Emerging approaches for ocular surface and corneal stromal regeneration: Recent advances and future perspectives

Abstract

Corneal blindness affects millions worldwide, with a particularly high burden in developing regions, especially in Asia and Africa. In India, the scarcity of donor corneal tissue and challenges in post-transplant care complicate efforts to address this issue. While enhancing the eye banking network and increasing surgical training remain important, corneal transplantation alone cannot address the problem, particularly for high-risk cases such as trauma, infections, and degenerative diseases, which often have poor long-term outcomes. Advances in regenerative medicine and bioengineering offer promising alternatives. Cell-based therapies, including cultivated limbal epithelial and mesenchymal stem cell treatments, aim to restore corneal function through the modulation of native cell behavior. Additionally, cell-free therapies, such as exosomes, decorin, and extracellular matrix derivatives, provide innovative, donor-independent options to reduce scarring and promote healing. Bioengineered corneas and hydrogel scaffolds further reduce dependence on donor tissue, expanding treatment possibilities and alleviating donor shortages. The successful integration of these therapies into clinical practice requires collaboration between research institutions and industry, along with localized manufacturing to ensure affordability and accessibility. To support these advancements, eye banks may need to evolve into comprehensive cell and tissue facilities, ultimately expanding care options for patients with corneal blindness in resource-limited settings.

According to the World Health Organization, corneal blindness constitutes around 5% of total blindness, affecting millions globally.[1] The problem of vision impairment and blindness due to corneal diseases is more prevalent in the developing world, particularly in Asia and Africa.[1] In India, the prevalence of moderate to severe visual impairment and blindness due to corneal diseases is the highest in the South Asian subcontinent.[2] The common causes of corneal blindness in this part of the world include trauma, infection, acquired and inherited corneal disorders, degenerative corneal diseases, chemical injuries, and ocular surface disorders.[2,3] Unfortunately, these etiologies inherently carry a suboptimal prognosis for the long-term survival of corneal grafts.[3] Therefore, when the prevalent high-risk indications are considered along with the global shortage of donor corneal tissue and trained corneal surgeons and the need for long-term follow-up and medications, it becomes apparent that corneal transplantation alone is not a sustainable way of addressing corneal blindness in much of the developing world.[4] Herein lies the need to find alternative approaches that can either circumvent or complement the limitations of corneal transplantation.

The last few decades have witnessed the development of innovative solutions to address the unmet need for corneal transplants and bridge the gap in the demand and supply chain. Apart from the evolution of lamellar corneal procedures, which aim to replace only the affected layers of the cornea and improve long-term survival by reducing the risk of immunological rejection, efforts also are being directed toward using regenerative approaches targeted specifically at the deranged elements within the corneal tissue.[5] Regenerative therapies essentially involve modulating the function of the native cells by using cell-based, molecular therapies or gene therapies.[5] Another approach is to use bioengineering techniques such as bio-fabricated tissue, lenticules, and hydrogels, which are designed to reduce the dependence on donor corneas and long-term medications, as well as improve outcomes.[6] This review focuses on the emerging novel strategies for the treatment of corneal stromal or ocular surface disorders, which are the major causes of corneal blindness and vision impairment in India and much of the developing world. Additionally, this review presents a perspective on how eye hospitals and institutions need to evolve to adapt to this future trend in ocular therapeutics.

Emerging Therapies: Regenerative and Bioengineering Approaches

Regenerative approaches

Advanced therapeutic medicinal products (ATMPs) are a group of diverse products that are at the forefront of novel therapies being developed for ocular disorders. These are generally classified as somatic cell therapy medicinal products (sCTMPs), tissue-engineered products (TEPs), gene therapy medicinal products (GTMPs), and combined ATMPs (cATMPs), which are a combination of two or more of the above.[7] ATMPs have also been developed for corneal and ocular surface disorders. Holoclar, which is a TEP, was the first ATMP to be approved in the EU for the treatment of ocular surface diseases.[8] A human somatic-cell-based product called “Nepic,” having a human autologous corneal derived epithelial sheet, has been approved in Japan for the treatment of limbal stem cell deficiency (LSCD).[9] Use of gene therapy has also been explored in Meesman’s corneal dystrophy to modify the expression of the mutant gene.[10]

Cell-based treatments for the ocular surface and corneal stroma

Ocular surface epithelial homeostasis is maintained by limbal epithelial stem cells (LSCs) in the palisades of Vogt.[11] Damage to the limbus, most commonly due to ocular burns, results in LSCD, which is medically irreversible.[11] LSCs can be transplanted from a healthy eye to restore the normal corneal phenotype and clarity. The conventional technique is called conjunctival-limbal autografting, which involves transplanting a conjunctival-limbal donor lenticule from the healthy fellow eye to the affected eye.[6] Cultivated limbal stem cell transplantation, or Holoclar, was the first ATMP to be approved for the treatment of LSCD. It involves cultivating a small amount of limbal tissue over a scaffold to form an epithelial sheet that is then transplanted. Another source of epithelial cells that has been used for ocular surface reconstruction using laboratory expansion is the oral mucosa. This approach, known as cultivated oral mucosal epithelial transplantation, was made popular by researchers in Japan and is approved for severe ocular surface disorders in their country.[12] In contrast, in most of the developing world, simple limbal epithelial transplantation (SLET) has gained more popularity, possibly owing to its economic and social benefits.[13]

Regenerative approaches are being developed to treat conditions affecting corneal stroma, particularly the ones resulting in corneal scarring and haze. Healthy corneal stroma is composed of collagen lamellae arranged parallelly to allow minimal interference and scattering to the passage of light. Interspersed within the lamellae are corneal stromal keratocytes (CSKs), which are the primary stromal cell type responsible for laying down the collagen and extracellular matrix (ECM).[14] Adult progenitor cells, the corneal stromal stem cells (CSSCs) are located at the anterior limbal stroma in the periphery. However, it is unclear whether CSSCs differentiate into CSKs in vivo.[15] Regenerative treatments to restore the normal function of CSKs are being developed for the treatment of corneal haze and scarring.

Native CSKs are difficult to propagate ex-vivo due to their propensity to differentiate into fibroblasts. However, studies have demonstrated limited expansion of CSKs in controlled conditions to inhibit TGF-beta-mediated profibrotic changes.[15] In vivo regenerative potential of CSSCs has been explored in animal models of corneal injury and scarring and has shown stromal regenerative and scar-inhibitory effects.[16] Another approach that has shown promise is the use of mesenchymal stem cells (MSCs) for stromal regeneration. MSCs are procured from ocular (limbus) as well as non-ocular sites (umbilical cord, adipose tissue, hair follicles, bone marrow, dental pulp) and have the capability to adapt to the features of a corneal stromal keratocyte.[17,18]

Human limbus-derived mesenchymal stem cells (hLMSCs) are unique MSCs found in the eye that have regenerative properties similar to MSCs from other sources, such as the bone marrow and adipose or dental pulp tissue.[19] Found in the peripheral corneal stroma, adjacent to the limbal epithelial stem cells, they are obtained from limbal corneal biopsies and are identified by cell markers such as ABCG2, Nestin, NGFR, Oct4, PAX6, and Sox2 [Fig. 1]. These also differentiate into keratocytes expressing characteristic marker genes (ALDH3A1, AQP1, KERA, and PTGDS).[19,20] hLMSCs help in maintenance of the normal corneal homeostasis. Given their regenerative and therapeutic potential, hLMSCs are being extensively explored for clinical applications. Previous studies confirm that MSCs obtained from limbal biopsies are functionally equivalent to corneal stromal stem cells in terms of mimicking the sphere shape, gene expression patterns, and the ability to construct the stromal ECM in vitro.[20] The recent exploration of hLMSCs’ remodeling, wound healing, and anti-inflammatory properties has opened up the possibility of developing a cell-based approach to treating corneal scars as an alternative to the existing invasive interventions.[21,22] Subsequent innovations in cultivating and transporting hLMSCs have made this approach even more promising. Damala et al.[23] proposed a cost-effective, safe, and efficient transportation method to address the high costs associated with live-cell transfer, which necessitates the maintenance of a cold chain. This method involves an alginate-based encapsulation of hLMSCs, which can then be transported at room temperatures even in Indian summer conditions for 3–5 days without losing cell viability or function, with the intention of making cell-based therapy accessible to remote areas at lower costs. Sahoo et al.[24] demonstrated the successful in vitro isolation, characterization, and culture optimization of hLMSCs in a serum-free environment. This is a significant step forward as it eliminates the need for any animal-derived products in processing hLMSCs, thus making it safer for human use. The hLMSCs, produced in facilities compliant with current good manufacturing practice (cGMP) guidelines, have undergone extensive preclinical animal safety and efficacy assessments based on which the product has received approval from the Central Drugs Standard Control Organization (CDSCO), which is India’s national regulatory body for human clinical trials.[23] Currently, two phase 1 clinical trials (CTRI/2021/07/035034 and CTRI/2020/07/026891) are ongoing, which are evaluating the safety of unpreserved and alginate-preserved hLMSCs in the treatment of corneal burns, scars, and non-infectious ulcers. Both trials have completed patient recruitment and are undertaking the 2-year follow-up to assess safety as mandated by the CDSCO. These are the first approved stem cell clinical trials in ophthalmology in India as per the New Drugs and Clinical Trial Rules, 2019.

Figure 1.

Schematic illustration of human limbus-derived mesenchymal stem cell cultivation and clinical application

The use of MSCs has also been explored in other corneal stromal diseases. MSCs secrete paracrine factors such as TGFβ-3, HGF, and PEDF, which affect stromal remodeling and promote healing and repair.[25] Studies have shown a significant increase in the stromal keratocyte count in patients with advanced keratoconus treated with adipose-derived MSCs. Although long-term results did not show an increase in stromal thickness, there was higher stromal cellularity, new collagen formation, and modulated stromal scarring.[26] By comprehensively studying how MSCs communicate with their surroundings, we can refine existing protocols and develop innovative strategies that ensure long-term effectiveness in treating immune-mediated diseases.[27]

Future research endeavors should be directed toward the investigation of different scaffolds acting as suitable carriers of these cells for further clinical interventions. Another important future direction can be toward exploring the paracrine signaling involved with hLMSCs. Gaining a better understanding of how MSC-based therapies work will make these treatments easier to replicate and apply to a wider range of eye conditions. Hence, future research should focus on exploring how MSCs interact with the unique environment of the eye. This knowledge could help us unlock the full potential of these therapies and bring them closer to becoming a standard part of eye care.

Cell-free therapies

Exosomes: Extracellular vesicles (EVs) or exosomes have recently gained a lot of interest for the treatment of various ocular and systemic diseases. These 70–200-nm sized vesicles are carriers of DNA, RNA, proteins, and lipids, and once endocytosed by a target cell, the contents are released to regulate various signaling cascades.[28] The use of exosomes has the advantage of being stable, safe, and less immunogenic and are easy to administer. Additionally, the dose of the exosomes can be titrated, and it is possible to administer multiple doses. Exosomes derived from MSCs and CSSCs have been shown to reduce stromal scarring and haze.[29,30] However, most studies are on animal models, and further studies are needed to ascertain their efficacy in human corneas.

Decorin: Decorin is a proteoglycan that plays an important role in maintaining corneal transparency. First, it binds to the collagen fibrils in the corneal stroma and maintains the interfibrillar spacing to minimize light scattering. Second, this leucin-rich molecule binds and sequesters TGF-β in the ECM, thereby inhibiting its pro-fibrotic activity. A murine keratitis model has demonstrated topically administered decorin to promote epithelial healing and reduce corneal opacification by reducing the levels of fibronectin, laminin, and α-SMA.[31]

Losartan: Losartan is an angiotensin-converting enzyme (ACE) II antagonist that downregulates the inflammatory response in the corneal stroma. It inhibits the pro-fibrotic TGF-β signaling and myofibroblast generation, thereby preventing corneal scarring.[32] Clinically, it has shown benefit in post-refractive surgery haze, causing significant improvement in patient’s vision and reducing haze when used topically for 4.5 months.[33] In a rabbit model, Sampaio and coworkers combined topical losartan and prednisolone acetate and reported reduced corneal stromal fibrosis in alkali burn.[34] Further trials are needed to understand the concentration and the dose to be used for clinical use.

ECM derivatives: Extracellular matrix provides the structural and functional integrity to tissues throughout the human body. By means of molecules such as matrix metalloproteinases (MMPs), integrins, and growth factors, it modulates cellular processes and signaling pathways to influence cell adhesion, migration, and wound healing.[35] Processed micro-particles from ECM have demonstrated reduced stromal fibrotic activity in rabbit corneal injury models.[36] Amniotic membrane-derived decellularized ECM has also shown the effects of increased wound healing and reduced corneal scarring.[37]

Bioengineering approaches

Bioengineered cornea

The approach of bioengineering focuses on addressing the scarcity of donor corneal tissue by developing a biomimetic substitute that replicates the structure and function of the natural cornea. Bioengineered corneas have caught on in India, and many groups are working on this approach. These bioengineered corneas can be developed by different synthetic and natural polymers such as collagen, gelatin, and silk fibroin, which are some of the materials that have been widely used.[38,39] There has been use of methods such as 3D bioprinting to produce corneal constructs that mimic the natural corneal tissue. In the last few years, Indian researchers have tried different materials and techniques to make the bioengineered cornea more biocompatible, transparent, and having more mechanical strength.[38] The outlook for bioengineered corneas in India is promising, with ongoing research focused on developing corneal substitutes that enhance regeneration using ECM, stem cells, and growth factors. Similarly, advancement in the methods of 3D bioprinting in the future will lead to improved efficiency in the production of engineered corneas in quantities sufficient to meet the unmet needs of the patients.[40] However, bioengineered corneas are still in development or undergoing preclinical validation and must fulfill regulatory requirements before receiving clinical approval. Overcoming these challenges will require collaboration among research institutions and innovations in cost-effective production technologies to enable their adoption in clinical practice.

Hydrogels

Hydrogels are three-dimensional structures consisting of a hydrophilic polymer network that has water swelling capacity and can hold large quantities of water. In Indian ophthalmology, these hydrogels occupy an important place because of their naturally available polymers, such as chitosan, hyaluronic acid, and alginate, and synthetic polymers such as polyvinyl alcohol and polydimethylsiloxane (PDMS), which are frequently cross-linked by physical, chemical, or ionic methods to provide improved mechanical strength and performance in ophthalmic applications.[41] Hydrogels are being developed for applications in ocular regeneration mainly on the basis of biocompatibility, transparency, and mechanical properties of hydrogel. Thus, the use of hydrogels in the field of ophthalmology is versatile and covers the areas of corneal therapeutics, pharmacotherapy, and regenerative medicine. Recent approaches have focused on investigating hydrogels as therapeutic delivery vehicles of growth factors, anti-inflammatory agents, and antibiotics. Notably, a human cornea-derived extracellular matrix hydrogel designed to prevent post-traumatic corneal scarring by mimicking the cornea’s native biochemical environment has been developed to promote natural healing and reduce the need for surgical interventions.[42,43] Various studies are being conducted on the fabrication of stimuli-responsive hydrogels that help in responding to physiological stimuli such as pH and temperature that are beneficial for targeted drug delivery in chronic ocular disorders such as glaucoma and retinal disorders.[44] Among the developments that have breakthrough potential is the incorporation of nanotechnology into the hydrogel system. Use of nanoparticles for nanocomposite hydrogels, enhances mechanical properties, transparency, and drug loading.[45] These exciting developments suggest that India holds great potential to lead global research in ophthalmic biomaterials and bioengineering. However, effective collaboration between Indian institutions will be crucial for advancing the prevention and treatment of ocular diseases by addressing critical challenges and gaps in clinical care and outcomes [Fig. 2].

Figure 2.

Schematic representation of hydrogel and bioengineered cornea preparation

Future Perspectives

Many developing countries, including India, face a substantial burden of corneal blindness, which cannot be fully addressed through corneal transplants alone. There is a pressing need to find ways to circumvent the limitations posed by keratoplasty, such as the risk of rejection and the need for donor corneal tissue, and find ways to use more targeted therapies. Researchers globally are working on regenerative and bioengineering approaches as alternative solutions. One of the ways to bring these novel therapeutic strategies into our practice is to import or locally license and manufacture internationally approved products or drugs in India. A good example of this is the collaboration between Astra-Zeneca and Serum Institute of India, facilitated by the central government, to provide COVID-19 vaccines in our country. The Indian government has now facilitated this further by waiving the need for local clinical trials for drugs already approved in the US, Australia, UK, EU, Canada, and Japan.[46] While this helps in expediting the availability of life-saving drugs, internationally developed therapies are fairly expensive and thus out of reach of most of the Indian population in need. For example, Luxturna, which is a gene therapy for inherited retinal dystrophy, costs more than INR 5,00,00,000 for a one-time treatment.[47]

The second approach is to invest in local research and development and manufacture the products in India, making them more affordable, easy to deliver, and accessible to patients in need. Collaborations among academic institutes and industries or start-ups manufacturing these products are essential to facilitate this process. The products made by the industry partners could be validated clinically in the ophthalmology institutes, and in the future, personalized medicine using these novel approaches could be developed. With the emerging developments in ocular therapeutics, there is a need for academic and research-oriented ophthalmology institutions to develop in-house capacity to develop, manufacture, and test these products. There is an opportunity for the eye bank to evolve into a biologics hub or an integrated cell, tissue, and eye bank from its traditional role of procuring, processing, and distributing corneal tissue[48] [Fig. 3]. Organizations having individual eye banks can upgrade their facilities to cGMP and good laboratory practice (GLP) compliance to equip themselves for these future avenues. It is also important to understand that ATMPs and other emerging therapies in ophthalmology are not going to be limited to the corneal or ocular surface but involve retinal or optic nerve therapies as well.[49] In the future, eye banks could truly live up to their name, serving the full spectrum of ocular tissues.

Figure 3.

Schematic representation of the proposed integrated cell-tissue eye bank serving the full spectrum of ocular tissues

In summary, the growing burden of corneal blindness in developing countries necessitates innovative approaches beyond traditional transplantation. Regenerative therapies and bioengineered solutions present promising alternatives to address the limitations of donor tissue availability and graft survival. As these technologies advance, integrating them into clinical practice will require collaboration between research institutions and industry, alongside the adaptation of eye banks into comprehensive cell and tissue facilities. Future efforts should focus on optimizing these therapies for widespread, cost-effective use, with the ultimate goal of making advanced corneal treatments accessible to the populations most in need.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Hyderabad Eye Research Foundation (HERF), Hyderabad, Telangana, India.