Immunophenotypical Characterization of Limbal Mesenchymal Stromal Cell Subsets during In Vitro Expansion.

Limbal mesenchymal stromal cells (LMSCs) reside in the limbal niche, supporting corneal integrity and facilitating regeneration. While mesenchymal stem/stromal cells (MSCs) are used in regenerative therapies, there is limited knowledge about LMSC subpopulations and their characteristics. This study characterized human LMSC subpopulations through the flow cytometric assessment of fifteen cell surface markers, including MSC, wound healing, immune regulation, ASC, endothelial, and differentiation markers. Primary LMSCs were established from remnant human corneal transplant specimens and passaged eight times to observe changes during subculture. The results showed the consistent expression of typical MSC markers and distinct subpopulations with the passage-dependent expression of wound healing, immune regulation, and differentiation markers. High CD166 and CD248 expressions indicated a crucial role in ocular surface repair. CD29 expression suggested an immunoregulatory role. Comparable pigment-epithelial-derived factor (PEDF) expression supported anti-inflammatory and anti-angiogenic roles. Sustained CD201 expression indicated maintained differentiation capability, while VEGFR2 expression suggested potential endothelial differentiation. LMSCs showed higher VEGF expression than fibroblasts and endothelial cells, suggesting a potential contribution to ocular surface regeneration through the modulation of angiogenesis and inflammation. These findings highlight the heterogeneity and multipotent potential of LMSC subpopulations during in vitro expansion, informing the development of standardized protocols for regenerative therapies and improving treatments for ocular surface disorders.

Mesenchymal Stem Cells and Exosomes: A Novel Therapeutic Approach for Corneal Diseases.

The cornea, with its delicate structure, is vulnerable to damage from physical, chemical, and genetic factors. Corneal transplantation, including penetrating and lamellar keratoplasties, can restore the functions of the cornea in cases of severe damage. However, the process of corneal transplantation presents considerable obstacles, including a shortage of available donors, the risk of severe graft rejection, and potentially life-threatening complications. Over the past few decades, mesenchymal stem cell (MSC) therapy has become a novel alternative approach to corneal regeneration. Numerous studies have demonstrated the potential of MSCs to differentiate into different corneal cell types, such as keratocytes, epithelial cells, and endothelial cells. MSCs are considered a suitable candidate for corneal regeneration because of their promising therapeutic perspective and beneficial properties. MSCs compromise unique immunomodulation, anti-angiogenesis, and anti-inflammatory properties and secrete various growth factors, thus promoting corneal reconstruction. These effects in corneal engineering are mediated by MSCs differentiating into different lineages and paracrine action via exosomes. Early studies have proven the roles of MSC-derived exosomes in corneal regeneration by reducing inflammation, inhibiting neovascularization, and angiogenesis, and by promoting cell proliferation. This review highlights the contribution of MSCs and MSC-derived exosomes, their current usage status to overcome corneal disease, and their potential to restore different corneal layers as novel therapeutic agents. It also discusses feasible future possibilities, applications, challenges, and opportunities for future research in this field.

Human SMILE-Derived Stromal Lenticule Scaffold for Regenerative Therapy: Review and Perspectives.

A transparent cornea is paramount for vision. Corneal opacity is one of the leading causes of blindness. Although conventional corneal transplantation has been successful in recovering patients' vision, the outcomes are challenged by a global lack of donor tissue availability. Bioengineered corneal tissues are gaining momentum as a new source for corneal wound healing and scar management. Extracellular matrix (ECM)-scaffold-based engineering offers a new perspective on corneal regenerative medicine. Ultrathin stromal laminar tissues obtained from lenticule-based refractive correction procedures, such as SMall Incision Lenticule Extraction (SMILE), are an accessible and novel source of collagen-rich ECM scaffolds with high mechanical strength, biocompatibility, and transparency. After customization (including decellularization), these lenticules can serve as an acellular scaffold niche to repopulate cells, including stromal keratocytes and stem cells, with functional phenotypes. The intrastromal transplantation of these cell/tissue composites can regenerate native-like corneal stromal tissue and restore corneal transparency. This review highlights the current status of ECM-scaffold-based engineering with cells, along with the development of drug and growth factor delivery systems, and elucidates the potential uses of stromal lenticule scaffolds in regenerative therapeutics.

PFN1 and integrin-ß1/mTOR axis involvement in cornea differentiation of fibroblast limbal stem cells.

Ex vivo limbal stem cell transplantation is the main therapeutic approach to address a complete and functional re-epithelialization in corneal blindness, the second most common eye disorder. Although important key points were defined, the molecular mechanisms involved in the epithelial phenotype determination are unclear. Our previous studies have demonstrated the pluripotency and immune-modulatory of fibroblast limbal stem cells (f-LSCs), isolated from the corneal limbus. We defined a proteomic profile especially enriched in wound healing and cytoskeleton-remodelling proteins, including Profilin-1 (PFN1). In this study we postulate that pfn-1 knock down promotes epithelial lineage by inhibiting the integrin-ß1(CD29)/mTOR pathway and subsequent NANOG down-expression. We showed that it is possible modulate pfn1 expression levels by treating f-LSCs with Resveratrol (RSV), a natural compound: pfn1 decline is accompanied with up-regulation of the specific differentiation epithelial genes pax6 (paired-box 6), sox17 (sex determining region Y-box 17) and ΔNp63-α (p63 splice variant), consistent with drop-down of the principle stem gene levels. These results contribute to understand the molecular biology of corneal epithelium development and suggest that pfn1 is a potential molecular target for the treatment of corneal blindness based on epithelial cell dysfunction.

Current Trends and Future Perspective of Mesenchymal Stem Cells and Exosomes in Corneal Diseases.

The corneal functions (transparency, refractivity and mechanical strength) deteriorate in many corneal diseases but can be restored after corneal transplantation (penetrating and lamellar keratoplasties). However, the global shortage of transplantable donor corneas remains significant and patients are subject to life-long risk of immune response and graft rejection. Various studies have shown the differentiation of multipotent mesenchymal stem cells (MSCs) into various corneal cell types. With the unique properties of immunomodulation, anti-angiogenesis and anti-inflammation, they offer the advantages in corneal reconstruction. These effects are widely mediated by MSC differentiation and paracrine signaling via exosomes. Besides the cell-free nature of exosomes in circumventing the problems of cell-fate control and tumorigenesis, the vesicle content can be genetically modified for optimal therapeutic affinity. The pharmacology and toxicology, xeno-free processing with sustained delivery, scale-up production in compliant to Good Manufacturing Practice regulations, and cost-effectiveness are the current foci of research. Routes of administration via injection, topical and/or engineered bioscaffolds are also explored for its applicability in treating corneal diseases.

Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method.

The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration.

In Situ-Forming, Bioorthogonally Cross-linked, Nanocluster-Reinforced Hydrogel for the Regeneration of Corneal Defects.

Corneal defects can lead to stromal scarring and vision loss, which is currently only treatable with a cadaveric corneal transplant. Although in situ-forming hydrogels have been shown to foster regeneration of the cornea in the setting of stromal defects, the cross-linking, biomechanical, and compositional parameters that optimize healing have not yet been established. This, Corneal defects are also almost universally inflamed, and their rapid closure without fibrosis are critical to preserving vision. Here, an in situ forming, bioorthogonally cross-linked, nanocluster (NC)-reinforced collagen and hyaluronic acid hydrogel (NCColHA hydrogel) with enhanced structural integrity and both pro-regenerative and anti-inflammatory effects was developed and tested within a corneal defect model in vivo. The NCs serve as bioorthogonal nanocross-linkers, providing higher cross-linking density than polymer-based alternatives. The NCs also serve as delivery vehicles for prednisolone (PRD) and the hepatocyte growth factor (HGF). NCColHA hydrogels rapidly gel within a few minutes upon administration and exhibit robust rheological properties, excellent transparency, and negligible swelling/deswelling behavior. The hydrogel's biocompatibility and capacity to support cell growth were assessed using primary human corneal epithelial cells. Re-epithelialization on the NCColHA hydrogel was clearly observed in rabbit eyes, both ex vivo and in vivo, with expression of normal epithelial biomarkers, including CD44, CK12, CK14, α-SMA, Tuj-1, and ZO-1, and stratified, multilayered morphology. The applied hydrogel maintained its structural integrity for at least 14 days and remodeled into a transparent stroma by 56 days.

Photoactivated growth factor release from bio-orthogonally crosslinked hydrogels for the regeneration of corneal defects.

In situ-forming hydrogels are an attractive option for corneal regeneration, and the delivery of growth factors from such constructs have the potential to improve re-epithelialization and stromal remodeling. However, challenges persist in controlling the release of therapeutic molecules from hydrogels. Here, an in situ-forming bio-orthogonally crosslinked hydrogel containing growth factors tethered via photocleavable linkages (PC-HACol hydrogel) was developed to accelerate corneal regeneration. Epidermal growth factor (EGF) was conjugated to the hydrogel backbone through photo-cleavable (PC) spacer arms and was released when exposed to mild intensity ultraviolet (UV) light (2-5 mW/cm2, 365 nm). The PC-HACol hydrogel rapidly gelled within a few minutes when applied to corneal defects, with excellent transparency and biocompatibility. After subsequent exposure to UV irradiation, the hydrogel promoted the proliferation and migration of corneal epithelial cells in vitro. The rate of re-epithelialization was positively correlated to the frequency of irradiation, verified through ex vivo rabbit cornea organ culture studies. In an in vivo rat corneal wound healing study, the PC-HACol hydrogel exposed to UV light significantly promoted re-epithelialization, the remodeling of stromal layers, and exhibited significant anti-scarring effects, with minimal α-SMA and robust ALDH3A1 expression. Normal differentiation of the regenerated epithelia after healing was evaluated by expression of the corneal epithelial biomarker, CK12. The remodeled cornea exhibited full recovery of corneal thickness and layer number without hyperplasia of the epithelium.

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.

Properties of Dual-Crosslinked Collagen-Based Membranes as Corneal Repair Material.

Corneal disease has become the second leading cause of blindness in the world. Corneal transplantation is currently considered to be one of the common treatments for vision loss. This paper presents a novel approach utilizing dual-crosslinked membranes composed of polyrotaxane multiple aldehydes (PRAs), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) in the development process. Collagen was crosslinked, respectively, by EDC/NHS and PRAs to form stable amide bonds and imine groups. Through the formation of a double interpenetrating network, dual-crosslinked (Col-EDC-PRA) membranes exhibited enhanced resistance to collagenase degradation and superior mechanical properties compared to membranes crosslinked with a single crosslinker. Furthermore, Col-EDC-PRA membranes display favorable light transmittance and water content characteristics. Cell experiments showed that Col-EDC-PRA membranes were noncytotoxic and were not significantly different from other membranes. In a rabbit keratoplasty model, corneal stromal repair occurred at 5 months, evidenced by the presence of stromal cells and neo-stroma, as depicted in hematoxylin-eosin-stained histologic sections and optical coherence tomography images of the anterior segment. Moreover, there was no inflammation and corneal neovascularization, as well as no corneal rejection reaction in the surgical area. Overall, the results demonstrated that the dual-crosslinked membranes served effectively for corneal tissue regeneration after corneal defect.

Elucidating the Corneal Endothelial Cell Proliferation Capacity through an Interspecies Transcriptome Comparison.

The regenerative capacity of corneal endothelial cells (CECs) differs between species; in bigger mammals, CECs are arrested in a non-proliferative state. Damage to these cells can compromise their function causing corneal opacity. Corneal transplantation is the current treatment for the recovery of clear eyesight, but the donor tissue demand is higher than the availability and there is a need to develop novel treatments. Interestingly, rabbit CECs retain a high proliferative profile and can repopulate the endothelium. There is a lack of fundamental knowledge to explain these differences. Gaining information on their transcriptomic variances could allow the identification of CEC proliferation drivers. In this study, human, sheep, and rabbit CECs are analyzed at the transcriptomic level. To understand the differences across each species, a pipeline for the analysis of pathways with different activities is generated. The results reveal that 52 pathways have different activity when comparing species with non-proliferative CECs (human and sheep) to species with proliferative CECs (rabbit). The results show that Notch and TGF-ß pathways have increased activity in species with non-proliferative CECs, which might be associated with their low proliferation. Overall, this study illustrates transcriptomic pathway-level differences that can provide leads to develop novel therapies to regenerate the corneal endothelium.

Biomimetic Convex Implant for Corneal Regeneration Through 3D Printing.

Blindness caused by corneal damage affects millions of people worldwide, and this number continues to rise. However, rapid epithelization and a stable epithelium process are the two biggest challenges for traditional corneal materials. These processes are related to corneal curvature, which is an important factor in determination of the corneal healing process and epithelial behavior during corneal damage. In this study, smooth 3D-printed convex corneal implants based on gelatin methacrylate and collagen are generated. As epithelium distribution and adhesion vary in different regions of the natural cornea, this work separates the surfaces into four regions and studies how cells sense topological cues on curvature. It is found that rabbit corneal epithelial cells (RCECs) seeded on steeper slope gradient surfaces on convex structures result in more aligned cell organization and tighter cell-substrate adhesion, which can also be verified through finite element simulation and signaling pathway analysis. In vivo transplantation of convex implants result in a better fit with adjacent tissue and stronger cell adhesion than flat implants, thereby accelerating corneal epithelialization and promoting collagen fibers and neural regeneration within 180 days. Taken together, printed convex corneal implants that facilitate corneal regeneration may offer a translational strategy for the treatment of corneal damage.

Human cornea-derived extracellular matrix hydrogel for prevention of post-traumatic corneal scarring: A translational approach.

Corneal scarring and opacification are a significant cause of blindness affecting millions worldwide. The current standard of care for corneal blindness is corneal transplantation, which suffers from several drawbacks. One alternative approach that has shown promise is the use of xenogeneic corneal extracellular matrix (ECM), but its clinical applicability is challenging due to safety concerns. This study reports the innovative use of human cornea-derived ECM to prevent post-traumatic corneal scarring. About 30 - 40% of corneas donated to the eye banks do not meet the standards defined for clinical use and are generally discarded, although they are completely screened for their safety. In this study, human cornea-derived decellularized ECM hydrogel was prepared from the non-transplantation grade human cadaveric corneas obtained from an accredited eye-bank. The prepared hydrogel was screened for its efficacy against corneal opacification following an injury in an animal model. Our in vivo study revealed that, the control collagen-treated group developed corneal opacification, while the prophylactic application of human cornea-derived hydrogel effectively prevented corneal scarring and opacification. The human hydrogel-treated corneas were indistinguishable from healthy corneas and comparable to those treated with the xenogeneic bovine corneal hydrogel. We also demonstrated that the application of the hydrogel retained the biological milieu including cell behavior, protein components, optical properties, curvature, and nerve regeneration by remodeling the corneal wound after injury. The hydrogel application is also sutureless, resulting in faster corneal healing. We envision that this human cornea-derived ECM-based hydrogel has potential clinical application in preventing scarring from corneal wounding. STATEMENT OF SIGNIFICANCE: There are significant challenges surrounding corneal regeneration after injury due to extensive scarring. Although there is substantial research on corneal regeneration, much of it uses synthetic materials with chemical cross-linking methods or xenogeneic tissue-based material devices which have to undergo exhaustive safety analysis before clinical trials. Herein, we demonstrate the potential application of a human corneal extracellular matrix hydrogel without any additional materials for scarless corneal tissue regeneration, and a method to reduce the wasting of donated allogenic corneal tissue from eye banks. We found no difference in efficacy between the usage of human tissues compared to xenogeneic sources. This may help ease clinical translation and can be used topically without sutures as an outpatient procedure.

ECM-Like Adhesive Hydrogel for the Regeneration of Large Corneal Stromal Defects.

The repair of large-diameter corneal stroma defects is a major clinical problem. Although some studies have attempted to use hydrogels to repair corneal damage, most of these hydrogels can only be used for focal stromal defects that are ≤3.5 mm in diameter due to poor hydrogel adhesion. Here, a photocurable adhesive hydrogel that mimics the extracellular matrix (ECM) with regard to composition for repairing 6 mm-diameter corneal stromal defects in rabbits is investigated. This ECM-like adhesive can be rapidly cured after light exposure, with high light transmittance and good mechanical properties. More importantly, this hydrogel maintains the viability and adhesion of cornea-derived cells and promotes their migration in vitro in 2D and 3D culture environments. Proteomics analysis confirms that the hydrogel promotes cell proliferation and ECM synthesis. Furthermore, in rabbit corneal stromal defect repair experiments, it is proven by histological and proteomic analysis that this hydrogel can effectively promote corneal stroma repair, reduce scar formation, and increase corneal stromal-neural regeneration at the six months follow-up. This work demonstrates the great application of ECM-like adhesive hydrogels for the regeneration of large-diameter corneal defects.

Digital light processing-bioprinted poly-NAGA-GelMA-based hydrogel lenticule for precise refractive errors correction.

Refractive disorder is the most prevalent cause of visual impairment worldwide. While treatment of refractive errors can bring improvement to quality of life and socio-economic benefits, there is a need for individualization, precision, convenience, and safety with the chosen method. Herein, we propose using pre-designed refractive lenticules based on poly-NAGA-GelMA (PNG) bio-inks photo-initiated by digital light processing (DLP)-bioprinting for correcting refractive errors. DLP-bioprinting allows PNG lenticules to have individualized physical dimensions with precision achievable to 10µm (µm). Material characteristics of PNG lenticules in tests included optical and biomechanical stability, biomimetical swelling and hydrophilic capability, nutritional and visual functionality, supporting its suitability as stromal implants. Cytocompatibility distinguished by morphology and function of corneal epithelial, stromal, and endothelial cells on PNG lenticules suggested firm adhesion, over 90% viability, phenotypic maintenance instead of excessive keratocyte-myofibroblast transformation.In-vitroimmune response analyzed by illumina RNA sequencing in human peripheral blood mononuclear cells indicated that PNG lenticules activated type-2 immunity, facilitating tissue regeneration and suppressing inflammation.In-vivoperformance assessed using intrastromal keratoplasty models in New Zealand white rabbits illustrated that implantation of PNG lenticules maintained stable optical pathway, induced controlled stromal bio-integration and regeneration, avoided complications such as stromal melt, interface scarring, etc, but exerted no adverse effects on the host. Postoperative follow-up examination on intraocular pressure, corneal sensitivity, and tear production remained unaffected by surgery up to 1-month post-implantation of PNG lenticules. DLP-bioprinted PNG lenticule is a bio-safe and functionally effective stromal implants with customizable physical dimensions, providing potential therapeutic strategies in correction of refractive errors.

Developing a Novel Platelet-Rich Plasma-Laden Bioadhesive Hydrogel Contact Lens for the Treatment of Ocular Surface Chemical Injuries.

Permanent injury to corneal limbal stem cells after ocular surface chemical and thermal injuries is a major cause of corneal blindness. In this study, a PRP-laden GelMA hydrogel contact lens is manufactured which is aimed to support the limbal niche after ocular surface insults thereby preventing limbal stem cell failure. GelMA with varying platelet-rich plasma (PRP) concentrations (5%, 10%, and 20%) is photopolymerized using a visible light crosslinking system followed by characterizations of mechanical properties, growth factor release, enzymatic degradation, and in vitro cytotoxicity. The addition of 10% PRP into 10% GelMA hydrogel precursor solution results in the highest tensile and compressive modulus (38 and 110 kPa, respectively) and burst pressure (251±37.66 mmHg). Degradation time varies according to the concentration of the collagenase enzyme tested (0, 2.5, 5, and 40 µg/mL) and is most prolonged with 20% PRP. EGF and TGF-ß release profiles suggest an initial burst release followed by sustained release, most consistent in the 10% PRP sample. Although cell viability decreases on day 1, rapid recovery is observed and is approximately 120% after day 21. PRP-laden GelMA in the form of a contact lens may be a promising biomaterial-based treatment approach for the maintenance of limbal epithelial stem cells after ocular surface insults.

Rho-kinase Inhibitors in Ocular Diseases: A Translational Research Journey.

Aim: This review summarizes current data on Rho-kinase (ROCK) inhibitors use in ocular diseases, primarily glaucoma. Background: Translational research over the last decade culminating in the development of ROCK inhibitors has provided a much-needed shot in the arm to glaucoma pharmacopeia. ROCK pathway is intricately involved in cytoskeletal modulation with action on cell morphology, cell motility, cell adhesion, cell apoptosis, and smooth muscle contraction. This cytoskeletal modulation property has been utilized to modify trabecular meshwork (TM) resistance, resulting in the discovery of ROCK inhibitors to increase trabecular outflow. Review results: Multicentric trials on ROCK inhibitors for antiglaucoma medications are summarized. The focus is on linking pharmacological action to the clinical utility of these drugs. While the Rho Kinase Elevated intraocular Pressure (IOP) Treatment (ROCKET) trials compared monotherapy with ROCK inhibitor netarsudil vs timolol, MERCURY trials compared a fixed dose combination of latanoprost and ROCK inhibitor netarsudil [fixed combination netarsudil-latanoprost (FCNL)] vs monotherapy with either and bimatoprost-timolol combination. While ROCKET trials showed ROCK inhibitors to be non-inferior to timolol, MERCURY trials showed FCNL achieving a much greater IOP reduction than monotherapy with either. Conjunctival hyperemia was the most common side effect reported with ROCK inhibitor use. Conclusion: Moderate efficacy of ROCK inhibitors with a common side effect of conjunctival hyperemia, makes it an adjunctive antiglaucoma drug of choice and not a first-line therapy. Clinical significance: ROCK inhibitors' action on diseased TM is more physiological compared to available antiglaucoma medications that either reduce aqueous secretion or enhance uveoscleral outflow. The property of ROCK inhibition to stabilize the endothelium of both retinal vasculature and cornea has opened a new chapter in the treatment of diabetic retinopathy and corneal decompensation. How to cite this article: Singh K, Singh A. Rho-kinase Inhibitors in Ocular Diseases: A Translational Research Journey. J Curr Glaucoma Pract 2023;17(1):44-48.

3D printed biomimetic epithelium/stroma bilayer hydrogel implant for corneal regeneration.

Corneal regeneration has always been a challenge due to its sophisticated structure and undesirable keratocyte-fibroblast transformation. Herein, we propose 3D printing of a biomimetic epithelium/stroma bilayer implant for corneal regeneration. Gelatin methacrylate (GelMA) and long-chain poly(ethylene glycol) diacrylate (PEGDA) are blended to form a two-component ink, which can be printed to different mechanically robust programmed PEGDA-GelMA objects by Digital Light Processing (DLP) printing technology, due to the toughening effect of crystalline crosslinks from long-chain PEGDA on GelMA hydrogel after photo-initiated copolymerization. The printed PEGDA-GelMA hydrogels support cell adhesion, proliferation, migration, meanwhile demonstrating a high light transmittance, and an appropriate swelling degree, nutrient permeation and degradation rate. A bi-layer dome-shaped corneal scaffold consisting of rabbit corneal epithelial cells (rCECs)-laden epithelia layer and rabbit adipose-derived mesenchymal stem cells (rASCs)-laden orthogonally aligned fibrous stroma layer can be printed out with a high fidelity and robustly surgical handling ability. This bi-layer cells-laden corneal scaffold is applied in a rabbit keratoplasty model. The post-operative outcome reveals efficient sealing of corneal defects, re-epithelialization and stromal regeneration. The concerted effects of microstructure of 3D printed corneal scaffold and precisely located cells in epithelia and stroma layer provide an optimal topographical and biological microenvironment for corneal regeneration.

Alginate-Based Composites for Corneal Regeneration: The Optimization of a Biomaterial to Overcome Its Limits.

For many years, corneal transplantation has been the first-choice treatment for irreversible damage affecting the anterior part of the eye. However, the low number of cornea donors and cases of graft rejection highlighted the need to replace donor corneas with new biomaterials. Tissue engineering plays a fundamental role in achieving this goal through challenging research into a construct that must reflect all the properties of the cornea that are essential to ensure correct vision. In this review, the anatomy and physiology of the cornea are described to point out the main roles of the corneal layers to be compensated and all the requirements expected from the material to be manufactured. Then, a deep investigation of alginate as a suitable alternative to donor tissue was conducted. Thanks to its adaptability, transparency and low immunogenicity, alginate has emerged as a promising candidate for the realization of bioengineered materials for corneal regeneration. Chemical modifications and the blending of alginate with other functional compounds allow the control of its mechanical, degradation and cell-proliferation features, enabling it to go beyond its limits, improving its functionality in the field of corneal tissue engineering and regenerative medicine.

Stability and Function of Extracellular Vesicles Derived from Immortalized Human Corneal Stromal Stem Cells: A Proof of Concept Study.

With significant advancement and development of extracellular vesicle (EV)-based therapies, there is a growing need to understand how their storage affects their physical and functional characteristics. EVs were isolated from the conditioned medium of a corneal stromal stem cell line (imCSSC) using Total Exosome isolation kit (TEI) and ultracentrifugation (UC) combined protocol. Purified EVs were stored at 4°C, - 80°C, room temperature (RT) after lyophilization with or without trehalose for 4 weeks. EVs stored at - 80°C and RT (lyophilization with trehalose) demonstrated a comparable morphology, while the freeze-dried samples without trehalose showed aggregation and degradation under a transmission electron microscope (TEM). Lyophilized samples without trehalose demonstrated a decreased particle concentration, recovery rate and protein concentration, which was remediated by the addition of trehalose. EVs stored at - 80℃ showed no change in the protein expression of CD9, CD63, and CD81. Regardless of the storage condition, all EV samples investigated reduced inflammation, as well as inhibited expression of fibrotic markers in vitro. Lyophilization of EVs with trehalose was a feasible storage method that retained the physical property and in vitro biological activities of EVs after 4 weeks of storage, while - 80°C offered the best retention of imCSSC-derived EV physical properties. For the first time, this data demonstrated a practical and translatable method for the storage of CSSC-derived EVs for clinical use.