCL nanofibrous scaffolds significantly increased light transmission compared to untreated equivalents.¹²² Secondly, blending different materials, particularly natural and synthetic polymers, proves effective.³⁵ Understanding the importance of keratocytes in maintaining corneal transparency, a third method involves keeping keratocytes in a quiescent state. Recent studies highlight the significance of intracellular protein expression, such as TKT and ALDH1A1, in achieving and preserving corneal transparency. Maintaining the quiescent phenotype is accomplished by providing the right topographical and chemical cues.²³⁸ For example, L. Samantha Wilson et al. demonstrated the critical role of topographical and chemical cues in reverting corneal fibroblasts to a keratocyte phenotype within 3D multi-layered constructs. Their study utilized aligned electrospun nanofiber meshes arranged orthogonally to mimic the stromal organization of the native cornea. These nanofibers aligned individual cells and facilitated interlayer migration, while also providing a mechanically robust structure with a higher initial modulus. The combination of serum-free media and insulin supplementation created an optimal chemical environment, reducing contraction and fibroblast activation, as evidenced by changes in elastic modulus, dimensional stability, and gene expression profiles. Constructs cultured under these conditions maintained a quiescent keratocyte phenotype, contrasting with those cultured in serum-containing media, which exhibited increased contraction and a fibroblast-like phenotype due to growth factor stimulation. The synergistic effect of nanofibers, serum-free media, and insulin supplementation offers a promising pathway for fabricating electrospun scaffolds tailored for CSE. This approach enhances mechanical strength, transparency, and cellular organization, addressing the challenges associated with developing in vitro tissue-engineered strategies for corneal repair. Optical coherence tomography and qPCR analyses further validated the constructs' capacity to revert fibroblasts to keratocytes, confirming their phenotypical and genotypical fidelity to native stromal tissue. These advances significantly contribute to the development of transparent, mechanically robust scaffolds that closely mimic the native cornea, opening new avenues for CSE applications (Fig. 9).²⁴⁸

Fig. 9 (A) Diagram illustrating the process of creating aligned, portable nanofiber meshes and structured multi-nanofiber mesh cell constructs. (a) SEM image showing aligned electrospun nanofibers (scale bar = 15 μm). (b) Cellulose acetate frames are affixed to the aligned nanofibers, and a scalpel is used to cut the frames from the collecting apparatus. (c) Portable fiber meshes are seeded with cells and arranged in an orthogonal pattern using a layer-by-layer technique, with acetate and filter paper as spacers. (d) After cells adhere for 2–3 hours, COL solution is introduced to infiltrate the fibers and secure them within the construct. (e) Excess fibers and acetate frames are removed, resulting in finalized nanofiber-cell constructs. Reproduced with permission.²⁴⁸ Copyright © 2024, Wiley. (B) (a) Aspect ratio for AHDCS cells in hydrogel constructs with and without nanofiber meshes after 7 and 14 days in F, K, and K* media. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (b)–(g) Representative actin-stained cells in COL hydrogel constructs without (b)–(d) and with (e)–(g) nanofiber meshes after 14 days in F, K, and K* media, respectively. In fiber-free constructs, cells in serum-containing F media exhibit a shorter, fusiform shape (b) compared to the elongated shape in cells cultured in serum-free K and K* media (c) and (d). The addition of nanofibers enhanced cellular alignment and promoted a more elongated cell morphology (e)–(g). Yellow arrows indicate fiber direction (scale bar = 50 μm). Reproduced with permission.²⁴⁸ Copyright © 2024, Wiley.

Beyond the imperative for high transparency and optimal refractive power, electrospun scaffolds must bear the mechanical stresses induced by intraocular pressure and eye movements.³ Achieving mechanical properties akin to natural corneal tissue is paramount. For example, Haleh Bakhshandeh et al. explored the development of a two-part artificial cornea designed to substitute for penetrating HCCK in patients with corneal blindness. The peripheral part, composed of plasma-treated electrospun PCL nanofibers, was engineered to enhance mechanical integrity and facilitate tissue biointegration. The Young's modulus of the PCL skirt was measured at 7.5 MPa, aligning well with the natural human cornea's elasticity range (0.3–7 MPa). This skirt was paired with a PVA hydrogel core as the optical center, exhibiting light transmittance greater than 85% across the 400–800 nm range, ensuring excellent optical properties. Morphological assessments revealed a highly porous PCL scaffold structure conducive to cell integration, while biocompatibility tests demonstrated robust adhesion and proliferation of rabbit limbal stem cells within 10 days of culture. These findings highlight the artificial cornea's potential for clinical application, with suitable mechanical properties, transparency, and epithelialization capabilities crucial for corneal regeneration.²⁴⁹ In another study, Bhattacharjee et al. reported that incorporating SF into PCL nanofibrous scaffolds, combined with plasma treatment, improved transparency, mechanical strength, and hydrophilicity, making them more suitable for corneal stromal reconstruction. The study demonstrated that PCL/SF blends remained stable for up to 6–8 hours without phase separation, allowing for uniform scaffold fabrication. Plasma treatment further enhanced cell attachment and proliferation, with significantly higher keratocyte adhesion observed after 5 hours and increased proliferation over 21 days (p < 0.05). The presence of SF not only improved cytocompatibility but also modified the nanoscale properties of the scaffolds, promoting the adsorption of adhesive molecules and optimizing cellular interactions. In addition, SF accelerated scaffold degradation (∼15 wt% weight loss in 12 hours), facilitating corneal tissue remodeling compared to the slow degradation of pure PCL. The study also confirmed that PCL/SF scaffolds supported higher expression of keratocyte-associated markers and corneal ECM-specific proteins (p < 0.01), indicating a more favorable environment for stromal regeneration. The increased fibroin content contributed to controlled degradation, enhanced oxygen permeability, and optimized repair processes. Overall, the findings highlight the potential of PCL/SF scaffolds as a promising biomaterial for CTE by balancing structural integrity, transparency, and biodegradation.²⁵⁰ Khaow Tonsomboon et al. presented an innovative strategy to address the mechanical limitations of hydrogel scaffolds in CSE by reinforcing ALG hydrogels with electrospun GEL nanofibers. This integration resulted in transparent, mechanically robust hydrogels that mimicked the structural and functional properties of native corneal tissue. The incorporation of non-crosslinked GEL nanofibers significantly enhanced the tensile elastic modulus of the hydrogels from 78 ± 19 kPa to 450 ± 100 kPa, demonstrating a marked improvement in mechanical strength while retaining optical clarity. Additionally, pre-crosslinking the nanofibers increased the modulus to 820 ± 210 kPa but slightly reduced transparency. This approach addresses the critical challenge of developing load-bearing, transparent scaffolds that meet the mechanical demands of CSE, offering a promising alternative to overcome the global shortage of donor corneas. The study underscores the potential of fiber-reinforced hydrogels as durable and functional materials for clinical applications in corneal transplantation (Fig. 10(A)).²⁵¹ Salehi et al. explored the mechanical properties of electrospun-aligned nanofibers made from varying blends of PGS and PCL for use in CSE. They fabricated fibers using different weight ratios of PGS and PCL, ranging from 1 : 1 to 4 : 1, and found that the elastic modulus of the fibers decreased as the PGS/PCL blend ratio increased. This change was attributed to the increasing amount of amorphous PGS in the composition, as confirmed by DSC and XRD measurements, which showed a reduction in overall crystallinity. Interestingly, when the surface modulus was measured through nano-indentation, it was found to be two orders of magnitude higher than the bulk elastic modulus, with a notable increase corresponding to higher PGS content. This phenomenon is believed to be due to the increased PGS content, which forces the PCL into more confined, cross-linked domains near the surface of the fibers, contributing to enhanced surface stiffness. This study highlights the complex interplay between material composition and mechanical properties, providing insights into designing nanofiber scaffolds for CSE.²⁵²

Fig. 10 (A) Fabrication process of a gelatin fiber-alginate hydrogel: (a)–(d) A gelatin mat is electrospun and infiltrated with alginate solution, (e) resulting in a gelatin fiber-alginate film after rinsing in distilled water, and (f) forming a gelatin fiber-alginate hydrogel after crosslinking in CaCl₂. Reprinted with permission.²⁵¹ Copyright © [2024], Elsevier. (B) Schematic procedure for creating chitosan-modified biomimetic nanofiber membranes (CBNMs) designed to replicate the microstructure and architecture of devitalized tissue membranes (e.g., HAM) for corneal repair. (a)–(c) Macroscopic morphology and nanoscale structure of HAM's base layer, with randomly arranged COL fibers. (d) Schematic overview of the CBNM fabrication process. Reproduced with permission.²⁵³ Copyright © The Royal Society of Chemistry. (C) SEM images of (a) aligned and (b) random electrospun fibrous scaffolds, with inset images showing lower magnifications. Scale bars indicate 2 μm for higher magnifications and 10 μm for lower magnifications. (c) Tensile properties of randomly oriented versus aligned scaffolds. Reproduced with permission.²⁵⁴ Copyright © 2024, Wiley.

Silva et al. reported that their C-MSC-laden GelNF-HA scaffold effectively mimicked corneal stromal tissue, supporting cellular adhesion, proliferation, and ECM remodeling. Their ex vivo organ culture studies demonstrated that corneas treated with the C-MSC-laden GelNF-HA scaffold exhibited a 68.3% reduction in stromal haze and a 72.5% decrease in α-SMA expression compared to the untreated keratectomy-only group. The scaffold also improved cell retention compared to the direct topical application of C-MSCs. Furthermore, the GelNF-HA scaffold exhibited a refractive index of 1.322, slightly lower than the human cornea (1.376), and a Young's modulus of 1.66 ± 0.59 MPa, aligning with the physiological range (1.3–5.9 MPa). These findings suggest that the GelNF-HA scaffold enhances corneal wound healing by providing structural support and maintaining an organized stromal architecture, reducing fibrosis and promoting transparency.²⁵⁵

Juan Ye et al. developed a highly effective method combining electrospinning and surface modification techniques to create biomimetic nanofibrous membranes of COL, HA, and PEO. These membranes demonstrated exceptional mechanical properties, with a tensile strength exceeding 20 MPa even in the wet state, indicating their robust structural stability. The electrospun membranes, designed to mimic the natural ECM, were optimized for their mechanical and biological performance. They showed superior results compared to traditional HAM, particularly in their ability to selectively promote the adhesion of epithelial cells and fibroblasts, which are crucial for CSE. In an in vivo rat model of alkali-burned corneal injury, the COL/HA/PEO membranes significantly enhanced re-epithelialization within just one week. This versatile approach holds great potential for biomedical applications, offering a promising alternative to HAM for corneal repair and other wound healing treatments while mitigating concerns related to infectious disease transmission (Fig. 10(B)).²⁵³

Jing Yan et al. investigated the impact of fiber alignment on the mechanical properties and cellular behaviors in a GEL/PLLA nanofibrous scaffold. Their study demonstrated that scaffolds with aligned fibers exhibited significantly improved mechanical properties compared to those with randomly oriented fibers. Specifically, aligned scaffolds showed a higher tensile modulus, greater break strength, and lower elongation at break, indicating the importance of fiber orientation in enhancing the mechanical robustness of the scaffold. Moreover, the research highlighted how fiber alignment influences cellular responses, with CKCs favoring alignment in the scaffolds for better adhesion and proliferation. In contrast, CECs showed enhanced interaction with randomly oriented scaffolds. This study underscores the potential of fiber alignment as a crucial factor in scaffold design, suggesting that scaffolds incorporating both aligned and randomly oriented fibers could be optimized to support the growth of different corneal cell types, a key consideration for the successful development of CSE applications (Fig. 10(C)).²⁵⁴

6.3. Other tissue engineering techniques

In addition to the aforementioned techniques for addressing CSE, various methods center around fabricating scaffolds characterized by distinct physicomechanical and chemical properties, as detailed in Table 5. We will provide an overview of these methods later.

Table 5 Summary of different experimental studies based on corneal stromal equivalent

Fabrication method	Material	Cell source	Results	Ref.
Cell sheet engineering	Silk/PolyNIPA GEL/PolyNIPA	GKCs	• Patterned substrates directed corneal cell alignment along the direction of substrate	256
			• Gene expression was higher over GEL-PolyNIPA matrix
	Fibroblast cells	HCCs	• Endothelial and epithelial cells attached to the reconstructed stroma	257
			• Epithelial cells formed epithelium layer, and endothelial cells formed endothelium layer
Self-assembly	Cyclodextrins/COL	HCCs	• The implants demonstrated tissue integration and supported re-epithelialization	258
			• Mimetic substitutes had progressive functional and structural properties
Microfabrication	Silk protein films	CFCs	• Improvement in alignment, proliferation and ECM expression profile on these films	114
			• Silk protein to produce a substrate with a well-defined topography developed the epithelial cells behavior toward directional epithelialization
	COL	CKCs	• Composite COL-based hydrogels with a transparent core and degradable peripheral skirt were developed to restore corneal transparency and act as reservoirs for cells and drugs.	130
			• The hydrogels supported cell populations in vitro and maintained corneal integrity in vivo, with controlled skirt degradation promoting host cell integration and nerve regeneration.
	COL	CKCs	• Orthogonally aligned multilayer COL fibril scaffolds, prepared using a high magnetic field, successfully recreated the organized architecture of the corneal stroma and supported the in vitro reconstruction of human hemi-corneas with differentiated epithelium and aligned keratocytes.	131
			• In vivo experiments in a rabbit model demonstrated the scaffolds' potential for anterior stromal repair, enabling re-epithelialization, restored transparency, and maintained ultrastructural organization.
	COL	CKCs	• COL film exhibits excellent mechanical properties, optical transparency, nutrient permeability, and stable morphology, making it suitable for diverse dimensions and easy fabrication.	132
			• In vivo corneal lamellar HCCK in rabbits demonstrated that the COL. film supports rapid re-epithelialization, restored transparency, and shows no rejection or neovascularization within two months, highlighting its potential for cost-effective corneal repair.

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In the context of corneal structure, cell sheet engineering in a bottom-up strategy proves promising for mimicking the ECM of the cornea and specific corneal functions. Ultrathin membranes, often porous layers of polymeric resources, serve as the physical carriers for cell sheets. This method is mainly helpful for selectively replacing damaged tissue in endothelium and epithelium layers due to their dimensions, especially for the endothelium layer (Table 6). To replace the corneal endothelium, the Descemet membrane of the endothelial cell carrier must also be replaced or repaired. Free-standing functionality on corneal endothelial cell sheets is achieved under specific conditions, often involving thermo-sensitive P(NIPAM-co-GMA) polymer membranes.^74,259,260 Several studies have explored the use of cell sheet engineering for keratocyte replacement and CSE. For example, Nara et al. utilized DWA to create microperiodic parallel patterns using silk–PolyNIPA and gelatin–PolyNIPA composites. These semi-interpenetrating networks demonstrated temperature-responsive properties suitable for cell sheet applications. At 37 °C, both hybrids displayed hydrophobic surfaces, with silk–PolyNIPA showing higher surface roughness compared to gelatin–PolyNIPA. When the temperature was lowered to 20 °C, surface roughness and contact angle values decreased, promoting better cell interactions. The parallel-patterned substrates aligned corneal cells along the patterns, enhancing COL-I and aggrecan gene expression, particularly in the gelatin–PolyNIPA matrix. Furthermore, gelatin–PolyNIPA promoted higher metabolic activity and vinculin expression, indicating improved biocompatibility. Although cell sheets detached quickly from planar films (within 10–30 minutes), intact recovery from patterned substrates remains challenging, highlighting the need for further optimization to develop adequate corneal stromal substitutes (Fig. 11(A)).²⁵⁶ Proulx et al. developed a tissue-engineered cornea by culturing fibroblasts in serum and ascorbic acid-enriched medium, leading to the formation of ECM sheets that were layered to reconstruct the corneal stroma. Endothelial and epithelial cells were then seeded on opposite sides of the stroma. After 10 days of air–liquid interface culture, the epithelial cells stratified into 4–5 layers, forming distinct basal and wing cells expressing Na +/K + -ATPase α1 and keratins. Endothelial cells also formed a tightly packed monolayer, expressing similar functional proteins. This fully biological corneal model closely resembled native corneal tissue, making it a promising tool for studying corneal pathologies and potentially for clinical applications (Fig. 11(B)).²⁵⁷

Table 6 Primary strategies for corneal treatment

Therapy technique	Pros	Cons	Ref.
Natural scaffolds	• Bioactivity	• Limited mechanical strength	261
	• Compatibility with living tissues	• Suboptimal optical transparency
	• Degradation through natural processes	• Invasive nature
	• Absence of cytotoxic effects	• Variability across diverse samples
	• Induction of biological recognition responses	• Potential for disease transmission
	• Promotion of cell adhesion and functionality	• Susceptibility to rejection
Synthetic scaffolds	• Abundant supply	• Lack of cellular recognition signals	262
	• Outstanding mechanical characteristics	• Minimal impact on proliferation, adhesion, and differentiation
	• Adjustable properties	• Potential for inflammatory reactions
	• Cost-effective	• Bio-toxicity concerns
	• Manageable degradability	• Limited biocompatibility
		• Invasive nature
Cell therapy	• Straightforward	• Uneven cell distribution	263–266
	• Minimally invasive	• Limited persistence
		• Inefficiency in repair
		• Requirement for prolonged patient stability in an eye-down position
		• Reduced adhesion at the injection site
		• Inappropriate cell migration
		• Restricted proliferation of primary HCECs
Xenograft transplantation	• Endless availability	• Potential transmission of zoonotic diseases	267
	• Comparable anatomical composition	• Risk of hyperacute rejection
		• Invasive nature
Autograft transplantation	• Presently considered the gold standard in treatment	• Limited donor availability	268
		• Potential for infection transmission
		• Invasive nature
		• Susceptibility to rejection
Cell sheet	• Preservation of ECM, microenvironment, and cell recognition signals	• Invasive nature	264
	• Impact on cellular proliferation, adhesion, and differentiation	• Time-intensive synthesis of cell sheets
	• Elimination of the need for sutures	• Suboptimal mechanical strength
	• Optical clarity

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Fig. 11 (A) Demonstration of DWA for fabricating microperiodic parallel patterns using silk–PolyNIPA and gelatin–PolyNIPA composites. Reproduced with permission.²⁵⁶ Copyright the Royal Society of Chemistry. (B) Macroscopic appearance of the bioengineered cornea: a. Arial 12 pt font dots visible through the bioengineered cornea. (b). The bioengineered cornea exhibits a slight haze. Tissue-engineered human cornea: c. Masson's Trichrome staining highlights a well-differentiated epithelial layer on the surface, a monolayer of endothelial cells at the base, and a self-assembled stromal matrix in between. (d). Transmission electron microscopy (TEM) of the epithelial basal membrane reveals numerous hemidesmosomes (arrows). (e). TEM of the corneal endothelium shows a monolayer of flattened cells. Scale bars: 50 μm (A), 1 μm (d), 0.5 μm (e) Reproduced with permission.²⁵⁷ Copyright © 2010 Molecular Vision.

Self-assembly is a fundamental technique in CSE, drawing inspiration from natural tissue formation processes. Mimicking the anabolism observed in in vitro organogenesis, self-assembly relies on structural motive forces, short-range interactions, and equilibrium conditions. This method facilitates the modular assembly of engineered tissues, replicating the interactions between the ECM and cells crucial for tissue formation. Although the precise assembly protocol for corneal fibers remains incompletely understood, it is believed to involve layers of COL aligned with the corneal tissue's anatomy and physiology, which are vital for optical transparency. In CSE, self-assembly offers adaptability in scaffold production by allowing adjustments in solvent, concentration, pH, temperature, and ionic strength to modify structural integrity. This reversible process, driven by non-covalent bonding mechanisms, occurs within controlled environments to yield diverse structures, reminiscent of natural processes like lipid organization, bone formation, and shell construction. Scaffold formation via self-assembly involves the aggregation of constituent fragments, initially maintaining separation between solvent and solute until reaching a critical solute concentration, leading to the formation of various micellar shapes.

Synthetic corneas have been successfully created using self-assembly, integrating three distinct cell types. Fibroblasts contribute to the fabrication of a stromal matrix resembling the ECM environment. At the same time, endothelial and epithelial cells are seeded on opposing sides of the self-assembled stroma to mimic the native corneal structure. Over 10 days, stratified epithelial and endothelial cells form a cohesive monolayer resembling native corneal tissue features. Beyond CTE, self-assembled scaffolds have widespread applications in drug delivery systems. They offer advantages such as mitigating drug side effects and enabling precise control over timing, solubility, and dosage at specific sites through modulation of the carrier's degradation rate. However, ongoing efforts are focused on enhancing self-assembled structures to minimize cytotoxicity and optimize functionality under specific environmental conditions.

Majumdar et al. explored the use of CDs to control COL fibrillogenesis and structure, creating transparent and mechanically robust COL vitrigel implants that replicate native corneal architecture. CDs interacted with COL during gelation and vitrification, guiding fibril formation into aligned lamellae stacks. Custom molds shaped these materials into implants that matched corneal curvature. In a rabbit keratectomy model, the implants showed good biocompatibility, and tissue integration and supported re-epithelialization, making them promising candidates for corneal reconstruction (Fig. 12).²⁵⁸

Fig. 12 (A) Role of cyclodextrins in modulating fibril formation and alignment in vitrified COL corneal substitutes through COL interactions: (a) illustration depicting how cyclodextrins interact with COL, influencing fibril arrangement and spacing. (b) Visual comparison of cyclodextrin-COL composites with vitrified COL layers of varying thicknesses. (c) Image showing 500 μm-thick implants fabricated using βCD-Suc and COL, highlighting their optical clarity. (d) Lamellar structures observed in the implants, were demonstrated by manually peeling individual layers. (e) Set up for suture pullout testing to evaluate the suture retention capacity of biomaterials. (f) Peak load data measuring suture retention strength of vitrified gels (n = 6). Results are presented as mean ± SD; statistical analysis by unpaired Student's t-test, ***P < 0.0001. (B) Functional performance and surgical outcomes of biomimetic COL-CD implants: a. Diagram illustrating the implantation of CD/COL materials in ex vivo rabbit corneas. (b) Time-lapse of corneal epithelial wound healing over 72 hours, visualized using fluorescein staining (yellow-green areas indicate non-epithelialized regions under blue light). (c) Epithelial proliferation across implants at 72 hours, assessed via H&E staining. (d) Immunohistochemical staining of ex vivo cornea sections using DAPI (blue) and K-14 (red) to assess epithelial cell maturation and functionality. (e) Custom molds for fabricating implants with the desired curvature. (f) Lenticular-shaped βCD/Col implants mimicked the curvature of healthy corneas. (g) Schematic of in vivo implantation of corneal implants, secured with interrupted sutures. (h) Gross image of ocular surgery in a rabbit model on the day of implantation. (i) Gross appearance of the ocular surface 31 days post-surgery. (j) Fluorescein staining (under blue light) on day 31 to evaluate reepithelialization of the βCD/Col implant. (k) Histological analysis using Masson's Trichrome staining to examine the implant at 14 days. (l) Immunostaining for laminin (red) and DAPI (blue) to assess epithelial cell activity in peripheral and central wound regions, showing laminin expression post-epithelial maturation. Reproduced with permission.²⁵⁸ Copyright © 2024, Wiley.

Inspired by geometrical patterns from ECM surface topography, microfabrication techniques aim to evaluate physiological functions and cell behavior on desired structures. The goal is to achieve the natural corneal architecture by utilizing information from surface effects on cells. This focus on scaffold construction aims to create the most effective topography for cell-scaffold interactions, leveraging insights gained from the influence of surface topography on cell behaviors such as adhesion, migration, and differentiation. In the context of CSE, mimicking topographies of various corneal layers is crucial for the cells in different layers. According to corneal anatomy, epithelial cells exhibit enhanced interaction on 2D surfaces, while keratocytes show better alignment in 3D scaffolds.²¹⁷ Lawrence et al. designed silk protein films to replicate the architecture of corneal stromal tissue. The films, measuring 2 μm in thickness, were surface patterned to guide cell alignment and featured pores (0.5–5.0 μm) to enhance nutrient diffusion and cell interactions. In both 2D and 3D cultures, the films supported human and rabbit corneal fibroblast proliferation, alignment, and ECM expression. Their favorable mechanical properties, optical clarity, and ability to support corneal cell functions make these silk films a promising biomaterial for CSE (Fig. 13(A and B)). Xiong et al. (265) developed micro-grooved COL films to study the impact of surface topography on corneal cell behavior and antifibrosis. The micro-grooved patterns significantly influenced cell orientation, proliferation, and migration, promoting epithelial cell movement and wound healing while reducing keratocyte fibrosis. The films demonstrated similar swelling, optical clarity, and biodegradability to native cornea, making them a promising option for CTE applications (Fig. 13(C and D)).¹¹⁴

Fig. 13 (A) (a) Grooved patterned silk films and (c). flat silk films with corresponding SEM images displaying their surface topography (b) and (d). (e). Silk films fabricated with pore sizes ranging from 500 nm to 5 mm, as shown from surface (f) and cross-sectional (g) perspectives. The films were 2 mm thick with pores extending throughout the cross-sectional area (b). Reprinted with permission.¹¹⁴ Copyright © [2024], Elsevier. (B) (a) Phase contrast image showing a GFP-rCF cellular process aligned along the groove axis of the patterned silk film (arrow) after 4 days of culture. (b) Fluorescent image confirming the cellular structure of the same process. (g) and (h) Actin filament staining on patterned and flat silk film surfaces, respectively, after 10 days of culture. False-color images depict actin filaments (red) and GFP fluorescence (green). (i) Statistical analysis revealed a significant difference in actin alignment between patterned and flat silk film surfaces (p < 0.05; error bars = SD; n = 3). (b), (c), (e), and (f) Fluorescent images illustrating actin filament organization at varying time points and cell densities for GFP-rCFs cultured on patterned (b) and (c) and flat (e) and (f) silk films. At lower cell densities, cells exhibited greater spreading, while higher densities led to more compact and aligned organization along the groove axis (c), compared to flat surfaces (f). (i) Increased cell density on patterned silk films significantly enhanced actin filament alignment (p < 0.05; error bars = SD; n = 3). Reprinted with permission.¹¹⁴ Copyright © [2024], Elsevier. (C) Microgrooved COL films were developed to evaluate their potential application in CTE. Reproduced with permission.²⁶⁵ Copyright the Royal Society of Chemistry. (D) Orientation of CECs and keratocytes cultured on COL films: a. Fluorescence image showing CECs and keratocytes after 48 hours of culture on COL films, stained for F-actin and nuclei (scale bar = 250 μm). (b) Alignment index indicating the percentage of aligned CECs among total cells. (c) Alignment index showing the percentage of aligned keratocytes among total cells (*p < 0.05, **p < 0.01). Reproduced with permission.²⁶⁵ Copyright the Royal Society of Chemistry. (E) Schematic illustration of the fabrication process for composite core-and-skirt hydrogels. (a) The setup for creating a mechanically compressed COL skirt involves COL without a cross-linker, placed between a nylon mesh template and compressed under a weight to facilitate fluid removal (indicated by white arrows). (b) A photograph of the COL skirt, with transparency adjustable based on COL concentration and thickness. (c) Central holes are created in the skirt to enable unobstructed vision. (d) The skirt (with holes) is embedded into a transparent COL core solution containing a cross-linker and allowed to cure. Scanning electron micrographs display the nanostructures of COL fibrils in the core, the core-and-skirt composite, and the skirt-only material. Reprinted with permission.¹³⁰ Copyright © [2024], Elsevier.

Rafat et al. addressed the critical challenges of corneal blindness and graft survival by developing innovative composite COL-based hydrogels designed to restore corneal transparency and serve as reservoirs for cells and drugs. These hydrogels feature a transparent core and a degradable peripheral skirt (Fig. 13(E)), enabling adjustable transparency and degradability. The skirt exhibited faster degradation in vitro and integrated seamlessly with the core to distribute mechanical loads. In vitro studies confirmed the hydrogels' support for HCCs populations, while in vivo transplantation in rabbit corneas demonstrated maintained corneal shape and integrity over three months. Controlled skirt degradation facilitated host cell migration, stromal integration, and nerve regeneration, highlighting the potential of these composites for clinical applications in corneal repair.¹³⁰

In another study, Shen et al. reported that a dual-crosslinked hybrid hydrogel composed of HAMA and pDCSM-G exhibited promising mechanical and biological properties for corneal defect repair. The hydrogel displayed over 80% light transmittance across the visible spectrum (380–800 nm), maintaining optical clarity. Mechanical assessments revealed that the hybrid hydrogel had a tensile strength of ∼43 kPa, demonstrating superior deformation resistance due to its high crosslink density. Biocompatibility evaluations showed that, unlike HAMA alone, it effectively supported corneal stromal cell adhesion, viability, and proliferation. Gene expression analysis indicated upregulation of keratocan and lumican, crucial for maintaining keratocyte phenotype and corneal transparency, while myofibroblast markers were downregulated, minimizing the risk of corneal scarring. In vivo results further confirmed rapid and complete re-epithelialization on hydrogel-treated corneas, with intact endothelial layers observed even eight weeks post-operation. Compared to HAMA and pDCSM-G alone, the hybrid hydrogel reduced corneal fibrosis and promoted long-term stromal regeneration, making it a potential candidate for CTE applications.²⁶⁶

Huang et al. reported that they fabricated GelMA hydrogels using gelatin derived from COL to mimic the ECM, providing a supportive scaffold for hAESC-CKCs. Their hydrogel demonstrated tunable mechanical properties, reinforcing the structural integrity of the corneal stroma in vitro. When applied in situ, it functioned as a bioactive platform promoting epithelial regeneration without the need for fibrin glue or sutures, significantly reducing the risk of postoperative complications. AS-OCT measurements confirmed seamless sealing of corneal incisions, ensuring proper integration between the hydrogel and host tissue for improved wound repair. Furthermore, their results aligned with previous studies showing that cell-laden COL matrices support epithelial stratification and corneal regeneration. In addition, their study demonstrated that the hydrogel effectively modulated the inflammatory response by inhibiting early immune activation, leading to the repression of TGF-β-mediated fibrosis and VEGF-induced neovascularization. The hAESC-CKCs exhibited potent immunomodulatory effects by secreting anti-inflammatory factors and inhibiting M1 macrophage polarization, fostering a favorable tissue microenvironment. Notably, the hydrogel exhibited superior anti-scarring effects, highlighting the critical role of keratocyte-like hAESC-CKCs in scarless corneal repair. These findings suggest that combining keratocytes with CSSCs could enhance therapeutic outcomes, though challenges remain in optimizing cell ratios and procedural consistency. Despite promising results, the study was limited by its short observation period, necessitating further long-term evaluations to assess its full therapeutic potential in preventing and treating corneal scarring.²⁶⁷

7. Clinical status of 3D scaffolds for corneal stroma equivalent

Tissue engineering offers a promising solution for CSE to address the significant challenges associated with corneal stromal damage. The primary goals of CSE are to restore corneal transparency and mechanical integrity, promote seamless integration with host tissue, and establish standardized clinical protocols.^268,269 The corneal stroma, a crucial layer for maintaining optical clarity and structural stability, can be damaged by injury, infection, or disease. Current treatment options, such as corneal transplantation, are limited by donor shortages, immune rejection, and inconsistent outcomes. Tissue-engineered CSE provides an innovative approach to address these limitations. By engineering biomaterial scaffolds that mimic the native corneal stroma, researchers aim to restore stromal function. These scaffolds, including collagen hydrogels, decellularized corneal matrices, and synthetic nanofibers, support cell proliferation and differentiation. Preclinical studies have demonstrated the potential of CSE to regenerate corneal tissue and restore vision.^268–271

Clinical trials evaluate the safety and efficacy of CSE. Key parameters include graft integration, optical clarity, and supporting epithelial and endothelial regeneration. A significant challenge in CSE is the development of biomaterials that possess the necessary mechanical properties and biocompatibility to promote tissue regeneration. For example, González-Andrades et al. initiated a clinical trial to evaluate the safety and efficacy of a bioengineered nanostructured fibrin-agarose corneal substitute. This substitute, incorporating allogeneic cells, aims to replicate the optical, mechanical, and biological properties of the native cornea.²⁶⁸ In another study, Xeroudaki et al. explored a collagen-based hydrogel for CSE. This transparent, mechanically stable hydrogel supports cell migration and maintains corneal thickness, offering a promising approach for corneal tissue replacement and regeneration (Fig. 14).²⁷¹

Fig. 14 (A) Properties of the BPC: SEM images show the surface morphology of the BPC, revealing long, parallel-aligned collagen fibers at both low (a) and high (b) magnifications. (c) Optical transmittance curve of the BPC hydrogel compared to the human cornea. (d) HCECs cultured in vitro without BPC and (e) cultured on the surface of BPC hydrogel, with live cells highlighted by green fluorescence. (f) In an ex vivo test, the BPC was sutured into a porcine eye using eight interrupted sutures. (g) SEM images show the interface between the sutured BPC hydrogel and the host cornea, highlighting microfractures caused by sutures cutting through both the BPC and corneal tissue (arrows). (B) Surgical Technique for Femtosecond Laser-Assisted ALK: (a) In the autograft procedure, a femtosecond laser is used to cut a stromal tissue disc, which is then reinserted into the bed and secured with eight interrupted sutures. (b) In the BPC hydrogel implantation, the native stromal tissue disc is removed and replaced with the BPC hydrogel, with mattress sutures placed to avoid suturing through the hydrogel. (c) Time-lapse images showing the laser cutting the stromal disc. (d) Time-lapse photos document the stromal disc's extraction, and its replacement with either the autograft tissue or BPC hydrogel, followed by the placement of interrupted or mattress sutures (e). Reproduced with permission.²⁷¹ Copyright © 2024, Springer Nature.

Building upon previous research, Rafat et al. introduce BPCDX, a novel cell-free engineered solution for CSE targeting visual impairment from corneal stromal diseases (Fig. 15). This minimally invasive approach, combined with a pilot feasibility study in India and Iran (NCT04653922), evaluated BPCDX implantation in 20 patients with advanced keratoconus. The study aimed to reshape the corneal stroma without tissue removal or sutures. Results demonstrated safety over a 24-month follow-up, with positive outcomes including increased corneal thickness (India: 209  ±  18 μm, Iran: 285  ±  99 μm), reduced keratometry (India: 13.9  ±  7.9 D, Iran: 11.2  ±  8.9 D), and improved visual acuity. Notably, contact lens-corrected acuity averaged 20/26 in India, while spectacle-corrected acuity reached 20/58 in Iran. Remarkably, all initially blind subjects regained vision (20/36 best corrected) and the ability to wear contact lenses. These findings suggest that BPCDX is a potentially safer, facile, and more accessible alternative to traditional corneal transplantation for vision restoration.²⁷⁰

Fig. 15 (A) Biomaterial properties of BPCDX. (a) Appearance of BPCDX demonstrating its transparency and refractive properties. (b) Light transmission through 550-μm-thick samples of BPCDX, single-crosslinked BPC, and human cornea. The human cornea, containing epithelial cells that absorb UV light, contrasts with the cell-free bioengineered materials. Data represent means and standard deviations from three independent samples. (c) Mechanical properties of BPCDX compared to single-crosslinked BPC and previously published data of bioengineered constructs made from porcine COL, with reference values from the human cornea for comparison. Measurements for BPCDX represent means and standard deviations from 22 independent samples (550-μm-thick ‘dog-bone’ specimens from different production batches). (d) SEM images showing the surface and bulk (cross-section) structures of BPCDX and porcine cornea, illustrating densely packed COL fibrils in BPCDX, with diameters slightly larger than those in the native porcine cornea. Representative images from three samples per cornea type show consistent results. (e) Degradation of BPCDX, single-crosslinked BPC, and human donor cornea in 1 mg mL⁻¹ collagenase, with data representing means and standard deviations from three independent samples for bioengineered materials and two independent samples of human donor cornea. (f) Attachment and growth of HCE-2 human corneal epithelial cells on BPCDX compared to control culture plates after 16 days. Cells attached to BPCDX, with NucBlue staining, indicating live, viable cell morphology. BPCDX demonstrated greater cell density than culture plasticware. Data from three control and six BPCDX samples, with error bars representing mean and standard deviation (P = 0.003, two-sided independent t-test). Scale bar, 100 μm. (B) Clinical data from a subject in India receiving BPCDX. (a) Pre-operative (left) and one-day post-operative slit-lamp photographs show immediate corneal thickness and curvature changes. (b) OCT scans illustrate sustained thickening and regularization of corneal curvature after implantation of 280-μm-thick BPCDX. Anterior and posterior surfaces of BPCDX are indicated by white arrows. (c) Topographic maps (left, keratometric power in diopters), anterior surface elevation maps (center, μm displacement from a best-fit sphere), and OCT pachymetric maps (right, thickness in μm) from the same subject, showing significant flattening of the steepest pre-operative region (black arrow) and a notable increase in corneal thickness post-operatively. The subject's initial BCLVA of 20/600 improved to 20/30 at 24 months. (C) Clinical data from subjects in Iran receiving BPCDX. (a) Keratometric and corneal thickness maps of the same subject show a thin, steep pre-operative cornea, which was thickened and flattened following intrastromal implantation of a 440-μm-thick BPCDX. OCT cross-sectional scans show corneal thickness and shape before and after implantation, with white arrows indicating the anterior and posterior borders of BPCDX. The subject's initial best spectacle-corrected visual acuity (BSCVA) of 20/200 improved to 20/50 at 24 months. (b) and (c) Photographs of eyes from two subjects showing maintenance of corneal transparency four months post-operatively. (d) and (e) In vivo confocal microscopy images from a single subject showing sub-basal nerve presence (d) and endothelial cell mosaic (e) at 6 months postoperatively. The endothelial cell density was 2222 ± 62 cells/mm². These images, obtained from one subject, may not be representative. Images in (d) and (e) are 400 × 400 μm². Reproduced with permission.²⁷⁰ Copyright © 2024, Springer Nature.

8. Future outlooks

The future of CSE hinges on addressing key scientific and clinical gaps related to bioengineered membranes. While advances in biomaterial science and scaffold fabrication have shown promise, clinical translation requires rigorous evaluation of biocompatibility, optical transparency, biomechanical integrity, and immune response within the complex corneal microenvironment. Current challenges include the lack of standardized fabrication methods to replicate the intricate structure and mechanical properties of native corneal stroma. Emerging techniques, such as 3D bioprinting and electrospinning, offer potential solutions but require further optimization. Integrating advanced drug delivery systems into scaffold designs can enhance therapeutic efficacy and reduce post-operative complications. A multidisciplinary approach, combining biomaterial engineering, cell biology, and ophthalmology, is crucial to accelerate progress in this field. Large-scale, multicenter clinical trials, coupled with advanced imaging techniques such as OCT and confocal microscopy, can provide valuable insights into stromal regeneration and facilitate regulatory approval for novel therapies. By addressing these challenges, we can develop scalable, clinically relevant solutions to restore vision and alleviate the global burden of corneal blindness.

9. Conclusions

The engineering of functional corneal stromal tissues remains a formidable challenge in regenerative medicine, imposing the convergence of innovative biomaterials and advanced fabrication technologies. While significant strides have been made, the optimization of scaffold design to more accurately replicate the ECM is essential for enhancing clinical efficacy. Emerging biomaterials, including collagen-based scaffolds and bioengineered ECM analogs, have demonstrated considerable potential in emulating the structural and biochemical complexity of the corneal stroma. Nevertheless, critical challenges persist, including scalability, mechanical durability, and long-term biocompatibility, which must be addressed to advance these technologies toward clinical application. Recent inventions in fabrication techniques, such as electrospinning and 3D bioprinting, have enabled the development of scaffolds with precisely tunable mechanical properties and microarchitectural features, significantly improving cell viability, integration, and tissue regeneration. Furthermore, the incorporation of bioactive modifications as gene-activated scaffolds and stimuli-responsive materials offers a transformative avenue for modulating host responses, mitigating inflammation, and promoting tissue repair. Despite these promising advancements, the path to clinical translation is hindered by regulatory, ethical, and manufacturing complexities that require careful consideration. Looking ahead, future research should focus on developing multifunctional scaffolds that incorporate bioactive molecules, such as growth factors and antimicrobial agents, to enhance regenerative outcomes. Advances in fabrication technologies, such as 4D bioprinting and smart biomaterials, hold promise for creating dynamic scaffolds that can adapt to the changing needs of the healing tissue. Furthermore, interdisciplinary collaborations between material scientists, biologists, and clinicians will be essential to accelerate the translation of these technologies into clinical practice. Overall, this review underscores the transformative potential of biomaterials and scaffold-based strategies in corneal stromal engineering. By addressing current limitations and leveraging emerging technologies, the field is poised to make significant advances toward achieving functional and sustainable corneal repair. We hope this work will inspire further innovation and collaboration, ultimately contributing to improved outcomes for patients with corneal injuries and diseases.

Abbreviation

CSE	Corneal stroma engineering
3D	Three-dimensional
WHO	World health organization
HCCs	Human corneal cells
LSCs	Limbus (Limbal) stem cells
CSCs	Corneal stromal cells
OH	Hydroxyl
rCEN	rabbit corneal endothelial
HCLE	Human corneal limbal epithelial
HCECs	Human corneal epithelial cells
PEG/PAA	Poly(ethylene glycol)/Poly(acrylic acid)
AM	Amniotic membrane
PGS	Poly glycerol sebacate
CAD	Computer-aided design
PEGDA	Poly ethylene glycol diacrylate
GEL	Gelatin
ALP	Alkaline phosphatase
FRESH	Freeform reversible embedding of suspended hydrogels
HA	Hyaluronic acid
dC	Decellularized cornea
PGA	Poly glycolic acid
PVAc	Poly vinyl acetate
PLLA	Poly-L-lactic acid
PHEMA	Poly(2-hydroxyethyl methacrylate)
TACs	Transient amplifying cells
LECs	Limbal epithelial cells
ALG	Alginate
CECs	Corneal endothelial cells
hTMSC	Human turbinate-derived mesenchymal stem cells
hASC-dCSKCs	hASC-derived corneal stromal keratocytes
ALDH1A1	Aldehyde dehydrogenase class 1A1
PNIPAAm	Poly(N-isopropylacrylamide)
GlMA	glycidyl methacrylate
He/O2	Helium–oxygen
CFCs	Corneal fibroblast cells
GKCs	Goat keratocyte cells
SS	Silk sericin
CHI	Chitosan
HPCHI	Hydroxypropyl chitosan
FIBn	Fibronectin
PdCT	Porcine decellularized corneal tissue
PEU	Poly(ester urethane)
RPE	Retinal pigment epithelium
hESC-LESCs	Human embryonic stem cell-derived limbal epithelial stem cells
FEM	Finite element method
HE	Hematoxylin and eosin
DWA	Direct-write assembly
ECM	Extracellular matrix
BPCDX	Bioengineered porcine construct double-crosslinked
BPC	Bioengineered porcine construct
MAPK	Mitogen-activated protein kinase
TGF- β	Transforming growth factor–β
miRNAs	microRNAs
IL-1	Interleukin-1
EBM	Epithelial basement membrane
BCLVA	Best contact lens-corrected visual acuity
ALD	Aldehyde
pDCSM	Porcine decellularized corneal stroma matrix
EB	Embryoid body
GelNF-HA	Gelatin nanofibers coated with hyaluronic acid
hAESCs	Human amniotic epithelial stem cells
2D	Two-dimensional
4D	Four-dimensional
HCCK	Human corneal cell keratoplasty
CHCCs	Cultured human corneal cells
CKCs	Corneal keratocyte cells
UV	Ultraviolet
BCECs	Bovine corneal epithelial cells
PDL	Poly-D-lysine
HECs	Human endothelial cells
FMN	Flavin-mononucleotide
HLSCs	Human limbal stem cells
LECs	Limbal epithelial cells
PCL	Polycaprolactone
MRI	Magnetic resonance imaging
GelMA	Gelatin methacrylate
LAP	Lithium phenyl (2,4,6-trimethyl benzoyl) phosphinate
OCT	Optical coherence tomography
SOALG	Sodium alginate
CS	Chondroitin sulphate
PLA	Poly lactic acid
PLGA	Poly lactide-co-glycolide
PVA	Poly vinyl alcohol
pNIPAM	Poly-N-isopropylacrylamide
PEGDA	Polyethylene (glycol) diacrylate
COL	Collagen
AG	Agarose
LSSCs	Limbal stromal stem cells
CEpCs	Corneal epithelial cells
hASCs	human adipose stem cells
TKT	Transketolase
hCFs	Human corneal fibroblasts
PIPAAm	Poly(N-isopropylacrylamide)
LCST	Lower critical solution temperature
AuNPs	Gold nanoparticles
PCECs	Primary corneal epithelial cells
SF	Silk fibroin
AVE	Aloe vera
HECTS	Hydroxyethyl chitosan
FIB	Fibrin
HdCT	Human decellularized corneal tissue
BdCT	Bovine decellularized corneal tissue
P(NIPAM-co-GMA)	Poly (N-Isopropylacrylamide-co-Glycidylmethacrylate)
ALK	Anterior lamellar keratoplasty
PET	Polyethylene terephthalate
DoD	Drop-on-demand
hTMSCs	Human turbinate-derived mesenchymal stem cells
CDs	Cyclodextrins
LASIK	Laser-assisted in situ keratomileusis
SEM	Scanning electron microscope
FAK	Focal adhesion kinase
CSSCs	Corneal stromal stem cells
Smad	Signal transduction by mothers against decapentaplegic
CTE	Corneal tissue engineering
PDGF- BB	Platelet-derived growth factor BB
αSMA	α-Smooth muscle actin
MC	Methylcellulose
HAMA	Methacrylated hyaluronic acid
hiPSCs	human-induced pluripotent stem cells
C-MSCs	Corneal Mesenchymal stromal cells
AS-OCT	Anterior segment optical coherence tomography

Data availability

The data presented in this study are available on request from the corresponding author.

Author contributions

A. O. M. S.: conceptualization, methodology, investigation, writing – original draft, validation, review & editing. M. S.: conceptualization, investigation, writing, review & editing. K. S. C. R.: conceptualization, investigation, writing, review & editing. W.O.L.: conceptualization, validation, writing, review & editing. N. M.: conceptualization, validation, supervision, writing, review & editing.

Conflicts of interest

A. O. M., M. S., K. S. C. R., W. O. L., and N. M. declare that they have no conflict of interest. This article does not contain any studies on human or animal subjects performed by any of the authors.

Acknowledgements

This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT) and Tecnológico de Monterrey. A. O. M. and K. S. C. R. acknowledge funding from CONACyT Graduate Studies Scholarships. N. M. acknowledges the School of Pharmacy at the University of Wisconsin-Madison, USA.

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Footnote

† These authors have contributed equally to this work.

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