Native Cornea Research Articles

Integrated environmental and health economic assessments of novel xeno-keratografts addressing a growing public health crisis

Tissue scarcity poses global challenges for corneal transplantation and public health. Xeno-keratoplasty using animal-derived tissues offers a potential solution, but its environmental and economic implications remain unclear. This study evaluated two xeno-keratoplasty procedures at a single institution: (1) native corneas (Option 1) and (2) tissue-engineered corneal scaffolds derived from slaughterhouse waste (Option 2). Life cycle assessment (LCA) quantified environmental impacts across 18 midpoint indicators, while cost-effectiveness analysis (CEA) incorporated cost and environmental impact using two approaches. Option 1 exhibited significantly lower environmental impact than Option 2 across most indicators, primarily due to the energy and equipment demands of cell culture in Option 2. Both CEA approaches (carbon offset pricing and utility decrement) demonstrated cost-effectiveness dominance for Option 1. Xeno-keratoplasty using native corneas (Option 1) appears more environmentally and economically favorable than tissue-engineered scaffolds (Option 2) in the current analysis. Future studies could explore diverse xeno-keratoplasty techniques for optimizing sustainability.

Preparation and characterization of peptide-modified core-shell fibrous substrates with UV-blocking properties for corneal regeneration applications

Effective UV protection is a key aspect of substrates directly exposed to UV radiation. Therefore, in the present study, fibrous substrates of core–shell morphology (PCL-core, PVP-shell) containing peptides based on tryptophan, tyrosine and cysteine (W6, YYC2 and YYC3) were prepared. Spectrophotometric studies showed UV absorption by peptides containing tyrosine and tryptophan in the UVB (up to 80%) and UVA (up to 40%) ranges. Cysteine, in turn, contributed to high antioxidant properties, confirmed by DPPH assay. The presence of peptides contributed to a nonwoven fabric characterized by the ability to absorb UV radiation and prevent the occurrence of oxidative stress (caused by the presence of free radicals). In turn, the increase in the surface zeta potential of the nonwoven after UV irradiation and higher thermal stability (demonstrated by DSC studies) indicated the crosslinking of the PVP layer under UVR, which further contributes to the increased protection of the nonwoven against its effects. In summary, obtained nonwoven exhibited functional similarity to the native cornea, providing a potential solution for enhancing corneal tissue engineering and regenerative medicine applications.

Liquid microlens compound structure for enhanced sub-micron resolution imaging and accelerated reconfigurable deformation manipulation

The optical observation of sub-micron structures encounters significant challenges due to the inadequate spatial shape matching of optical devices and the limitations imposed by diffraction. These factors result in suboptimal imaging resolution and the introduction of defocus distortion. One approach to mitigating these limitations involves utilizing a liquid microlens (LML) assisted microscope, which offers real-time and localized control capabilities. However, the dynamic tuning of LML is subject to volatility and instability, adversely affecting sub-micron resolution imaging. To address this issue, a contour-following coating inspired by the natural cornea is applied to the droplet's surface, resulting in the formation of liquid microlens compound structures (LMLCS). Firstly, photoresist microholes are fabricated on the optical disc. Then, liquid self-assembly technology is used to fill glycerol in the microholes. At last, a conformal spreading method of UV-curable adhesive precursor film is developed to ensure the combination of the microlens bilayer structure. Through the application of optical information transmission theory, finite difference time domain (FDTD) method, and the principle of Abbe microscopic imaging, the imaging magnification process and sub-micron resolution characteristic of the LMLCS are revealed. Remarkably, the obtained focal width at half maximum (FWHM) of 1.54 μm is smaller than the theoretical value, enabling reconfigurable sub-micron resolution observation and 1.63 times imaging magnification of 226 nm grating lines under the optical microscope. Compared with unencapsulated LML, this compound structure exhibits better sub-micron resolution capability, greater imaging magnification, and faster adaptive dynamic response characteristics. Consequently, the LMLCS introduces exciting prospects for exploring optical sub-micron measurement and sensing devices with exceptional performance.

Corneal stromal structure replicating humanized hydrogel patch for sutureless repair of deep anterior-corneal defect

A critical shortage of donor corneas exists worldwide. Hydrogel patches with a biological architecture and functions that simulate those of native corneas have garnered considerable attention. This study introduces a stromal structure replicating corneal patch (SRCP) composed of a decellularized cornea-templated nanotubular skeleton, recombinant human collagen, and methacrylated gelatin, exhibiting a similar ultrastructure and transmittance (above 80 %) to natural cornea. The SRCP is superior to the conventional recombinant human collagen patch in terms of biomechanical properties and resistance to enzymatic degradation. Additionally, SRCP promotes corneal epithelial and stromal cell migration while preventing the trans-differentiation of stromal cells into myofibroblasts. When applied to an ocular surface (37 °C), SRCP releases methacrylated gelatin, which robustly binds SRCP to the corneal stroma after activation by 405 nm light. Compared to gelatin-based photocurable hydrogel, the SRCP better supports the restoration of normal corneal curvature and withstands deformation under an elevated intraocular pressure (100 mmHg). In an in vivo deep anterior-corneal defect model, SRCP facilitated epithelial healing and vision recovery within 2 weeks, maintained graft structural stability, and inhibited stromal scarring at 4 weeks post-operation. The ideal performance of the SRCP makes it a promising humanized corneal equivalent for sutureless clinical applications.

Hydrogel-based hybrid membrane enhances in vitro ophthalmic drug evaluation in the OphthalMimic device

Envisaging to improve the evaluation of ophthalmic drug products while minimizing the need for animal testing, our group developed the OphthalMimic device, a 3D-printed device that incorporates an artificial lacrimal flow, a cul-de-sac area, a moving eyelid, and a surface that interacts effectively with ophthalmic formulations, thereby providing a close representation of human ocular conditions. An important application of such a device would be its use as a platform for dissolution/release tests that closely mimic in vivo conditions. However, the surface that artificially simulates the cornea should have a higher resistance (10 min) than the previously described polymeric films (5 min). For this key assay upgrade, we describe the process of obtaining and thoroughly characterizing a hydrogel-based hybrid membrane to be used as a platform base to simulate the cornea artificially. Also, the OphthalMimic device suffered design improvements to fit the new membrane and incorporate the moving eyelid. The results confirmed the successful synthesis of the hydrogel components. The membrane’s water content (86.25 ± 0.35 %) closely mirrored the human cornea (72 to 85 %). Furthermore, morphological analysis supported the membrane’s comparability to the natural cornea. Finally, the performance of different formulations was analysed, demonstrating that the device could differentiate their drainage profile through the viscosity of PLX 14 (79 ± 5 %), PLX 16 (72 ± 4 %), and PLX 20 (57 ± 14 %), and mucoadhesion of PLXCS0.5 (69 ± 1 %), PLX16CS1.0 (65 ± 3 %), PLX16CS1.25 (67 ± 3 %), and the solution (97 ± 8 %). In conclusion, using the hydrogel-based hybrid membrane in the OphthalMimic device represents a significant advancement in the field of ophthalmic drug evaluation, providing a valuable platform for dissolution/release tests. Such a platform aligns with the ethical mandate to reduce animal testing and promises to accelerate the development of safer and more effective ophthalmic drugs.

Impact of keratocyte differentiation on corneal opacity resolution and visual function recovery in male rats

Intrastromal cell therapy utilizing quiescent corneal stromal keratocytes (qCSKs) from human donor corneas emerges as a promising treatment for corneal opacities, aiming to overcome limitations of traditional surgeries by reducing procedural complexity and donor dependency. This investigation demonstrates the therapeutic efficacy of qCSKs in a male rat model of corneal stromal opacity, underscoring the significance of cell-delivery quality and keratocyte differentiation in mediating corneal opacity resolution and visual function recovery. Quiescent CSKs-treated rats display improvements in escape latency and efficiency compared to wounded, non-treated rats in a Morris water maze, demonstrating improved visual acuity, while stromal fibroblasts-treated rats do not. Advanced imaging, including multiphoton microscopy, small-angle X-ray scattering, and transmission electron microscopy, revealed that qCSK therapy replicates the native cornea’s collagen fibril morphometry, matrix order, and ultrastructural architecture. These findings, supported by the expression of keratan sulfate proteoglycans, validate qCSKs as a potential therapeutic solution for corneal opacities.

A sustainable approach to derive sheep corneal scaffolds from stored slaughterhouse waste.

Aim: The escalating demand for corneal transplants significantly surpasses the available supply. To bridge this gap,we concentrated on ethical and sustainable corneal grafting sources. Our objective was to create viable corneal scaffolds from preserved slaughterhouse waste.Materials & methods: Corneas were extracted and decellularized from eyeballs that had been refrigerated for several days. These scaffolds underwent evaluation through DNA quantification, histological analysis, surface tension measurement, light propagation testing,and tensile strength assessment.Results: Both the native and acellular corneas (with~90% DNA removed using a cost-effective and environmentally friendly surfactant) maintained essential optical and biomechanical properties for potential clinical use.Conclusion: Our method of repurposing slaughterhouse waste, stored at 4°C for several days, to develop corneal scaffolds offers a sustainable and economical alternative xenograft model.

Design and Biocompatibility of a Novel, Flexible Artificial Cornea.

We sought to introduce the materials, design, and biocompatibility of a flexible and suturable artificial corneal device. Single-piece, fully synthetic, optic-skirt design devices were made from compact perfluoroalkoxy alkane. The skirt and the optic wall surfaces were lined with a porous tissue ingrowth material using expanded polytetrafluoroethylene. Full-thickness macroapertures around the skirt perimeter were placed to facilitate nutrition of the recipient cornea. Material properties including the skirt's modulus of elasticity and bending stiffness, optic light transmission, wetting behavior, topical drug penetrance, and degradation profile were evaluated. The final prototype suitable for human use has a transparent optic with a diameter of 4.60mm anteriorly, 4.28mm posteriorly, and a skirt outer diameter of 6.8mm. The biomechanical and optical properties of the device closely align with the native human cornea with an average normalized device skirt-bending stiffness of 4.7kPa·mm4 and light transmission in the visible spectrum ranging between 92% and 96%. No optical damage was seen in the 36 devices tested in fouling experiments. No significant difference was observed in topical drug penetrance into the anterior chamber of the device implanted eye compared with the naïve rabbit eye. The flexibility and biocompatibility of our artificial cornea device may offer enhanced tissue integration and decreased inflammation, leading to improved retention compared with rigid keratoprosthesis designs. We have developed a fully synthetic, flexible, suturable, optic-skirt design prototype artificial cornea that is ready to be tested in early human feasibility studies.

Investigation of mechanical strength and structure of corneal graft-host junction

Dehiscence is a common complication of corneal transplant surgery involving separating the graft from the host eye. The present article aims to investigate fundamental insights into the mechanical and structural aspects of the graft-host junction (GHJ) of a graft that survived in a patient for 13 years after penetrating keratoplasty (PK). Additionally, it adopts the sutur retention strength (SRS) test procedure defined in ISO:7198-2016 and aims to provide a comprehensive test protocol to study the biomechanics of the GHJ in extracted PK buttons. A 9 mm corneal button with GHJ was extracted from a 46-year-old patient who underwent PK 13 years back. The strength of the GHJ was quantified using the SRS test. Corresponding control results were obtained from the SRS tests of a corneoscleral button with no history of any refractive procedure. Birefringence, histological, and scanning electron microscopy (SEM) imaging were used to visualize the microstructural details of the GHJ. The strength of the GHJ was observed to be ten times lower than the native cornea. Histopathological features, such as fragmented Bowman's layer, and fibrosis with a clear demarcation line between host and graft tissue, were observed at the GHJ, suggesting a weak bond across the GHJ. The low strength of the GHJ in PK indicates the high susceptibility of the GHJ towards wound dehiscence.

Mimicking the Physicochemical Properties of the Cornea: A Low-Cost Approximation Using Highly Available Biopolymers.

Corneal diseases represent a significant global health challenge, often resulting in blindness, for which penetrating keratoplasty is the clinical gold standard. However, in cases involving compromised ocular surfaces or graft failure, osteo-odonto keratoprosthesis (OOKP) emerges as a vital yet costly and complex alternative. Thus, there is an urgent need to introduce soft biomaterials that mimic the corneal tissue, considering its translation's physicochemical, biological, and economic costs. This study introduces a cross-linked mixture of economically viable biomaterials, including gelatin, chitosan, and poly-D-lysine, that mimic corneal properties. The physicochemical evaluation of certain mixtures, specifically gelatin, chitosan, and poly-D-lysine cross-linked with 0.10% glutaraldehyde, demonstrates that properties such as swelling, optical transmittance, and thermal degradation are comparable to those of native corneas. Additionally, constructs fabricated with poly-D-lysine exhibit good cytocompatibility with fibroblasts at 72 h. These findings suggest that low-cost biopolymers, particularly those incorporating poly-D-lysine, mimic specific corneal characteristics and have the potential to foster fibroblast survival. While further studies are required to reach a final corneal-mimicking solution, this study contributes to positioning low-cost reagents as possible alternatives to develop biomaterials with physicochemical properties like those of the human cornea.

Patterned collagen films loaded with miR-133b@MBG-NH2 for potential applications in corneal stromal injury repair

Corneal stromal injury is a common surgical disease. With the development of tissue engineering materials, many artificial corneal scaffolds have been developed to replace allograft corneal transplantation and solve the problem of corneal donor shortage. However, few researchers have paid attention to corneal stromal wound healing. Herein, a nanocomposite of amino modified mesoporous bioactive glass (MBG-NH2) and microRNA-133b (miR-133b) was introduced into the patterned collagen films to achieve corneal stromal injury repair. MBG-NH2 nanoparticles as a nano delivery carrier could efficiently load miR-133b and achieve the slow release of miR-133b. The physicochemical properties of collagen films were characterized and found the microgrooved collagen films loaded with miR-133b@MBG-NH2 nanoparticles possessed similar swelling properties, optical clarity, and biodegradability to the natural cornea. In vitro cell experiments were also conducted and proved that the patterned collagen films with miR-133b@MBG-NH2 possessed good biocompatibility, and miR-133b@MBG-NH2 nanoparticles could be significantly uptake by rabbit corneal stromal cells (RCSCs) and have a significant impact on the orientation, proliferation, migration, and gene expression of RCSCs. More importantly, the patterned collagen films with miR-133b@MBG-NH2 could effectively promote the migration of RCSCs and accelerate wound healing process, and down-regulate the expression levels of α-SMA, COL-I, and CTGF genes associated with myofibroblast differentiation of corneal stromal cells, which has a potential application prospect in the repair of corneal stromal injury.

Use of discarded corneo-scleral rims to create cornea-like tissue.

Corneal disease is a major cause of blindness. Transplantation of cadaver-derived corneas (keratoplasty) is still the current therapy of choice; however, the global shortage of donor corneas continues to drive a search for alternatives. To this end, biosynthetic corneal substitutes have recently begun to gain importance. Here, we present a novel method for the generation of a cornea-like tissue (CLT), using corneo-scleral rims discarded after keratoplasty. Type I collagen was polymerized within the corneo-scleral rim, which functioned as a 'host' mould, directing the 'guest' collagen to polymerize into disc-shaped cornea-like material (CLM), displaying the shape, curvature, thickness, and transparency of normal cornea. This polymerization of collagen appears to derive from some morphogenetic influence exerted by the corneo-scleral rim. Once the CLM had formed naturally, we used collagen crosslinking to fortify it, and then introduced cells to generate a stratified epithelial layer to create cornea-like tissue (CLT) displaying characteristics of native cornea. Through the excision and reuse of rims, each rim turned out to be useful for the generation of multiple cornea-shaped CLTs. The approach effectively helps to shorten the gap between demand and supply of CLMs/CLTs for transplantation. We are exploring the surgical transplantation of this CLT into animal eyes, as keratoprostheses, as a precursor to future applications involving human eyes. It is possible to use either the CLM or CLT, for patients with varying corneal blinding diseases.

A pilot study for decellularizing human and porcine cornea for future use in corneal regeneration

Purpose: To effectively decellularize human and porcine corneas for future use in corneal regeneration by using an already established decellularization technique being efficient in decellularizing blood vessels.Methods: One human and 4 porcine corneas were decellularized by using various washing steps with 3‐[(3‐cholamidopropyl) dimethylammonio]‐1‐propanesulfonate (CHAPS) detergent, Benzonase, Ethylenediaminetetraacetic acid (EDTA), 5% Dextran or 5% D‐Mannitol. The quality of the decellularization process was assessed by quantitative and qualitative measurement of DNA content, glycosaminoglycans (GAGs), immunofluorescent staining for Collagen I, V and keratocan, and Transmission Electron Microscopy (TEM) to observe the structure of the collagen fibrils was used.Results: The decellularization process showed 99,94% and 99,52% DNA content reduction in the human and porcine corneas when using Dextran, respectively, while more DNA remained in the porcine corneas when D‐Mannitol was used. Similar pattern was observed in the preservation of GAGs, with the highest amount being found in the human cornea, and less in the porcine corneas. H&E and Immunofluorescent staining showed no presence of cell nuclei when compared to the control native corneas, while Alcian blue staining qualitatively confirmed the presence of GAGs. Collagen I, V and keratocan were still present in the decellularized corneas when compared to the native ones, while TEM microscopy further confirmed the similar patterns of the collagen fibrils in the decellularized‐, compared to the native‐ corneas.Conclusions: This pilot study showed that our decellularization method is effective in decellularizing human and porcine corneas, with very high amount of DNA being removed from the corneas, while the GAGs were preserved to an acceptable extent, and the structure and pattern of the collagen fibrils maintained.

A Versatile Hydrogel with Antibacterial and Sequential Drug-Releasing Capability for the Programmable Healing of Infectious Keratitis.

Hydrogels have attracted tremendous attention as favorable corneal substitutes for treating severe infectious keratitis (IK). However, current hydrogel-based corneal substitutes were majorly designed to promote the single stage of corneal regeneration, which falls short in meeting the clinical management needs of severe IK including the multiple phases of corneal wound healing. Herein, we introduce a versatile hybrid hydrogel (SQPV) composed of silk fibroin and chitosan, which exhibits spatiotemporal properties for drug release. The SQPV is fabricated by incorporating verteporfin-loaded poly(lactic-co-glycolic)-polyethylene glycol-o-nitrobenzene micelles into a hydrogel network, which is formed from methacrylate silk fibroin and glycidyl methacrylate functionalized quaternized chitosan containing polydeoxyribonucleotide. This double network approach results in a material with exceptional anti-inflammatory, antibacterial, and proliferative stimulation and tissue remodeling regulation capabilities. Furthermore, SQPV showcases mechanical strength and transparency akin to those of native cornea. Extensive in vitro and in vivo studies validate SQPV's ability to effectively eliminate residual bacteria, mitigate inflammation, foster regeneration of corneal epithelium and stroma, prevent corneal scarring, and ultimately expedite wound healing. In summary, the SF/CS-based hybrid hydrogel may represent a promising substitute for comprehensive corneal repair and regeneration in severe IK.

An artificially-intelligent cornea with tactile sensation enables sensory expansion and interaction

We demonstrate an artificially-intelligent cornea that can assume the functions of the native human cornea such as protection, tactile perception, and light refraction, and possesses sensory expansion and interactive functions. These functions are realized by an artificial corneal reflex arc that is constructed to implement mechanical and light information coding, information processing, and the regulation of transmitted light. Digitally-aligned, long and continuous zinc tin oxide (ZTO) semiconductor fabric patterns were fabricated as the active channels of the artificial synapse, which are non-toxic, heavy-metal-free, low-cost, and ensure superior comprehensive optical properties (transmittance >99.89%, haze <0.36%). Precisely-tuned crystal-phase structures of the ZTO fibers enabled reconfigurable synaptic plasticity, which is applicable to encrypted communication and associative learning. This work suggests new strategies for the tuning of synaptic plasticity and the design of visual neuroprosthetics, and has important implications for the development of neuromorphic electronics and for visual restoration.

A Bioengineering‐Regenerative Medicine Approach for Ocular Surface Reconstruction Using a Functionalized Native Cornea‐Derived Bio‐Scaffold

AbstractLimbal stem cell deficiency (LSCD) is characterized by the loss of limbal epithelial stem cells (LESCs) which compromises corneal transparency, leading to blindness. It cannot be treated with pharmacological or corneal transplantation interventions, instead a specialized stem cell (SC) therapy is needed to restore eye health and sight. Herein, a native cornea‐derived biomaterial, a by‐product of a laser refractive surgical procedure called small incision lenticule extraction is identified as a new cell delivery matrix. Culture conditions are optimized to facilitate LESC attachment, expansion and stratification, and their identity is immunophenotyped. Using electron microscopy, bio‐constructs display stratification, similar to the architectural and cellular organization of a native mammalian cornea with formation of a basement membrane and an orderly array of collagen fibrils. Neuronal growth and depleted CD45+/CD14+ leukocytes on lenticules are also shown, suggesting that in transplantation experiments, they will re‐innervate and not trigger a host‐mediated immune response. Finally, human lenticules are geometrically customized to successfully fit them over a LSCD murine cornea ex vivo, during which they maintain curvature. The authors are poised to conduced similar studies in live mice using these and other carriers currently used in the clinic to compare SC therapy outcomes.

Preparation and Characterization of a Photo-Crosslinked Methacryloyl-Collagen Composite Film to Promote Corneal Nerve Regeneration via Surface Grafting of Taurine Molecules.

Blindness is frequently caused by corneal abnormalities, and corneal transplantation is the most effective treatment method. It is extremely important to develop high-quality artificial corneas because there are not enough donor corneas accessible for cornea transplantation. One of the most-often utilized materials is collagen, which is the primary component of natural cornea. Collagen-based corneal repair materials have good physicochemical properties and excellent biocompatibility, but how to promote the regeneration of the corneal nerve after keratoplasty is still a big challenge. In this research, in order to promote the growth of nerve cells on a collagen (Col) substrate, a novel collagen-based material was synthesized starting from the functionalization of collagen with unsaturated methacryloyl groups that three-dimensionally photopolymerize to a 3D network of chemically crosslinked collagen (ColMA), onto which taurine molecules were eventually grafted (ColMA-Tr). The physicochemical properties and biocompatibility of the Col, ColMA and ColMA-Tr films were evaluated. By analyzing the results, we found that all the three samples had good moisture retention and aq high covalent attachment of methacryloyl groups followed by their photopolymerization improved the mechanical properties of the ColMA and ColMA-Tr. Most importantly, compared with ColMA, the taurine-modified collagen-MA film significantly promoted the growth of nerve cells and corneal epithelial cells on its surface. Our preliminary results suggest that this novel ColMA-Tr film may have potential use in cornea tissue engineering in the future.

Construction and In Vitro Evaluation of Collagen/Bioactive Glass Composite Membranes as a Corneal Implant

Collagen (Col), the major component of native cornea, has been extensively employed for constructing corneal repair materials. However, inflammation impacts the process of corneal repair, even leading to transplant failure. Bioactive glass (BG) exhibits an excellent anti-inflammatory effect and immunomodulatory function in soft tissue repair. Herein, in this study, the potential of incorporating BG into collagen-based membranes for corneal repair was explored. BG was incorporated at 1, 5, and 7 wt %. The physicochemical properties of the membranes were determined, and in vitro cell-based analyses were also conducted. The results revealed that the Col-BG membranes exhibited desirable light transmission properties, mechanical properties, and good biocompatibility, which meet the requirements of a corneal substitute. Furthermore, Col membranes with 5 and 7% ratio of BG effectively induced corneal epithelial cell adhesion and migration. The Col-BG membranes displayed anti-inflammatory effects as confirmed via polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) analysis. Among the different BG concentrations tested, the Col-BG membrane containing 5 wt % BG displayed the most favorable combination of physical and biological properties in vitro. Thus, this study described a corneal repair material and provides evidence regarding the feasibility of using Col-BG membranes for corneal repair.

A scalable corneal xenograft platform: simultaneous opportunities for tissue engineering and circular economic sustainability by repurposing slaughterhouse waste.

Introduction: Corneal disease is a leading cause of blindness globally that stems from various etiologies. High-throughput platforms that can generate substantial quantities of corneal grafts will be invaluable in addressing the existing global demand for keratoplasty. Slaughterhouses generate substantial quantities of underutilized biological waste that can be repurposed to reduce current environmentally unfriendly practices. Such efforts to support sustainability can simultaneously drive the development of bioartificial keratoprostheses. Methods: Scores of discarded eyes from the prominent Arabian sheep breeds in our surrounding region of the United Arab Emirates (UAE) were repurposed to generate native and acellular corneal keratoprostheses. Acellular corneal scaffolds were created using a whole-eye immersion/agitation-based decellularization technique with a widely available, eco-friendly, and inexpensive 4% zwitterionic biosurfactant solution (Ecover, Malle, Belgium). Conventional approaches like DNA quantification, ECM fibril organization, scaffold dimensions, ocular transparency and transmittance, surface tension measurements, and Fourier-transform infrared (FTIR) spectroscopy were used to examine corneal scaffold composition. Results: Using this high-throughput system, we effectively removed over 95% of the native DNA from native corneas while retaining the innate microarchitecture that supported substantial light transmission (over 70%) after reversing opacity, a well-established hallmark of decellularization and long-term native corneal storage, with glycerol. FTIR data revealed the absence of spectral peaks in the frequency range 2849cm-1 to 3075cm-1, indicating the effective removal of the residual biosurfactant post-decellularization. Surface tension studies confirmed the FTIR data by capturing the surfactant's progressive and effectual removal through tension measurements ranging from approximately 35mN/m for the 4% decellularizing agent to 70mN/m for elutes highlighting the effective removal of the detergent. Discussion: To our knowledge, this is the first dataset to be generated outlining a platform that can produce dozens of ovine acellular corneal scaffolds that effectively preserve ocular transparency, transmittance, and ECM components using an eco-friendly surfactant. Analogously, decellularization technologies can support corneal regeneration with attributes comparable to native xenografts. Thus, this study presents a simplified, inexpensive, and scalable high-throughput corneal xenograft platform to support tissue engineering, regenerative medicine, and circular economic sustainability.

Development of stromal differentiation patterns in heterotypical models of artificial corneas generated by tissue engineering.

Purpose: We carried out a histological characterization analysis of the stromal layer of human heterotypic cornea substitutes generated with extra-corneal cells to determine their putative usefulness in tissue engineering. Methods: Human bioartificial corneas were generated using nanostructured fibrin-agarose biomaterials with corneal stromal cells immersed within. To generate heterotypical corneas, umbilical cord Wharton's jelly stem cells (HWJSC) were cultured on the surface of the stromal substitutes to obtain an epithelial-like layer. These bioartificial corneas were compared with control native human corneas and with orthotypical corneas generated with human corneal epithelial cells on top of the stromal substitute. Both the corneal stroma and the basement membrane were analyzed using histological, histochemical and immunohistochemical methods in samples kept in culture and grafted in vivo for 12months in the rabbit cornea. Results: Our results showed that the stroma of the bioartificial corneas kept ex vivo showed very low levels of fibrillar and non-fibrillar components of the tissue extracellular matrix. However, in vivo implantation resulted in a significant increase of the contents of collagen, proteoglycans, decorin, keratocan and lumican in the corneal stroma, showing higher levels of maturation and spatial organization of these components. Heterotypical corneas grafted in vivo for 12months showed significantly higher contents of collagen fibers, proteoglycans and keratocan. When the basement membrane was analyzed, we found that all corneas grafted in vivo showed intense PAS signal and higher contents of nidogen-1, although the levels found in human native corneas was not reached, and a rudimentary basement membrane was observed using transmission electron microscopy. At the epithelial level, HWJSC used to generate an epithelial-like layer in ex vivo corneas were mostly negative for p63, whereas orthotypical corneas and heterotypical corneas grafted in vivo were positive. Conclusion: These results support the possibility of generating bioengineered artificial corneas using non-corneal HWJSC. Although heterotypical corneas were not completely biomimetic to the native human corneas, especially ex vivo, in vivo grafted corneas demonstrated to be highly biocompatible, and the animal cornea became properly differentiated at the stroma and basement membrane compartments. These findings open the door to the future clinical use of these bioartificial corneas.