Scaffold-free and scaffold-based cellular strategies and opportunities for cornea tissue engineering

The human cornea is a dome-shaped, multilaminar, transparent, immune-privileged membrane in the front of the eye’s optical system. The cornea protects the intraocular parts and letting light into the interior for an image formation on the retina through a crystalline lens. Infection, inflammation, traumatic disorders, and systematic diseases mediate structural changes in the cornea, resulting in irreversible blindness. Up to 12 million patients are awaiting treatment. The inconsistencies in corneal transplantation and artificial implant side effects underscore the need for tissue engineering to address this gap. This study outlines limitations in current corneal treatment and highlights the self-healing potential of each corneal cellular layer to frame necessities for cell-based bioengineering strategies. This study discusses principles, current progress, construct design, and opportunities of scaffold-free and scaffold-based cellular strategies towards repairing epithelium, endothelium, stroma, and full-thickness cornea.


Introduction
The cornea is a transparent, dome-shaped, multilaminar, immune-privileged membrane in the front of the eye's optical system.The cornea diameter is around 11.04-12.50mm in adult males and 10.70-12.58mm in adult females.It acts as an air-liquid interface on the surface of the eyeball and prohibits foreign materials entry [1,2].The cornea guides incident light towards the pupil in a forward direction and supplies two-thirds of the refractive power for an image formation on the retina through a crystalline lens.The oxygen and nutrients needs are met through diffusion from the aqueous humor, tears film, and limbal capillaries (figure 1(A)) [1,3].

Current treatment and shortcomings
The human cornea is immune-privileged, accessible, avascular, and less prone to rejection than vascular organs.Full-thickness cornea transplantation (penetrating keratoplasty (PK)) from a deceased human donor is a common treatment to improve visual function [9].The recent progress in surgical techniques opens possibilities to correct specific corneal damaged parts (lamellar keratoplasty).It reduces allogeneic tissue, leads to earlier rehabilitation and better patient satisfaction than PK (figure 1(D)) [10,11].Despite progress, there persist inconsistencies in transplantation, i.e. corneal grafting induces scarring at the recipient interface, showed poor outcomes in high-risk recipients, results in incomplete nerve regeneration, and up to 25% of the transplanted cornea failed just a half-decade [12,13].The marked decline in endothelium cell number upon transplantation has been reported and is a risk factor for later graft failure [14,15].The increased population aging, adoption of laser corneal surgery, and rising prevalence of transmissible diseases render about one-third of donated corneas unusable for transplantation [16].Unfortunately, many developing parts of the world lack eye banking infrastructure, and in addition, tissue procurement became more challenging during the recent coronavirus pandemic.These factors limit the donor pool, and it is estimated that up to half of the patients, most of whom live in developing countries, have no access to corneal transplantation [17,18].
Keratoprostheses are transparent optical artificial implants studied to replace the central cornea and are easy to manufacture in large quantities.The clinical studies indicate that prostheses have a poor clinical success rate, insufficient integration into the host tissue, and need immunosuppressant medications.They are only recommended for end-stage corneal diseases, and biological compliance remains a fundamental problem, i.e. most prostheses do not allow for re-epithelialization, re-innervation, and recellularization [19,20].Furthermore, their long-term outcomes are uncertain and are prone to rejection, retinal detachment, corneal calcification, recurrent glaucoma, stromal melting, and persistent inflammation [21,22].The shortcomings in current treatment options and the exorbitant cost of cornea healthcare underscore the need for regenerative strategies to treat corneal issues.

Cell-based tissue engineering strategies
Tissue engineering is a multidisciplinary field.It combines cell biology, chemistry, medicine, materials science, mathematical modeling, nanotechnology, and engineering principles to improve tissue reconstruction.Cornea bioengineering focuses on regenerative strategies involving suitable cell types, bio-cues to guide cell response, and polymeric materials for functional support.Stem cells hold therapeutic potential against various injuries and offer sophisticated modes of action that chemical compounds cannot imitate.Since each corneal cellular layer holds unique cellular compositions, thus more attention is given to cell-based reconstruction instead of cell-free scaffolds.Both corneal and non-corneal sources have been introduced for corneal bioengineering [23].One crucial aspect for translational application is the delivery method in a format that should support cell retention, cell survival, tissue integration, and minimum side effects.Scaffold-based and scaffold-free are two main cell-based bioengineering strategies for tissue reconstruction and have undergone exciting progress in the recent decade (figure 2) [24,25].Scaffold-free strategies (i.e.cell-suspension, cell-sheet, cellular spheroids, and cell printing) use stem cells to create a self-producing matrix [24].The cell suspension injection via engineering strategy is less invasiveness, easy to prepare cell stock, avoided artificial substrate, minimal surgical problems, and could be performed quickly.Cell sheet engineering uses responsive surfaces that enable cells to detach in the form of a cell sheet.It preserves matrix components, cell-to-matrix contact, cell-to-cell junctions and could hold bioadhesive properties [26].Cellular spheroids have recently been recognized as building blocks for bioengineering and could provide a three-dimensional (3D) physicochemical environment, facilitate cell-cell communication, and maximize cell-matrix interaction [27].Cell bioprinting can assemble different cells in a pattern resembling natural tissues and later creates a matrix microenvironment in a confined space [28].The scaffold-free construct could mimic the target tissue, lessen cytotoxicity concerns, and possibly eliminate the need for a sutures process due to adhesive matrix components and the absence of exogenous biomaterials [29].
Scaffold-based strategies merge biomaterials, cells, and molecular factors in the form of a functional scaffold that can support cellular function and model tissue fabrication.The design, mechanical properties, surface chemistry, adhesive properties, and biological functionalities of scaffolds depend on inherent materials properties (natural membrane, natural materials, synthetic materials, composite materials), manufacturing processes (acellular membrane, cast film, hydrogel, nanofibers, bioprinting), and correct use of cells, molecular factor, and crosslinking agents [30].The scaffold-based construct can influence cell attachment, cell expression, cell proliferation, matrix formation and be designed to support targeted bio-integration [31].
Cornea bioengineering presents unique challenges and necessitates that cell-based bioengineering substitutes be mass-produced, have good mechanical properties, integrate into host tissue, hold sufficient optical properties and biomimetic composition [32].This review summarizes cornea biology and regenerative potential to frames the necessities for corneal and non-corneal stem cell applications.This study discusses principles, current progress, construct design, and opportunities of cell-friendly scaffold-free and scaffold-based bioengineering strategies towards repairing epithelium, endothelium, stroma, and full-thickness cornea.

Cornea epithelium tissue engineering
The corneal and conjunctival epithelium is separated by a transitional zone called limbus (1-2 mm).It is composed of ∼10-12 cell layers and prevents the conjunctivalization and neovascularization of the cornea [33].The fibrovascular ridges of the limbus called limbal palisades of Vogt.It is surrounded by Langerhans cells, melanocytes, T-lymphocytes, and blood vessels.Most importantly, it serves as a niche for LESCs plus CSSCs [34][35][36].The limbus zone protects stem cells from UV radiation plus stress-inducing conditions and provides essential cues to maintain stemness properties [33,37,38].The cornea epithelium (40-60 µm) comprises stratified squamous non-keratinized epithelial cells and occupies 10% corneal thickness.It can be sub-divided into three sub-layers, i.e. tri-layered superficial cells, tri-layered wing cells, and monolayered basal cells.The superficial layer consists of flat polygonal cells that form tight matrix junctions and prohibit foreign materials entry [39].The uppermost superficial cells have numerous microplica, which increases the surface area for oxygen absorption and tear film (∼3-6 µm) attachment.The superficial cells have a short life span (7-10 d) and regularly desquamate into the tear film [40].The epithelial shedding stimulates dormant LESCs, which divide asymmetrically into the transient amplifying cells (TACs) and stem cells (remain in the niche).TAC migrates centripetally and anteriorly towards the central zone, undergoes multiple rounds of replication, loses stemness properties, and is committed into columnar basal epithelial cells.The basal columnar cells adhered to the basal lamina (40-60 nm), modulating the migration and proliferation of cells into wing cells and superficial cells.The epithelium mass remains stable since basal cell growth rate equals the rate of cell desquamation, and this homeostasis process persists throughout the organism's lifetime (figure 3) [41][42][43].
The cornea epithelium is densely innervated (2500 nerve terminals/mm 2 ) and up to 300 times more pain-sensitive than skin.Most nerves bundles originate from the trigeminal nerve of the ophthalmic branch.Nerve bundles sacrifice their perineurium and myelin sheaths in the limbus zone and radially join the anterior stroma [44,45] and form a sub-basal nerve plexus beneath the basal lamina.Before penetrating the Bowman's membrane, nerve fibers transform into right angles, branch onto numerous small branches, and terminate near the superficial sub-layer of the epithelium.The corneal nerves secrete many trophic factors that regulate tear production, blinking response, pain perception, and proliferation of cornea epithelium [46][47][48].
Numerous factors, i.e. chemical burns, inflammation, traumatic injuries, microbial infections, and systemic disease, can damage the epithelium, limbus, and corneal nerves [49].Limbus damage often leads to LESCs deficiency (LESCD), impairs epithelium self-healing, epithelium net loss and thus allows conjunctival tissue to colonize the corneal surface.These changes can cause corneal opacification, inflammation, neovascularization, permanent epithelial abnormalities, and blindness [50,51].On the other hand, the damaged corneal nerve diminishes the sensation and could result in tearing dysfunction, surface scarring, pain episodes, and a delayed healing process [52,53].PK replaces the central cornea but cannot restore the limbal zone and nerve regrowth, resulting in poor outcomes.Healthy limbus pieces from patients' unaffected eyes, living relatives, or deceased donors may be grafted to replenish the stem cell population but is not exempt from limitations of donor site morbidity and lack of autologous graft (in bilateral conditions).These factors necessitate cell-based bioengineering strategies to restore LESCs and nerve function to ensure long-term ocular surface protection [54,55].

Cell source for epithelium bioengineering
LESCs can be expanded from a minimal biopsy sample, preferably taken from the superior and inferior limbus zone.It possesses a high nuclear to cytoplasmic ratio.The candidate stem cell biomarker includes but not limited to ABCG2 [56], ABCB5 [57], N-cadherin [58], ∆NP63α, C/EBPσ, OCT4, αvβ5-integrin, because some biomarkers are also selective for TACs and terminally differentiated cells [59].LESCs preferred to be cultured on a feeder cells layer that provides stemness-supporting factors and enables cells to propagate for up to 14 passages before senescence [60,61].Autologous LESCs are gaining popularity to treat unilateral ocular surface defects.The allogenic LESCs preferred to reconstruct bilateral cases or when more than half of the cornea is affected.The potential shortcomings in corneal cell sources include donor site morbidity, limited cell passage number, potential contamination from cell feeder, and high expected clinical cost [62,63].
Non-corneal stem cells, i.e.MSCs, OMECs, and iPSCs, have been recognized as an alternate option for repairing bilateral LESCD.These cells hold self-renew potential, ease expanded, transform into corneal phenotypes, and reduce product costs.Progress has been made to trans-differentiating different stem cells into the epithelial cells' lineage, but most published studies used stem cells from animal sources, with only a few claiming in vivo success [64][65][66][67][68]. Non-corneal stem cells do not express many anti-angiogenic factors.Further research is required to unravel homing, immunomodulation, and trans-differentiation mechanisms and minimize immune rejection and neovascularization concerns [69].There is a need to revise protocols to avoid feeder-free and xeno-free culturing supplements in compliance with a good manufacturing practice.Furthermore, long-term clinical trials are expected before translating the benefits because damaged tissue holds different microenvironments, leading to undesired morphologic changes [70,71].

Scaffold-free epithelium bioengineering
The ocular surface of unilateral LESCD patients could be reconstructed using expanded autologous LESCs in the form of a cell sheet.LESCs sheets were implanted onto the patient's damaged cornea (n = 2), and the treatment was effective in regenerating the corneal epithelium and improving visual acuity [72].The autologous OMECs have been cultured with and without using a feeder layer on temperature-responsive surfaces, and resultant cell sheets were implanted onto the damaged corneal surfaces of rabbit models and human patients.The results showed that corneal surfaces were re-epithelialized, remained transparent, had no ocular complications, and had a reasonable long-term prognosis [73][74][75][76].These studies demonstrated that LESCs and OMECs based ultra-thin cell sheets might be beneficial to maintain sufficient cell count and reshaping the ocular surface.Due to the delicate mechanical and structural properties, it is difficult to handle cell sheets for direct transplantation into damaged ocular surfaces and thus necessitate sub-micron scale carrier material for ease transplantation and clinical integration [77].

Scaffold-based epithelium bioengineering
The amniotic membrane is semi-transparent and possesses inherent growth factors, antiangiogenic, antimicrobial, anti-inflammatory, and antiviral properties.It is used as a cell-sheet carrier and provides a biological matrix to promote cell expansion [78].The autologous OMECs cultured with and without fibroblast feeder on the amniotic membrane and resultant bio-membrane resembled cornea epithelium morphology.They could improve LESCD ocular surface reconstruction and visual acuity [79,80].LESCs have been expanded on the amniotic membrane following good manufacturing protocol and implanted onto the LESCD corneal surface of patients.The results showed that corneal surfaces become transparent, remained stable, and applied treatment is safe since most patients do not experience inflammation in the transplanted region [81,82].Despite progress, the clinical use of an amniotic membrane is far from optimal due to numbers of limitations include growth factor content variation, unknown mode of action, poor biodegradation rate, inter-donor batch variation, suboptimal transparency, granulomatous reaction, and concerns about infectious pathogen transmission [83][84][85].The chemical cross-linking and biomimetic functionalization could improve the membrane's biological performance [86].
Electrospun nanofibers could serve as excellent substrates for LESCs adhesion and expansion, as well as a tissue-like structure [87].The nanofibers expand both corneal and non-corneal cells, and resultant cell/scaffold constructs were then implanted onto the injured ocular surface of rabbits.The results demonstrated that cellular scaffolds integrate onto the damaged tissue surface, enhanced ocular tissue reconstruction, and improved corneal transparency [88].Some studies tried to recapitulate the limbus crypts using the nanofibrous system to provide the natural microenvironment for cells.The results showed that bioengineered limbal crypts showed protective properties, allowed LESCs expansion, produced basement membrane proteins, stimulate directional migration, and induce epithelization in ex vivo rabbit corneas [89,90].
The autologous LESCs were cultured or transplanted into fibrin scaffolds before being transplanted into the patient's damaged cornea, and results reported that up to 2/3 of the patients' corneal surfaces remained stable.The cultured cells that express 3.0% ∆Np63α holoclones have a high success rate, whereas cultures with less than ∆Np63α + cells have a lower success rate.In recent, LESCs expanded on the 3T3-J2 based feeder layer and then grown on fibrin hydrogel.The resultant transparent circular sheet system containing 79 000-316 000 cells cm −2 corneal epithelial cells and gained clinical traction to treat moderate-to-severe LESCD patients [91][92][93].The bacterial nano-cellulose film functionalized with laminin plus collagen holds superior mechanical, semi-transparent, biomimetic, and cell-scaffold interface properties.The ex vivo results demonstrated that construct support LESCs expansion and maintain self-renewal and stemness properties, which is crucial for repairing injured ocular surfaces [94].The polymethacrylate hydrogels surface has been modified with amines and matrix components (i.e.collagen, fibronectin, and laminin).The modified hydrogel membrane was shown to improve cell growth and promote epithelium reconstruction [95].These studies indicated that nanofiber, hydrogels, and film-based scaffolds could expand and deliver cells, and the cell scaffold interface interaction is important to maintaining a functional cell population.
Corneal nerve integrity is essential in maintaining a healthy ocular surface.A few attempts have been made to include neuron development cues in bioengineered scaffolds despite their functional importance.The mechanically stable, transparent, and laminin functionalized hydrogel-based matrices have been designed and implanted into pigs' cornea.The results demonstrated that resultant scaffolds promoted cell adhesion, epithelial stratification, and neurite in-growth [96].The silk protein-based scaffold has been used to support the epithelium, stroma, and neural networks formation.The neural networks innervated both stromal and epithelial layers and increased cellular function.The neural innervated scaffold had a beneficial impact on epithelial maturation, and the proposed in vitro tissue model could be used in drug development, disease intervention, and physiological research [97].The collagen and methacryloyloxyethyl phosphorylcholine were crosslinked, shaped into corneal dimensions, and resultant scaffolds were implanted into pigs' corneas.The findings showed that implants improved corneal cell regeneration, nerve density, and tear film production [98].These studies demonstrate that corneal nerves regeneration should be part of bioengineering strategies to ensure long-term graft survival.The schematic cell-based bioengineering strategies for ocular surface reconstruction and mode of cell-scaffold implantation are summarized in figure 4.

Cornea endothelium tissue engineering
The endothelium is made up of a monolayer (4-6 m) of non-proliferating flattened CEnCs.These cells are derived from the neural crest and are packed in a hexagonal mosaic pattern.The apical side of CEnCs facing aqueous humor while the basal side is slightly irregular and adhering to the DM [99].Sodium/potassium ATPase pump and sodium/bicarbonate cotransporter present at the basolateral side of plasma membranes function to transport water and ions into the anterior chamber side from the corneal stroma.Intercellular junctional complexes (zonula occludens (ZO-1)) near the apical domains function as a partial barrier and allows passive nutrients movement from the anterior chamber towards the stroma.The pump-leak mechanism is essential for nourishing the cornea, controlling stromal deturgescence, and maintaining cornea clarity and thickness [100,101].
CEnCs density is higher at birth (5000-6000 cells mm −2 ).The means thickness, diameter, and area of CEnCs are around 5 µm, 20 µm, and 250 µm 2 , respectively [102].Mature CEnCs have a normal component of telomeres but are non-mitotic and locked in the quiescent G1 phase due to numerous factors, i.e. cell-cell contact inhibition, a cyclin-dependent kinase inhibitor, high concentrations of negative growth factors transforming growth factor-ß (TGF-ß), the compositional change in basement components, and reactive oxygen species-mediated stress-induced senescence [103,104].CEnCs density is inversely proportional to age, i.e. declines to about 4250 cells mm −2 in the first two years from birth due to normal growth in corneal diameter and then decreases (0.3%-0.6% per annum) in the central zone with age.The normal CEnCs loss throughout life complement through sliding and enlargement of adjacent cells (instead of cell division) [105] but at the cost of increased cell size (polymegathism) and a decline in the proportion of hexagonal cell shape (pleomorphism) [106].The age-related CEnCs are not a concern, and normal reserve density (2300 cells mm −2 after eight decades) is sufficient for normal endothelium function [107].
Numerous factors, i.e. trauma, inflammation, FECD, pseudophakic bullous keratopathy, intraocular surgeries, and previous corneal transplantation, often threaten CEnCs density.Once CEnCs count is reduced below a limit of 500 cells mm −2 , the endothelium fails to perform the pump-leak function and results in cornea clouding, edema, corneal blindness (figure 5) [108].It is a more severe problem since it lacks regenerative capabilities and cannot replace defective cells due to its mitotic arrest.Up to 30% of corneal transplants are undertaken to reconstruct endothelium degeneration.The higher post-surgical CEnCs loss has been noticed in the transplanted cornea than the age-related decline rate.It can lead to later graft failure and limit donor pool since corneas with reduced CEnCs count (<2000 cells mm −2 ) are unsuitable for transplantation.These shortcomings necessitate cell-based bioengineering strategies to restore endothelium [6,[109][110][111].
CEnCs are separated from donor corneas by peeling the DM and then digesting the tissues with enzymes.The prolonged enzymatic incubation step could damage the CEnCs and possible cross-contamination with keratocytes.Various methods have been introduced, i.e. sphere-forming assay, magnetically activated cell sorting, density-gradient centrifugation, and fluorescence-activated cell sorting to purify CEnCs for clinical use [119][120][121].The detached CEnCs are cultured using the mitogen-rich medium, molecular inhibitor (rho-associated protein kinase (ROCK) inhibitor, p38MAPK, p120 catenin inhibitor), and basement membrane components to overcome the mitotic block and to promote cell expansion [122][123][124][125][126]. Furthermore, co-culturing the CEnCs with the i3T3 feeder could preserve morphological and functional characteristics [127].
CEnCs respond in an age-dependent manner.Cells from older donors show minimum proliferation potential and less response to mitogenic stimuli due to high expression of senescence-associated markers compared to adult donors' CEnCs [128,129].In addition, sporadic karyotypic aberrations in cultured CEnCs have been reported, possibly due to cellular distress during the culturing, and are common in cells taken from elderly donors [130].The cell propagation is a challenge because CEnCs lead to earlier senescence in the mitogen-reduced medium and can become more heterogeneous in cell enlargement, intercellular junctions, and matrix protein expression after each passage in a mitogen-rich medium.Multiple strategies such as inhibition of TGFβ blockage, p120-catenin, matrix metalloproteinase have been introduced to minimize phenotypic changes [131][132][133][134].
The scarcity of young donor tissues, poor expansion of cells, difficulties in maintaining cells phenotypes, and stringent quality control requirements are the main shortcomings that limit the translational application of CEnCs-based therapies [135,136].These factors support the use of non-corneal cell lineages for endothelium reconstruction.To date, only a few reports claim successful differentiation of ESCs [137][138][139], NCSCs [140], and iPSCs [141][142][143][144] into CEnCs lineage under different supplements and conditioned medium.Most published studies used stem cells from an animal source, and only a few reported in vivo successes.There is a need to revise protocols to avoid xeno-free supplements following good manufacturing practice conditions.The non-corneal stem cells could raise concerns about genomic alteration and malignant transformation; therefore, detailed studies are needed to unravel the mechanisms of homing, immunomodulation, programmed cell cycle (after implantation), and cell senescence before developing in vivo strategies [145,146].

Scaffold-free endothelium bioengineering 3.2.1. Cell injection engineering
CEnCs injection into the anterior chamber has garnered growing attention because of its benefits, including reduced invasiveness, avoided artificial substrate, minimal surgical problems, and ease of repeat treatment when necessary.The cells delivered via simple injection have a poor cellular attachment to DM, and non-targeted delivery could blockage Schlemm's canal, increase intraocular pressure, guttae formation (figure 6(A)).These shortcomings necessitate engineering strategies to minimize these side effects.ROCK inhibitor supplementation, ferromagnetic induction, mini-sheet injection, laminin 511 pre-coating on damaged DM membrane, and transplantation of transparent gel sheets before cell injection have been investigated to promote the engraftment of injected cells into the DM of the recipient cornea.
The ROCK inhibitor suppresses apoptosis, stimulates intracellular adhesion, promotes cell proliferation, lowers intraocular pressures, and effectively treats endothelial diseases [147,148].A mixture of ROCK inhibitor and CEnCs has been injected into the anterior chamber of animal models.A prone positioning technique (face-down position) is used to enhance cell attachment to recipient DM.The applied treatment resulted in a normal corneal clarity and decreased central corneal thickness compared to injection of CEnCs alone [149,150].Furthermore, this combination has been used to treat bullous keratopathy patients, and results showed reversal of corneal edema and clinical outcomes being stable in 10/11 patients for up to half-decade.The author claims that one donor cornea can be used to treat up to hundreds of patients (figure 6(B)) [151,152].
The endocytosis of superparamagnetic microspheres or magnetic nanoparticles into CEnCs does not affect the optical properties, proliferation capacity, and pump-leak function of cultured CEnCs.The intracameral injection of superparamagnetic CEnCs following an external magnetic field reduced corneal edema.It promoted the cell's adhesion in the form of a monolayer to the intact DM in animal models [153,154].The immunomagnetic nanoparticles labeled umbilical cord blood endothelial progenitor cells were intercalated in the rabbit's anterior chamber.A magnetic field was applied to the eyelid to force the cells' adhesion to the DM.The magnetic field-based treatment promoted rapid adhesion on the DM and improved corneal thickness and endothelial pump function compared to counterpart non-magnetic field treatment [155].Many others' studies found that superparamagnetic CEnCs led to the functional monolayer formation on the DM, minimize concerns related to non-targeted delivery in the trabecular meshwork, require less time on DM compared to other methods, and decreased the corneal thickness in animals' models (figure 6(C)).The potential concern of self-aggregation of the cells, rise in intraocular pressure, and iron leakage from the loaded cells need detailed studies to confirm the long-term effectiveness of ferromagnetic induced treatment [153,[156][157][158].
The injection of mini CEnCs sheets (contained 4-10 cells) into the anterior chamber of rabbit (n = 60) promoted cell adhesion, tight junction formation, organized cell distribution, and leads to improved clarity recovery compared to single-cell suspensions (figure 6(D)) [159].The transparent nanocomposite gel sheet is inserted (via incisions) into the anterior chamber and keeps it close to the damaged endothelium of patients (n = 3).The small holes in the gel sheet could aid the circulation of aqueous humor.CEnCs were then injected in between the gel sheet and posterior cornea.The gel sheet is used as a temporary supporting material that could ease removed and proposed to avoid non-targeted cell adhesion and boost cell settlement on DM.The applied treatment showed no adverse effect, and bullae in the cornea disappeared in all patients (figure 6(E)) [160,161].
The cell injection strategies necessitate intact DM, restricting their effectiveness for late-stage endothelial diseases and other pathophysiological conditions.The pre-injection of DM components (laminin 511) into the anterior chamber of the rabbit (n = 26) could improve the micro-environment of posterior damaged DM.The laminin pre-coating did not increase the intraocular pressure, promote the expression of pump-leak markers, and facilitates the attachment of injected CEnCs on DM.These properties, in return, could promote rapid endothelium regeneration and better corneal clarity compared to the counterpart control (figure 6(F)) [162].Overall, each delivery method has its pros and cons.The availability of different engineering modalities to deliver CEnCs is important for applicability to varying scenarios of endothelial diseases.

Cell sheet transplantation
Nitschke et al used NiPAAm-DEGMA copolymer-based thermo-responsive surfaces to generate CEnCs sheets.The copolymer sheet's phase transition temperatures are similar to physiological ranges.The fewer temperature shocks were required to detach the cell sheet, and thus preserving tight junctions, extracellular matrix (ECM) components, and resembling an endothelium monolayer [163].The chelating agents and physical pressure have been applied to harvest the CEnCs sheets.The proposed method maintains matrix proteins, supports endothelium functional reconstruction, and improves the clarity of the rabbit cornea [164].Sumida et al develop a CEnCs sheet on collagen-coated dishes using poly(N-isopropylacrylamide) (PNIPAAm) based thermo-responsive surfaces.The resultant cell sheet displays hexagonal morphology, numerous microvilli, and improves corneal thickness once grafted into in rabbit model [165].Lai et al created CEnCs sheets on PNIPAAm surfaces and used gelatin disks as carrier materials to implant fragile cell sheets in a rabbit model's intraocular space.The results showed that CEnCs sheets adhere to the model cornea, but gelatin disc could obstruct the normal flow of aqueous humor.The same research group introduced porous gelatin as a carrier to overcome nutrient transfer limitations and to ensure the normal flow of aqueous humor [166,167].The ultra-thin cell sheet beneficial to maintain cell count, balanced cell distribution, and adhesive function, but their reduced mechanical properties necessitate sub-micron scale scaffolding support to facilitate clinical integration [77].

Scaffold-based endothelium bioengineering
Human amniotic membrane, decellularized crystalline lens capsules, and decellularized animal-derived corneal scaffolds are among the biological membrane scaffolds that have been introduced for endothelium reconstruction.The amniotic membrane serves as a carrier scaffold to promotes the CEnCs expansion.The seeded cells change the membrane's physiological properties, and the resultant cellular construct becomes clearer than the counterpart membrane.The cellular membrane maintains corneal thickness and improves the visual outcomes in animal model studies [168,169].The decellularized animal posterior lamellae and DM serve as a natural CEnCs substrate.They contain essential matrix components such as collagen, laminin, and fibronectin, which promote CEnCs morphological and physiological properties.The pre-clinical results demonstrated that cell/scaffold constructs well-integrated in host corneas, increased corneal clarity, and restored endothelium function [170][171][172][173].The human anterior lens capsule, rich in collagen (I and IV) and laminin content, has been recognized as a scaffold for CEnCs expansion, confluence monolayer formation, and function to maintain both barriers and pump function [174,175].Natural membranes hold several drawbacks, including the risk of disease transmission, the lack of standardized preparation protocols, the reduced biodegradation, semi-opaque properties, high variability in biochemical properties, and donor dependency [83].
The biomaterial-based engineered scaffolds are simples to mass-produce, hold tailored mechanical, structural, biocompatible, adhesive, and optical properties.Casted films, hydrogel, nanofibers, and bio-printed scaffolds have been used in endothelium bioengineering and gained considerable attention in the recent decade.The transparent, thin, and flexible heparin-functionalized scaffolding membrane could absorb and release basic fibroblast growth factors and function to improve CEnCs survival, maintain cell morphological properties, improve pumping function, and integrated into the host tissue [176].Composite of PDDLA/gelatins were assembled in a transparent, semipermeable, and ultrathin (<1 µm) sheet using multistep spin-coating, and scaffold surface maintain morphological and functional properties of cultured CEnCs [177].Ultra-thin, clear, and elastic chitosan/PEG-based transparent hydrogel films hold potential for glucose plus albumin permeability and promote CEnCs expansion and hexagonal morphology [178].Poly-ε-lysine based cross-linked hydrogel could promote CEnCs expansion in confluent monolayer, and expanded cells retain pump-leak characteristics and hexagonal morphological properties.The resultant cellular hydrogel is transparent, provides good mechanical properties, and could enable ease surgical transplanted [179].The nano-pillars patterned substrate using crosslinked bio-functional gelatin methacrylate improved CEnCs density, tight junction expression and holds enough mechanical strength, which could allow ease implantation [180].
The collagen sheet expands and maintained CEnCs hexagonal morphology and pump-leak function.The cellular construct implanted near the posterior DM of monkey models and results showed enhanced corneal clarity and decreased corneal thickness.The transplanted cells maintain sufficient density for up to four years [181].The aloe vera functionalized silk fibroin film scaffolds are transparent, support CEnCs proliferation, and provide a suitable environment for maintaining their morphology and critical functions.The in vivo results showed that scaffolds are better for blurred vision and minimal refraction alteration in the rabbit eyes [182].The cultured CEnCs have been seeded on spherical gelatin hydrogel, and expanded cells expressed tight junction plus ion pumps protein, indicating normal cell physiology.The in vivo results involving the bullous keratopathy monkey model demonstrated that the transplanted cell scaffold ease adhered to the posterior cornea and maintained transparency and thickness [183].Kim et al embedded ribonuclease five-overexpressed CEnCs in gelatin-RGD (Arg-Gly-Asp) based hydrogel and bio-printed it using an extrusion-based method on the surface of decellularized amniotic membrane.The cell-laden graft maintained a sufficient cell survival rate, confluent cell monolayer, higher expression of pump function, and normal hexagonal morphology.The resultant cell construct was implanted into the rabbit's cornea, and it normalized the corneal transparency and thickness profile [184].
The current research on cellular-based biomaterial scaffolds is more focused on supportive, mechanical, adhesive, transparent, and carrier potential, but nano-structural properties, cell-scaffold interface interaction, and scaffold degradation studies are less researched.Since DM is a natural CEnCs substrate and plays an important role in damaged endothelium regeneration, and necessitate that scaffolds contain sufficient patterning cues (e.g.collagen, laminin) and resemble the DM physicochemical properties to control optimal interface interactions to guide cell adhesion, cell growth, orientation, barrier properties, and inhibition of endothelial-to mesenchymal transition [162,170,185].Since stem cell fate after transplantation remains obscure, the future progress in imaging techniques could predict the success of different cell-based therapies and boost corneal medical treatments [186].

Cornea stroma tissue engineering
The corneal stroma (∼500 µm) is the core layer and accounts for up to 85% corneal thickness.It contains collagen fibrils, matrix metalloproteinases, glycosaminoglycans (GAGs), spindle-shaped stromal cells (called keratocytes), and nerve fibers [187].Collagen comprises up to 70% dry weight of cornea stroma and primarily consists of collagen type I (but also other collagen types in minute quantity) in the form of fibrils.Collagen fibrils are uniform in diameter (20-30 nm) and arranged parallel into organized sheets of lamellae.The human cornea contains up to 250-500 collagen lamellae, and each lamellar sheet is about 2 µm thick and 200-250 µm broad.These sheets are arranged orthogonal to each under a confined space perpendicular to the path light.The lamellae organization and orientation are more interlaced in the anterior 1/3 stromal part than the posterior 2/3 stromal part [188,189].
Around 65% of corneal GAGs are keratin sulfate, with the others being dermatan sulfate and chondroitin sulfate.The distribution of these components differs throughout the stroma, e.g. the posterior stromal contains more hydrophilic keratan sulfate, and the anterior region contains dermatan sulfate, which is far less hydrophilic.GAGs help maintain the cornea's hydration properties and regulate the spacing and orientation between collagen fibrils (30-40 nm) and lamellae sheets [190].The interweaving lamellae orientation and regular spacing minimize light scatter, maintains refractive power, and contribute to corneal strength, resilience, proper curvature, and hydration properties [191,192].
Keratocytes comprise 3%-5% of the stromal volume and are mitotically quiescent under normal conditions throughout adult life.These cells are flattened in shape and sparsely interspersed at low density (2.5 × 10 6 ) between lamellae.These cells contact each other via long cytoplasmic processes [193].Keratocytes are responsible for producing stromal matrix and influencing the fibril spacing, organization, and balance of stromal substances.These cells express specific crystalline proteins (i.e.transketolase (TKT) and aldehyde dehydrogenase class 1A1(ALDH1)) that are crucial to maintaining the refractive index [194,195].Many reports confirmed that the corneal nerves regulate keratocytes density and stromal regeneration [196].
The corneal keratocytes hold reduced repair capacity due to insufficient cell density plus quiescence nature, and some keratocytes undergo apoptosis at the wounded area.Other cells at the peripheral wound area become activated, lose their dendritic shape, and differentiated into the fibroblastic cells, probably under the action of corneal epithelial cells secreted tumor necrosis factor-alpha (TNF-α) and Interleukin-1 (IL-1) [195].The stromal fibroblast aggressive proliferates and increases the deposition of fibrotic ECM components necessary to regenerate the wound area.Matrix metalloproteinases are thought to control matrix components turnover.The cells in the healed stroma may not have the same features as normal keratocytes.The complete remodeling of tissue might take several years to restore the proper lamellar structure, corneal strength, and transparency [197,198].
Under severe stromal injuries, surface damaged epithelium and basement membrane release of platelet-derived growth factor (PDGF) and TGF-β which penetrate the injured stroma and function to differentiated fibroblast onto alpha-smooth muscle actin (αSMA) containing myofibroblast [199][200][201].The stromal myofibroblast is more proliferative, adopts a fusiform shape, uses contact guidance for migration, and secret disorganized matrix components to aid wound contraction.Long-term fibroblastic conversion decreases crystallins protein expression (TKT and ALDH1), disorganized matrix deposition, disturbs lamellae organization and leads to long-lasting opacification, fibrotic scar formation, lower biomechanical properties (than normal cornea), and sometimes blindness (figure 7) [202][203][204].The damaged epithelium and basement membrane release cytokines and chemokines which enabling apoptosis (of some keratocytes), activation, and differentiation of adjacent keratocytes into fibroblasts and myofibroblasts in the injured stroma.These fibroblastic cell types are proliferative, adopt fusiform morphology, use contact guidance for migration, and secret higher disorganized matrix components to aid stromal wound contraction at the cost of scar formation and decrease crystallins protein expression.The scarring changes the corneal light refractive property, impedes light rays' transmission, and could lead to irreversible corneal opacification.Abbreviation: TNF-α, tumor necrosis factor-alpha; IL-1, interleukin 1; αSMA, alpha-smooth muscle actin; PDGF, platelet-derived growth factor; TGF-β, transforming growth factor-beta.

Cell source for stoma bioengineering
The inhibition and prevention of scar tissue formation depend upon the normal morphological and physiological properties of keratocytes.These cells can be characterized by keratocan, TKT, lumican, CD133, CD34, and ALDH3A1 expression.Due to low cell densities, autologous keratocyte extraction is not possible, and in vitro propagation is challenging due to their limited proliferation potential and insufficient cell passage number.Furthermore, cultured cells could transform into fibroblasts, and a change in gene expression could be detrimental to corneal transparency due to heterogeneous matrix deposition [195,205].
The CSSCs present close to the anterior stromal-limbal junction could be a source for stroma bioengineering.The limbal stroma zone is a nutritional-rich area and contains distinct ECM components, maintaining the stem cell population [206][207][208].The CSSCs express progenitor markers (Pax6, Six2, ABCB56, and ABCG2) and some MSCs markers (CD73 and CD90), indicating that stromal stem cells that hold self-renewal potential can be differentiated into other cell types.There persist challenges in controlling CSSCs expression, i.e. quiescent stromal stem cells upon culturing, losing their dendritic shape, hold a bipolar morphology, producing stress fibers, and transforming into stromal fibroblasts.Some research studies demonstrated that CSSCs could be differentiated into keratocytes phenotype under various chemical stimuli.The differentiated cells induce high expression of keratocytes markers (keratocan, TKT, ALDH3A1), showed similar matrix expression, prevent scar tissue formation, and induce stromal lamellar structure regeneration [209][210][211][212][213]. CSSCs are a promising cell source for stromal reconstruction, but significant challenges restrict their translational.For example, stem cells isolation is difficult due to the deeper cell niche, and progress is needed to regulate CSSCs differentiation into functional keratocytes following good manufacturing protocols [214,215].
Stem cells from non-corneal sources such as MSCs, iPSCs, and ADSCs could differentiate into keratocytes phenotype under specific differentiation conditions and have gained attention for stroma reconstruction.The transplantation of umbilical cord MSCs to the stromal portion of cornea in lumican-null mice resulted in low rejection risk, long-term cell viability, and successful differentiation into keratocytes phenotype under in vivo conditions [216].BM-MSCs could differentiate into keratocytes-like cells, express their characteristic markers, express negligible α-smooth muscle actin markers, and showed no sign of immune response when injected into the damaged stroma of the animal model [217][218][219].ADSCs could express the main components of the corneal matrix.It can be differentiated into functional keratocytes.It does not induce an inflammatory response and hold the potential to modulate preexisting scars and increase corneal transparency [220].These studies show that non-corneal stem cells can be used for stromal bioengineering, but only a few animal studies have recorded in vivo success so far.More systematic

Scaffold-free stroma bioengineering
The repopulation of keratocytes is an effective option for restoring stromal clarity and reducing the expression of disorganized ECM.Once implanted into the stroma of the rabbit model, the ADSCs should be differentiated into functional keratocytes, express stroma matrix components, and keratocytes-specific markers without causing an inflammatory response [221].The CSSCs have been injected into the stroma of Lumican-deficient mice's corneas to help with stromal regeneration, scar prevention, lamellae organization, and corneal clarity restoration, and keratocytes components production [222].In keratoconus patients, autologous ADSCs were implanted into stromal and pre-clinical studies show that direct intrastromal stem cell implantation can develop a new matrix and is a promising treatment option for corneal dystrophies.The injection of stromal cells is a simple strategy for restoring stroma transparency (figure 8(A)), but it can disrupt the fibril architecture [223,224].Furthermore, this procedure is ineffective in restoring highly damaged stroma due to a lack of structural matrix support and poor cell localization.
The micro-grooved substrates allowed parallel CSSCs alignment, matrix organization, stromal matrix components expression, mimic the stroma lamellae, and increased corneal clarity once implanted into mouse stromal pockets [225].The self-producing scaffold-free stromal transparent construct has been fabricated by stacking CSSCs cell sheets.The resultant construct guide stromal cell alignment, anisotropic collagen fibrils arrangement and remained transparent with no adverse effects when incorporated into rabbit stromal pockets models [226].Muse spheroids have been activated by the dynamic rotary cell culture system, which improved stemness, adherence, and protein expression properties compared to static control.The activated spheroids enabled differentiation into stromal cells, prevent corneal scarring, increased re-epithelialization, increased nerve regrowth, and reduced inflammation in impaired mouse cornea [227].Scaffold-free strategies are appealing because they are biomimetic and minimized inflammatory reactions.Much time, resources, stringent quality requirements, and comprehensive in vivo studies are needed to control the intricate matrix alignment (figure 8(B)).

Scaffold-based stroma bioengineering
Decellularized corneal stroma from animal sources has received attention in stromal engineering because it comprises the basement components, and their supply is abundant to meet the demand.The LESCs and CEnCs have been expanded onto the anterior and posterior corneal surfaces, respectively, while CSSCs were injected into the stromal segment of the decellularized porcine cornea.The in vitro results depicted that each corneal cell type remained functional.More research is required to see whether the xeno-corneas and immunogenic epitope reduction can sustain stromal properties over time [228].The decellularized donor cornea stromal sheets were injected in vitro with cultivated ADSCs to re-cellularize the transplant.The cellular grafts were inserted into the rabbit model's stromal pocket.The result demonstrated that the graft remained stable, transparent, and cells express the human keratocytes-specific protein, with no signs of inflammation [229].Porcine corneas have been decellularized for stroma reconstruction because they are readily available and closely match the dimensional properties of the human cornea.Since the proliferation of keratocytes inside the decellularized corneal stroma matrix is challenging, limiting its effectiveness.Ma et al defined a system in which keratocytes were seeded on thin acellular porcine cornea sheets (20 µm).The same process was repeated until five stacked cell sheets were deposited.The cell sandwich multilayered construct was implanted into the rabbit's damaged stromal region, and it improved transparency and stromal performance [230] (figure 8(C)).In particular, the application of decellularized scaffolds for stromal reconstruction is useful when both epithelium and endothelium are functional.The common limitation of biological membrane scaffolds includes inter-donor batch variation, screening for infectious pathogen transmission, and dependency on deceased donors.
Hydrogels are high-water-content porous scaffolds and are useful for replicating the cornea environment.Collagen is the important structural component of the cornea stroma, and collagen-based biodegradable scaffolds are ideal biomaterial for corneal reconstruction.Since collagen hydrogel holds poor water solubility and holds reduced mechanical strength, thus various manufacturing strategies have been introduced to improve scaffolds mechanical properties, i.e. plastic compression strategies, fiber-reinforced hydrogel, composite material, and cross-linking strategies [231].External load and absorbing pads are used in plastic compression strategies to remove excess fluid.These strategies could increase about 100-200 fold collagen matrices density.The stromal cells remain alive and buried inside the scaffold matrix, resulting in a thin collagen structure that resembles soft tissues.The compressed cellular collagen-based disc could ease inserted into the damaged cornea [232].Simple hydrogels hold poor mechanical properties, uncontrolled drug release due to high water content, lack the potential to guide cell differentiation, and these factors limit their applications.The addition of fibrous structures in hydrogels could be a potential solution to promoting cell-scaffold interaction and guiding matrix deposition [233].The mixture of collagen with other biomaterials has been used to improve scaffold stiffness and biological properties.The patterned silk films and collagen gel have been used to construct a biomimetic stomal model and demonstrate that the topographical and dome-shaped mechanical strain drove keratocytes alignment, keratocytes marker expression, and matrix arrangement [234].
The damaged cornea often necessitates the reconstruction of both stroma and cornea epithelium at the same time.The in situ forming collagen cross-linked hydrogel has greater transparency and good mechanical strength, promoting keratocytes encapsulation and expansion within crosslinked gels.Furthermore, the keratinocytes expanded on the gel surface, and it can be ease applied to rabbit corneas.The results demonstrated that it could be a promising candidate for both epithelium and stromal reconstruction [235].The hydrazone-crosslinked hyaluronic acid-functionalized with dopamine moieties are adhesive and hold the potential to encapsulate ADSCs into its 3D internal structure and, at the same time, promote the expansion of LESCs.Both cells express their functional properties, and the resultant cell/scaffold construct could ease implanted into the porcine corneal organ culture model due to their adhesive properties.It could minimize the need for suturing for implantation [236].
The fibrous structure can resemble soft tissue fibers and provide hierarchical, mechanical, and morphological cues to induce cell differentiation and development.Most studies in common depicted that the aligned nanofibrous scaffolds could induce cultured cells alignment.The surface chemistry and surface patterned scaffolds hold more potential to control cell proliferation, migration, and differentiation than random fibers.The fibrous-based functionalized and oriented surfaces could hold the potential for converting corneal fibroblasts to corneal keratocytes.These scaffolding properties are crucial to reduced myofibroblast phenotype expression, control scar formation, and thus could aid in stromal reconstruction.Overall, these studies indicate that the interface interaction between cells and scaffolds plays a crucial role in cell maturation and matrix production.The biodegradation rate, potential inflammatory responses, and impact of 2D scaffold on in vivo reconstruction of stroma need detailed studies [237][238][239][240][241] (figure 8(D)).
3D bioprinting has opened new possibilities to fabricate complex structures and disease-specific scaffolds using hydrogel as a bio-ink.It could offer unique benefits, including automation, precise fabrication, multi-material integration, placement of biologics (i.e.cells, growth factors, genes) to recapitulate hetero-cellular tissue biology, rapid fabrication of scalable tissue constructs, and has raised hopes for the specific treatment [242].Only a few studies used additive manufacturing strategies to reconstruct stroma.Combining polycaprolactone/polyethylene glycol nanofibers and gelatin methacrylate-based gel improved the mechanical properties, and the resultant construct is transparent, regulating the high expression of CSSCs markers.The preliminary studies have raised some questions about inflammation [233].Extrusion-based printing methods have been used for stromal bioengineering using gelatin methacrylate and collagen-based hydrogel.The resultant scaffold was biomimetic, hold good optical properties, and scaffolds construct support cellular properties and alignment, which could be crucial for stromal reconstruction [243,244].The recombinant laminin and human-sourced collagen-based composite bioink have been used to develop a layered stroma-mimicking structure using laser-based bioprinting.It supports the expansion, differentiation, and organization of human ADSCs that have been used to create an organized stroma [245].More recently, the decellularized stromal ECM hydrogel has been used for cell 3D printing in the form of a transparent construct.The bio-printed structure mimics the stromal collagen-aligned architect and holds high cellular alignment capability, which could be important for stromal reconstruction [246].Despite progress, the balance between materials properties and patterned scaffold surfaces is a significant concern because many biomimetic materials are challenging to bioprinted into transparent and mechanical stable bioscaffolds.There is a need to improve nanoscale filament resolution, the biomimetic composition of bioink, biomechanical properties, and sophisticated design to control the ability of cells to deposit ECM in a controllable manner [247] (figure 8(E)).
Overall, current constructs are far from replicating the biochemical, mechanical, structural, optical, and hierarchical alignment of corneal stroma in the single construct.The synthetic materials are mostly used in scaffold fabrication, raising concerns about solid inflammatory responses upon micro-environmental changes and biodegradation, which could delay the healing process.Most studies do not meet clinical applications' requirements.There remain substantial gaps in controlling cellular expression, bio-integration, biodegradation, in vivo fate of transplanted cells, and managing the inflammatory response.For example, most studies determine the effect of surface chemistry and surface morphology using 2D scaffolds while stromal cells in the native cornea are surrounded by a complex 3D functional microenvironment (figure 8(F)).Nonetheless, stromal reconstruction is beginning, and future synergism of scaffold-free and scaffold-based biomimetic technologies, including polymer grafting, cell patterning, cell sheet layering, and additive manufacturing, could advance scaffold functionalities for stromal reconstruction [24,248].

Conclusions and future perspectives
Each corneal cellular layer is unique in cellular composition and poses complex challenges in tissue reconstruction.Progress in corneal and non-corneal stem cell research has opened new prospects in bioengineering.Corneal stem cells minimized the risk of rejection and potentially allowed multiple patients to be treated from a single donated cornea; nevertheless, substantial progress and source are needed to expand cells in a reproducible fashion.Non-corneal stem cells could meet the increasing demand and reduce treatment costs, but current progress is still under unraveling homing, immunomodulation, and trans-differentiation mechanisms using good manufacturing practices.Furthermore, translational application of cells is dependent on scaffold-free and scaffold-based strategies that can support cell expansion, mimic matrix composition, ease delivery route and accelerate the reconstruction process.
Scaffold-free strategies do not present inflammatory responses, could deliver rich cellular density in the target site and are more suited for epithelium and endothelium reconstruction since these layers do not contain complex matrix components.On the other hand, scaffold-based construct could easier to mass-produce, and current research on cellular-based construct more focused on supportive, mechanical, adhesive, transparent, and carrier potential, but nano-structural properties, cell-scaffold interface interaction, and scaffold degradation studies are less researched.Numerous scaffold-free (cell injection, cell sheet, spheroids, cell printing) and scaffold-based cellular constructs (biological membrane, hydrogel, films, nanofibers, bioprinting) have been developed, which is important for treating the spectrum of corneal disease conditions.
The cellular bioengineering strategies are in the right direction to address epithelium plus endothelium problems, and future progress could mark a paradigm shift in transplantation.Despite progress, existing scaffolds are far from replicating the biochemical, mechanical, structural, optical, and hierarchical alignment of corneal stroma in a single construct.Furthermore, the cornea equivalent scaffold is important for in vitro drug screening, toxicological testing, cellular interactions study, and direct usage as a transplant.Still, limited research has been done for engineering partial or full-thickness cornea because multilayer cornea damage offers more significant challenges and cellular-based bioengineering requirements (figure 9).The parallel advancements in cornea biology, stem cell fields, designed surfaces, scaffold innervation, cell patterning, interface maintenance, and synergism of scaffold-free plus scaffold-based strategies are required to solve multiple bottlenecks towards partial and full-thickness cornea reconstruction.

Figure 1 .
Figure 1.The human cornea.(A) The schematic eye structure and location of the cornea.(B) The cross-sectional schematic diagram of the full-thickness cornea.(C) The defect in each corneal cellular layer leads to specific diseases.(D) Surgical strategies for donor corneal tissue transplantation.Abbreviation: DALK, deep anterior lamellar keratoplasty; FECD, fuchs endothelial corneal dystrophy; LESCD, limbal epithelium stem-cell deficiency; PK, penetrating keratoplasty; MCD, macular corneal dystrophy; DMEK, DM endothelial keratoplasty.

Figure 3 .
Figure 3.The schematic illustration showed that the limbus zone separates the corneal and conjunctival epithelium.Palisades of Vogt is a vascularized limbal region and acts as a niche for stem cells.The epithelial shedding stimulates dormant LESCs.The sum of X (LESCs division) and Y (TAC centripetal migration) must equal Z (shedding of surface cells) to maintain ocular health (X + Y = Z).The cornea epithelium is densely innervated, and nerve endings terminate near the superficial epithelium.

Figure 4 .
Figure 4. (A) The schematic cell-based bioengineering strategies for unilateral and bilateral ocular surface reconstruction.(B) The schematic diagram showed that the construct could be implanted onto the ocular surface using different techniques.Note: In inlay strategy, cellular constructs grafted into the damaged epithelium under the limbus tissue and surface-attached cells (upward position) function as substitutes for the lost tissue.In overlay strategy, cellular constructs function as a temporary scaffold over the denuded cornea and limbal tissues.It protects the wound against environmental damage, and surface-attached cells (downward position) can integrate and release biochemical factors to promote epithelium healing.In overlay/inlay strategy, construct placed beneath and on top of the limbal grafts, and transplanted cells are expanded between the scaffolds to reconstruct the epithelium.

Figure 5 .
Figure 5. (A) The hexagon appearance of the CEnCs apical surface depend upon close interaction between tight junctions (ZO-1 (green)) and actomyosin (myosin IIa (red)) network.Scale = 10 µm.(B) and (C) A simplified 3D model showed that CEnCs apical cell surface is hexagonal in shape, while basal cell surface is irregular flower-like in shape.(A)-(C) Reproduced from [99].CC BY 4.0.(D) In normal endothelium, CEnCs are smaller in dimension, packed in a hexagonal mosaic pattern, and maintain cornea clarity.(E) In damaged endothelium, CEnCs increase cell size, decrease hexagonal proportion, reduce cell density, contribute to numerous guttae, and promote cornea clouding.

Figure 6 .
Figure 6.CEnCs injection into the anterior chamber and engineering-based methods to promote the engraftment of injected cells into the DM of recipient cornea.(A) A simple injection of cells in the upward eye position.(B) ROCK inhibitor supplementation, (C) ferromagnetic induction, (D) mini-sheet injection, (E) transplantation of transparent gel sheet before cell injection, (F) laminin pre-coating on damaged DM membrane before cell injection.

Figure 7 .
Figure7.The damaged epithelium and basement membrane release cytokines and chemokines which enabling apoptosis (of some keratocytes), activation, and differentiation of adjacent keratocytes into fibroblasts and myofibroblasts in the injured stroma.These fibroblastic cell types are proliferative, adopt fusiform morphology, use contact guidance for migration, and secret higher disorganized matrix components to aid stromal wound contraction at the cost of scar formation and decrease crystallins protein expression.The scarring changes the corneal light refractive property, impedes light rays' transmission, and could lead to irreversible corneal opacification.Abbreviation: TNF-α, tumor necrosis factor-alpha; IL-1, interleukin 1; αSMA, alpha-smooth muscle actin; PDGF, platelet-derived growth factor; TGF-β, transforming growth factor-beta.

Figure 8 .
Figure 8. Bioengineering strategies used in stromal reconstruction: (A) intrastromal injection of cell suspension.(B) Scaffold-free construct transplantation.(C) Repeated stacking of cell-free scaffold transplantation and cell seeding via cell suspension drop.(D) 2D cell/scaffold constructs transplantation.(E) 3D cell/scaffold constructs transplantation.(F) Corneal cells in normal tissue are surrounded by complex 3D matrix components which modulate cell function.

Figure 9 .
Figure 9.The schematic of the cornea tissue engineering model ranges from a simple monolayer system (i.e.epithelium, stroma, and endothelium) to a bilayer system (epithelium-stroma, stroma-endothelium), and cornea equivalent model.As the corneal system becomes complicated, the cost, new cell source, cell culture sustainability, engineering-based problems, and clinical significance rise.